Evaluating Immunoreactivity of Polyclonal Antibodies Developed against AU-565 Cell Line for Diagnosis and Immunotherapy of Breast Cancer

Evaluating Immunoreactivity of Polyclonal Antibodies Developed against AU-565 Cell Line for Diagnosis and Immunotherapy of Breast Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluating Immunoreactivity of Polyclonal Antibodies Developed against AU-565 Cell Line for Diagnosis and Immunotherapy of Breast Cancer Murat IHLAMUR, Atıfcan DEMİRCİOĞLU, Aslı Pınar ZORBA, Emrah Şefik ABAMOR, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3966637/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Breast cancer is the most commonly diagnosed cancer type in women and approximately 700 thousand people around the world lose their lives due to breast cancer every year. Mammography and ultrasound are the techniques that are frequently applied for the diagnosis of breast cancer. However they involve several limitations such as low sensitivity and exposing to high radiation. Additionally, false negative and false positive results could be obtained in conventional diagnostic methods for breast cancer. So it is crucial to generate new diagnostic kits which enable rapid and accurate detection of breast cancer. Antibodies created using hybridoma technology can be considered in the diagnostic kits since they are important tools to bind cancer cell antigens. Although monoclonal antibodies are usually utilized in antibody-mediated diagnostic kits and they possess high specificity in diagnosis and treatment, they conversely indicate low avidity to tumor antigens in comparison to polyclonal antibodies because they can only bind to a single epitope region. Therefore, polyclonal antibodies display a pivotal role in recognition of many epitopes of breast cancer cells. The major aim of this study is to create polyclonal antibodies against whole cell lysate of AU-565 cell line by hybridoma technology and examine their diagnostic value by comparing with conventional antibodies. The acquired tumor cell antigens were supplemented with two distinct adjuvants Complete Freund’s Adjuvant (CFA) and Polyoxidonium (PO) while preparing the formulations for immunization. Thus we also evaluated in vivo immunogenic properties of antigen-adjuvant combinations and compared immunostimulatory efficacies of CFA and PO over prepared antigens. The outputs revealed that whole cell antigens reinforced with CFA demonstrated robust immunostimulatory activities, in vivo by enhancing the produced antibody levels in mice excessively. Polyclonal antibodies that were obtained from spleens of mice immunized with AU-565 cell antigens and CFA combinations were highly effective to capture the antigens that were isolated from different breast cancer cell line. It was detected that obtained polyclonal antibodies exhibited stronger immune reactions with breast cancer antigens when compared with conventional antibodies. Consequently, considerable immunostimulatory performance of AU-565 cell antigens and CFA combination was shown as a vaccine candidate and high diagnostic value of polyclonal antibodies produced in response to vaccination with mentioned formulation was established for the first time in the present study. Breast cancer polyclonal antibodies hybridoma diagnosis immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introductıon Cancer occurs as a result of uncontrolled division of cells. Although there are many types of cancer, the most frequently diagnosed cancer in women worldwide is breast cancer with more than 2 million new cases in 2020. Breast cancer is the second most common cause of cancer death among women in the world. At the same time, its incidence and mortality rates have been increasing steadily over the past three decades. It is estimated that 2.7 million new breast cancer cases will be detected worldwide in 2030, and deaths from breast cancer will reach 870 thousand [ 1 ]. Treatment methods such as surgery, chemotherapy and radiotherapy are involved in the treatment of breast cancer. With the development of immunotherapy and targeted drug treatment approaches, more effective results could be obtained in breast cancer treatments [ 2 ]. Cancer immunotherapy, as an alternative treatment method, has developed significantly in recent years. Unlike other therapeutic concepts, immunotherapy primarily aims at preventing the metastatic spread of the disease and improving the life quality of affected individuals. Moreover, by introducing cancer cell antigens to B and T lymphocytes, immunotherapy can induce immunity on individuals before the onset of the disease, and provide a faster response than current treatments. Furthermore, therapeutic vaccination is also accepted as a part of immunotherapy since administration of antibodies can recognize and destroy cancer cells. However, no effective and reliable vaccine formulation has been created against breast cancer so far. Another important limitation of breast cancer therapy is the inadequacy of present diagnostic methods [ 3 ]. Mammography and ultrasound are the techniques that are frequently applied for breast cancer diagnosis. Acquisition of false positive and false negative results is the major limitation of mammography applications. Moreover, suspicious individuals are exposed to high radiation during diagnosis with mammography. On the other side, some small solid tumors can miss from ultrasound screening despite the fact that this technique does not require radiation. In addition, sensitivity of ultrasound is not high as mammograpghy for screening of breast cancer cells[ 4 ]. Finally, some breast cancer types are not diagnosed by screening and invasive technique such as biopsy is the only option that would be successful to provide accurate diagnosis in that cases[ 5 ]. Owing to limitations of current diagnostic methods, it is very important to develop new diagnostic kits for the accurate and rapid identification of breast cancer. Polyclonal and monoclonal antibodies which are produced by hybridoma technology have been used in the diagnosis of many diseases in recent years. The biggest advantage of hybridoma technology-based diagnostic kits is that they demonstrate high specificity to capture special antigens[ 6 ]. In addition design of the most appropriate protocol to produce hybridoma technology-based monoclonal antibodies is so important. The most suitable adjuvant should be selected to improve immunogenicities of used antigens during immunization of animals because this directly influences the specificity of produced antibodies to capture special antigens[ 7 ]. However, the diagnostic values of hybridoma-based antibodies that have been created to date was not adequate enough so they have failed to be used for preparation of diagnostic kits against breast cancer. We assume that diagnostic performances of produced mono or polyclonal antibodies could be advanced by administration of a strong adjuvant in immunization. In the scope of this study, we explored the assistant performances of one microorganism based adjuvant such as Complete Freund's and one polymeric adjuvant called as Polyoxidonium (PO) in order to check their capacities to improve immunogenicities of whole breast cancer cell antigens. In this study, the importance and effects of adjuvants in hybridoma technology were also investigated. Although in some studies it was stated that Complete Freund's adjuvant is more toxic and not active enough compared to other adjuvants there are also several studies in which Complete Freund's adjuvant (CFA) was used and positive results were obtained in stimulation of humoral and cellular immune responses [ 8 – 10 ]. However, until now, studies examining the antibody production of hybridomas created using breast cancer cell antigens in conjunction with Complete Freund's adjuvant and the determination of the diagnostic valence of the obtained antibodies are quite insufficient. There is also no study in the literature that invesitigates diagnostic and immunogenic features of antibodies acquired by hybridoma technology that are produced against breast cancer cell antigens supported by PO as an adjuvant. Hence the main goal of the present study was to generate hybridoma-based polyclonal antibodies against AU-565 breast cancer cell line antigens accompanied by CFA or PO as an adjuvant and explore the comparative efficacies of these two adjuvants over diagnostic values of produed polyclonal antibodies. The immunogenic features of antigens in combinations with CFA or PO was also compared in current study. Material and method Cell Culture AU-565 breast cancer cell line culture used for antigen production was grown in RPMI-1640 medium containing 10% FBS, 1% Pen-step. For cytotoxicity and nitric oxide studies, L929 Fibroblast cell line culture was grown in RPMI-1640 medium containing 10% FBS, 1% Pen-step, and J774 macrophage cell line culture was grown in RPMI-1640 medium containing 10% FBS, 1% Pen-step. P3-X63-Ag8.653 myeloma cell line used for fusion was cultured. P3-X63-Ag8.653 myeloma cell line was grown in with RPMI-1640 containing 20% FBS and 1% Pen-Strep and treated with 20 µg 8-azaguanine/mL prior to the hybridoma protocol [ 11 ]. Preparation of Breast Cancer Antigens Sonication procedure was applied to obtain AU-565 breast cancer antigens. The cell pellet was attached to the sonicator probe. The sonicator was set to 40 seconds and 3 Cycles. The power potentiometer of the sonicator generator was set to 40%. Sonication was repeated 5 times. The resulting lysate was centrifuged at 10,000 rpm for 3 minutes. The supernatant was taken and measured in UV-Vis spectrophotometer. To determine the protein content inside the lysate, the Warburg-Christian method was used with a UV-vis spectrometer under 280 and 260 nm wavelengths [ 12 ]. NO ve MTT Analysis The amount of nitric oxide (NO) produced by the cells was determined by the Griess method in order to determine the immunostimulatory activities of the vaccine formulations prepared at different concentrations by combining the antigens obtained by sonication method alone or in combination with different adjuvants in macrophage cell culture systems. Antigen prepared at seven different concentrations (10 µg/ml, 20 µg/ml, 40 µg/ml, 80 µg/ml, 100 µg/ml120 µg/ml, 160 µg/ml) after incubation of 1*10 5 cells/ml macrophage cells in an oven at 37⁰C containing 5% CO 2 for 24 hours, and antigen + adjuvant (Complete Freund's (40 µg/ml) and PO (100 µg/ml) combinations were added. Supernatants were collected after 48 hours of incubation. Griess reagent was added to the supernatants and reacted. For Griess reagent, 2.5 ml of phosphoric acid, 0.1 g of N-(1-Naphthyl) Ethylenediamine and 1 g of Sulfanilamide were prepared. It was added to 100 ml of distilled water. Then, 50 µl of the culture medium was taken and added to the 96-well plates for NO measurement. Griess reagent was added to 96-well plates. It was incubated for 10 minutes at room temperature. Absorbance values were measured at 540 nm on an ELISA Reader[ 13 ]. For cell viability analysis, antigen prepared in seven different concentrations (10 µg/ml, 20 µg/ml, 40 µg/ml, 80 µg/ml, 100 µg/ml, 120 µg/ml, 160 µg/ml) and antigen + adjuvant (Complete Freund's (40 µg/ml) and PO (100 µg/ml) were used. Then MTT reactant was applied to the fibroblast cells incubated for 48 hours. Cell viability rates were evaluated with MTT salt (5 mg/ml stock solution) containing 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltatrazilium bromide. 10 µl of MTT solution was added to each well containing sample in 96-well plates. Cells in the well plate were incubated for 3 hours at 37°C in the dark. After the incubation, the liquids containing the MTT solution were aspirated and removed. 100 µl of dimethylsulfoxide (DMSO) was added to each well. Afterwards, the well plates were kept in the dark at room temperature for 30 minutes. Cell viability analysis was performed by measuring at a wavelength of 570 nm [ 14 ]. Experimental Animals Vaccination procedures and blood collection procedures to be applied in animals were carried out at Bezm-i Alem University Experimental Animals Research Center after the approval of Istanbul Bezm-i Alem University Experimental Animals Ethics Committee (Ethical number: 2018.05). Experimental animal studies were determined by statistical calculation and started with 3 female Balb/c mice (6 weeks old) in each group. 3 groups were created. Approximately 0.2 mL of blood samples were taken from each group for pre-vaccination tail titer testing. The first group was determined as the PO group. In the first group, a mixture of 100 µg sonicated breast cancer antigen and 250 µg PO adjuvant was formed and intraperitoneally administered with a volume of 0.2 mL (27). The second group was determined as Complete Freund's group. In the second group, a 1:1 mixture of 100 µg sonicated breast cancer antigen and 100 µg Complete Freund's complete adjuvant was formed and administered intraperitoneally with a volume of 0.2 mL[ 15 ]. The third group is the control group. Mice were vaccinated in one-week period until antibody response was approximately 10 times higher than control. Blood samples were isolated from animals before each vaccination. Except for the control group, only breast cancer antigen was used in the last booster dose. Cell Fusion and Cloning 10 week old Balb/c mice from the control group were sacrificed to collect peritoneal macrophages as the feeder layer for hybridoma cells prior to cell fusion. Peritoneal macrophages were harvested in RPMI-1460 medium. One of the Balb/C mice with ELISA titers 10 times higher than the control was sacrificed. Spleen cells of the sacrificed mouse were collected and fused with myeloma cell (10:1) by adding PEG1500 (Sigma-Aldrich®) in RPMI-1640 containing 20% FBS mixture. The fused cells were centrifuged at 1250 rpm for 10 minutes. After centrifugation, cells were resuspended in RPMI-1640 medium supplemented with 20% FBS and HAT (hypoxantine-aminopterin-thymidine). The diluted fused cell solution was seeded into a 96-well plate. The plate was incubated for 14 days at 37°C under 5% CO 2 (28). On the 14th day, the media were taken from the medium in which the cells were found, and the antibody level was determined. At the following days HAT medium was replaced with HT (hypoxanthine-thymidine) medium and the cells were incubated. Antibody measurements were performed by taking samples from the supernatant of hybridomas at certain intervals [ 16 ]. Enzyme-Linked Immunosorbent Assay (ELISA) Breast cancer antigen produced by sonication method was prepared in 0.05 M carbonate coating solution (pH: 9.6). 100 µL of antigen solution was added to 96 well plates, with 10 µg/ml antigen in each well. The prepared well plate was incubated at 4°C overnight. After incubation, the well plate was washed three times with phosphate buffered saline (PBS) containing 0.05% Tween-20. Each well was coated with 2% powdered milk and incubated for 1 hour at 37°C. Serum samples were diluted 1:50 with 2% PBS/Tween 20/Milk. After rinsing prepared serum samples were added to the wells and incubated for 1 hour at 37°C. Washing was done after incubation. The alkaline phosphatase conjugated IgG antibody was diluted 1:1000 with PBS/Tween20. 100 µL of diluted antibodies were included to each well and the well plate was incubated at 37°C for 1 hour. Following to washing step, 10 mg of substrate (p Nitrophenyl Phosphate, Sigma-Aldrich) was dissolved in 200 mL of prepared substrate buffer (0.02 g ZnCl2; 0.04 g MgCl2; 1.5 g Glycine, pH: 10.4). Prepared substrate solution was added to each well as 100 µL. The well plate was incubated for 30 minutes at room temperature in the dark. At the end of these procedures, the well plate was measured at 405 nm in the ELISA Reader [ 17 ]. Statistical Analysis GraphPad Prism 9 software was used for statistical analyses. The data was analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests. Results Determination of NO Activity It is well known that nitric oxide (NO) is an important component of immunity and has two main roles in the immune response: destroying pathogens and regulating immune pathways. NO is the key molecule in modulating Th1 and Th2 responses. Low NO concentrations can stimulate Th2, while high NO concentrations can promote Th1 immune response. In addition, Acosta et al. showed the correlation between the increased NO response of vaccinated animals and the level of protection against related pathogens [ 18 ]. Therefore, we investigated the effect of obtained vaccine formulations on concentrations of NO released by J774 macrophages to assess in vitro immunogenicity of vaccine formulations. Secreted NO level is an important tool for determining the probable stimulatory effects of vaccine formulations. In different studies it was established that vaccine candidates have ability to increase NO activity at a high rate[ 19 ]. Initially enhanced concentrations of sonicated antigens were applied on macrophages in order to detect the most appropriate antigen dosage that stimulated NO response. The results indicate that the highest NO level was detected when macrophages exposed to 40 µg/ml antigen concentration. At this concentration, there was a 1.2-fold increase compared to the control group (Fig. 1 ). According to outputs of this assay, it was decided to use 40 µg/ml concentration of sonicated antigens for further investigations. Adjuvants are substances that act to improve the immunogenicity of antigens for leading to high immune responses after vaccine administrations[ 20 ]. Here, we calculated the in vitro immunogenic features of antigen-adjuvant combinations in terms of secreted NO levels from treated macrophages. Antigens were combined with Freund’s and PO adjuvants and stimulatory activities of combinations were compared with sole antigens. It was exerted that produced NO amounts were significantly high in the groups where macrophages were treated with antigen + adjuvant combinations rather than macrophages exposed to pure antigens alone. In the study, the NO activity of antigen + adjuvant combinations is higher than pure antigens. Approximately 1.47-fold increase was recorded in the group treated with combination of antigen and Complete Freund's adjuvant, while association of antigens with PO adjuvant stimulated 1.3-fold advancement at secreted NO levels in contrast to use of antigens alone (Fig. 2 ). MTT assay was conducted for establishment of biocompability ratios of prepared vaccine formulations. Viability degrees of treated macrophages were evaluated and compared for pure antigen and antigen-adjuvant combinations. Figure 3 shows the cell viability results of J774 macrophage cells exposed to different vaccine formulations. As can be seen, the antigens did not exhibit significant toxicity against macrophages at investigated concentrations. In antigen-adjuvant combinations, the 40 µg/ml antigen-40 µg/ml adjuvant combination with the highest NO response gave the ideal toxicity response. Therefore, further immunization studies were designed with the vaccine formulation including 40 µg/ml antigen-40 µg/ml adjuvant since this mixture is superior in regards to stimulating in vitro NO responses and being non-toxic for macrophages. Overall, biocompatibility results of the current paper are consistent with our previous study in which antigen and antigen-adjuvant combinations were non-toxic against L929 cell lines [ 21 ]. Evaluation of Antibody Titers after Immunization For detection of in vivo immunostimulatory performances of selected formulations, Balb/c mice were vaccinated with antigen-adjuvant mixtures at concentrations, described above. Antibody responses against sonicated breast cancer antigens remarkably enhanced in immunized mice compared to the control when they were applied together with Complete Freund's and PO adjuvants. Nevertheless the most significant increment was recorded in the group immunized with the combination including sonicated antigens and Complete Freund's adjuvant. As it is clearly seen in Fig. 4 , initial ELISA values measured in sera from control groups were between 0.05 and 0.06. In negative controls, these values were measured between 0.04 and 0.05. After the 5th vaccination, antibody levels have been started to raise in Complete Freund's adjuvant group. Following to 10th vaccination with antigen-Complete Freund adjuvant combination, measured specific antibody levels against breast cancer antigens were about 10-times higher than the control group (P < 0.05). The massive improvement detected in serum samples of this group were considered as sufficient for further hybridoma applications. On the other hand, only a 2-fold increase in antibody levels was observed after the 11th vaccination of antigens accompanied by PO adjuvant (Fig. 4 ). These outputs reflect that Complete Freund’s adjuvant were more vigorous to induce the immunogenic features of breast cancer antigens in comparison to PO adjuvant. Morphological Screening of Hybridoma Cells Following to determination of high murine antibody titers in response to vaccination with antigen and Complete Freund’s adjuvant combinations, immunized mice in this group were sacrificed and their spleens were collected safely for further hybridoma applications. Subsequently, splenocytes isolated from the organs of immunized mice were fused with myeloma cells and appearances of hybridoma cells were monitored for 55 days. Figure 5 indicates the images that were taken from the flasks where hybridoma process was conducted in various time slots. As it is obviously observed from the images, first hybridoma colonies were established at 21st day of the culture and the number of colonies notably augmented in the upcoming days. Rapid and excessive growth of colonies in HAT medium within examined time intervals was the demonstration of successfully maintenance of hybridoma procedure (Fig. 5). Antibody Levels Produced by Hybridoma Cell The antibody levels in hybridoma cells that were produced against breast cancer antigens were assessed with ELISA methodology conducted on supernatants isolated from the culture at 14., 21., 33., 45. and 55. days of fusion. It was discovered that the produced antibody amounts in hybridoma cells accelerated until the 55th day of incubation. The antibody level of the hybridoma culture exhibited approximately 1.25-fold raise at day 55 compared to day 14 (P < 0.001). Additionally, nearly 2-times increment was recorded in antibody levels generated by hybridoma cells at the 55th day of incubation in comparsion to control. These outcomes are the signs implying that concentrations of antibodies secreted from the hybridomas outstandingly improve at upcoming days of incubation. Comparison of produced polyclonal and commercial antibodies and their diagnostic valence Diagnostic valences of produced polyclonal antibodies and commercially obtained antibodies against different breast cancer antigens were compared using ELISA technique. It was determined that the produced polyclonal antibody exhibited 2.9-times higher diagnostic value against AU-565 breast cancer antigens compared to commercial antibodies (P < 0.001). It was also established that the produced polyclonal antibody elicited 2.3-times greater diagnostic value against MCF-7 breast cancer antigens in contrast to commercial antibodies (Fig. 7 ). These ouputs exert that produced polyclonal antibodies are effective to capture the antigens of both MCF-7 and AU-565 cell lines. Discussion Many treatment approaches are currently being developed against breast cancer. However, it is believed that immunization of the body against breast cancer could be more powerful for prevention of this highly deadful disease[ 22 ]. For this purpose, vaccine formulations that can create immunity against many types of breast cancer have been tried to be developed, so far. Whole tumor cell lysates, immunogenic synthetic peptides derived from tumor antigens, DNA-based and dendritic cell-based vaccines are the most frequently used tools for stimulation of immunity against breast cancer[ 23 ]. Despite of many advantages, there are some limitations of biotechnological cancer vaccine candidates [ 24 ]. For instance, most peptide vaccines are only influential over cancer types that are positive for human leukocyte antigen (HLA) and therefore patients who do not express common HLA classes cannot be treated with the vaccine [ 25 ]. Hence, there is a huge necessity for development of a comprehensive vaccine that could cover all breast cancer subtypes in general. Accordingly, using whole cell lysates including entire immunogenic cancer antigens is one of the most ideal vaccine approach to combat breast cancer. Vaccination with whole cell cancer lysates could trigger the formation of specific antibodies against various antigens and substantial acquired immunity can be provided against breast cancer. In previous studies, experimental animals were exposed to peptides of breast cancer proteins in conjuction with Incomplete Freund adjuvant (IFA) and successful outcomes were achieved [ 26 – 28 ]. The choice of adjuvant is very substantial in the emergence of humoral and cellular immune response. Although many adjuvants could activate either Th1 or Th2 response, Complete Freund's adjuvant (CFA) is an efficient instrument for stimulation of both Th1 and Th2 cells [ 29 ]. However, Complete Freund adjuvant has some disadvantages due to its toxic effect. In recent years, the use of more effective, non-toxic adjuvants have attracted attention in vaccine studies. For this purpose, many targeted vaccine delivery vehicles have been designed and polymeric adjuvants capable of regional degradation have also started to be used in vaccine applications. In one of the vaccine study against Leishmaniasis, it was reported that PO adjuvant was highly effective in regards to enhancement of immunogenic features of antigens used together while demonstrating no toxicity and therefore this adjuvant was suggested to be utilized in vaccine researches [ 16 ]. Accordingly, this study is the first in the literature to compare the effectiveness of Complete Freund's adjuvant with PO adjuvant as an alternative in production of polyclonal antibodies by hybridoma technology against breast cancer. We initially evaluated the secreted nitric oxide amounts from macrophages treated with antigen and adjuvant combinations. This assay was followed with the calculation of in vivo antibody levels in mice immunized with different vaccine formulations prepared with two distinct adjuvants and their immunostimulatory performances were compared. The vaccine formulation with higher scores was selected and examined for further studies including detection of diagnostic values of produced polyclonal antibodies. According to the results obtained, it was discovered that whole cancer cell antigens prepared with sonication method readily achieved the induction of antibody production in immunized mice independent from type of utilized adjuvants. However, we calculated that the vaccine formulation prepared with Complete Freund's adjuvant lead to 5-times higher antibody production than the vaccine formulation prepared with PO adjuvant. Therefore, in further examinations involving creations of polyclonal antibodies with hybridoma technology, it was planned to consider the vaccine formulations produced with antigens and Complete Freund's adjuvant. The emerged hybridoma cells were discovered to release notable levels of polyclonal antibodies and produced antibody concentration in supernatant was ascertained to increase daily until 55th day of incubation. The generated polyclonal antibodies are supposed to be used for immunotherapy of breast cancer since they have ability to conjuct specifically with tumor cells. Besides, it can be also thought that antibodies obtained with hybridoma technology can be used for diagnostic purposes against breast cancer. In the diagnosis of breast cancer, the mostly applied methods are mammography, ultrasound and biopsy [ 30 ]. However, these techniques require either surgical intervention or long time to obtain accurate results. On contrary, antibody-mediated serological diagnostic kits enable the detection of breast cancer antigens with a more rapid, reliable and sensitive way [ 31 ]. Additionally, the biggest advantage of diagnostic kits involving polyclonal antibodies produced with hybridoma technology may be that they are specialized to capture the breast cancer-specific antigenic molecule in the sample, not the antibody. This approach can help to overcome the serious disadvantage of alternative serological kits whose working principle is relying on the arrest of antibodies in blood sample since tumor specific antibodies are rarely present in blood of breast cancer patients. Currently, diagnostic kits coated with antibodies are being developed against breast cancer, but no effective results have been obtained so far. The fundamental reason for low sensitivity of newly formed kits is the application of monoclonal antibodies during production. Although monoclonal antibodies indicate high potential in diagnosis and treatment of various cancers, their action is interfered with specifically binding to a single epitope region of tumor antigens, resulting in low avidity results compared to polyclonal antibodies[ 32 , 33 ]. Accordingly, the use of polyclonal antibodies in development of diagnotic kits could be considered more advantageous in contrast to monoclonal antibodies. At the final step of our study, the avidity properties of polyclonal antibodies derived from hybridomas that were developed against sonicated breast cancer cell antigens were explored and their performances were also compared with commercially available monoclonal antibodies. Eventually it was established that polyclonal antibodies produced from hybridomas demonstrated superior ability to interact with breast cancer antigens acquired from AU-565 cells in contrast to commercial antibodies. Interestingly, we also decipher that currently generated polyclonal antibodies also indicated robust immunoreactivity to antigens obtained from MCF-7 cells while commercial antibodies did not induce such an effect. These outputs reveal that diagnostic value of currently produced polyclonal antibodies were enormously higher than conventional antibodies and they could be used for diagnosis of breast cancer cases originated from different sources. Furthermore it is predicted that these newly generated polyclonal antibodies can readily detect metastic forms of breast cancer as well as its non-metastatic types and offer a great potential to be used in diagnostic kits which will recognize many antigenic epitopes of various cancer cells. Conclusion Consequently, sonicated breast cancer lysates obtained from AU-565 cell line were combined with two separate adjuvants in order to produce innovative vaccine candidates within the scope of this research. The in vitro immunostimulatory efficacies of formulations were proven since they greatly ameliorated NO levels secreted from macrophage cells. The in vivo immunostimulatory activities of vaccine candidates were determined by evaluation of antibody titers upon immunization. According to the results combination including breast cancer antigens and Complete Freund's adjuvant was more influential in terms of stimulating humoral response and increasing antibody levels compared to combination formulized with PO adjuvant. Polyclonal antibodies were produced from splenocytes of mice immunized with breast cancer antigens and Complete Freund's adjuvant with hybridoma technology and improved amounts of antibodies were assessed within hybridoma cell culture upon long-term incubation. Finally, it was demonstrated that isolated polyclonal antibodies elicited strong interaction with antigens that were obtained from both AU-565 and MCF-7 cell lines. In conclusion, immunostimulatory activities of breast cancer antigens that were acquired with sonication method applied on AU-565 cell line were indicated for the first time in this study. It was also revealed that efficacy of antigens could be advanced by the use of adjuvants. Although Complete Freund's adjuvant was more powerful in regards to influencing B lymphocytes to create high titers of antibodies, PO adjuvant also induce the macrophages to secrete great amounts of NO. Hence, we foreseen that both formulation could be further used as immunotherapeutic vaccine candidate against breast cancer, however combination involving Complete Freund's adjuvant would be more promising. Moreover, obtained polyclonal antibodies could be further utilized for development of reliable and sensitive diagnostic kits for identification of different breast cancer types since they exerted strong immune reaction with breast cancer antigens with distinct behaviors. So, we predict that gathered outputs in this study will further make a huge contribution to researches related to development of immunotherapy approaches and diagnostic kits against breast cancer. Declarations Declaration of Competing Interest The authors declared that they had no conflict of interest. Author Contribution Conceptualization, M. I. and E.S.A.; methodology, software, formal analysis and investigation, M. I., A. D., A.P.Z.; writing—original draft preparation, M. I. and E.S.A.; review and editing, A. A., M. B, and E.S.A.; visualization, supervision, project administration, M. I. All authors have read and agreed to the published version of the manuscript. Acknowledgement This work was supported by the Yildiz Technical University Scientific Research Project Coordination Department (Project No: FBA-2018-3359). References Łukasiewicz S, Czeczelewski M, Forma A, Baj J, Sitarz R, Stanisławek A. 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Signal Transduction and Targeted Therapy. 2023;8(1):283. Ihlamur M, BaŞAri H, ZengİN Y, Abamor EŞ. Evaluation Of Immunostimulant/Cytotoxic Activity Of Human Breast Cancer Prepared By Different Antigen Preparation Methods With Adjuvants Combination. Süleyman Demirel University Faculty of Arts and Science Journal of Science. 2022;17(1):96-110. Disis ML, Cecil DL. Breast cancer vaccines for treatment and prevention. Breast Cancer Res Treat. 2022;191(3):481-9. Liu J, Fu M, Wang M, Wan D, Wei Y, Wei X. Cancer vaccines as promising immuno-therapeutics: platforms and current progress. J Hematol Oncol. 2022;15(1):28. Kaczmarek M, Poznańska J, Fechner F, Michalska N, Paszkowska S, Napierała A, et al. Cancer Vaccine Therapeutics: Limitations and Effectiveness-A Literature Review. Cells. 2023;12(17). Nordin ML, Azemi AK, Nordin AH, Nabgan W, Ng PY, Yusoff K, et al. Peptide-Based Vaccine against Breast Cancer: Recent Advances and Prospects. Pharmaceuticals (Basel). 2023;16(7). Fatima GN, Fatma H, Saraf SK. Vaccines in Breast Cancer: Challenges and Breakthroughs. Diagnostics (Basel). 2023;13(13). Donninger H, Li C, Eaton JW, Yaddanapudi K. Cancer Vaccines: Promising Therapeutics or an Unattainable Dream. Vaccines (Basel). 2021;9(6). Fan T, Zhang M, Yang J, Zhu Z, Cao W, Dong C. Therapeutic cancer vaccines: advancements, challenges, and prospects. Signal Transduction and Targeted Therapy. 2023;8(1):450. Cribbs DH, Ghochikyan A, Vasilevko V, Tran M, Petrushina I, Sadzikava N, et al. Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with beta-amyloid. Int Immunol. 2003;15(4):505-14. He Z, Chen Z, Tan M, Elingarami S, Liu Y, Li T, et al. A review on methods for diagnosis of breast cancer cells and tissues. Cell Prolif. 2020;53(7):e12822. Lengfeld J, Zhang H, Stoesz S, Murali R, Pass F, Greene MI, et al. Challenges in Detection of Serum Oncoprotein: Relevance to Breast Cancer Diagnostics. Breast Cancer (Dove Med Press). 2021;13:575-93. Zahavi D, Weiner L. Monoclonal Antibodies in Cancer Therapy. Antibodies (Basel). 2020;9(3). Ritter MA. Polyclonal and monoclonal antibodies. Methods Mol Med. 2000;40:23-34. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3966637","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273578390,"identity":"ec74cb9b-f99f-49d6-b414-eb45b1791252","order_by":0,"name":"Murat IHLAMUR","email":"","orcid":"","institution":"Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yildiz Technical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Murat","middleName":"","lastName":"IHLAMUR","suffix":""},{"id":273578391,"identity":"3faa3642-f324-4243-8c52-d3aee1a2c6a6","order_by":1,"name":"Atıfcan DEMİRCİOĞLU","email":"","orcid":"","institution":"Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yildiz Technical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Atıfcan","middleName":"","lastName":"DEMİRCİOĞLU","suffix":""},{"id":273578392,"identity":"b9cdafd0-8e8c-40fb-9a7e-3866d84cf142","order_by":2,"name":"Aslı Pınar ZORBA","email":"","orcid":"","institution":"Department of Medical Services and Techniques, School of Vocational of Healty, Istinye University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aslı","middleName":"Pınar","lastName":"ZORBA","suffix":""},{"id":273578393,"identity":"3f92ac18-4fd1-4237-bc66-f0d26c8c0a80","order_by":3,"name":"Emrah Şefik ABAMOR","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYFACHjApx8DM3CCRAGIeIFKLMQMzI1wLYwMxWhIbgAolGIjRws9+9uCnGxX30jccZ2y88eAPgxzfjQT2xxV4tEj25CVL55wpzt1wmLHZIoGHwVjyRgJj4xk8WgwO5BhI57YlgLS0SSRIMCRuAGnB5zL782+MfwO1pBuAtRgw1BPUYiCRYwayJQGiJYEhwYCQFokb79Ksc84kGM4E++WAhOHMMw8bZ+LTwt+fe/h2TkWCPN/5wwdv/vhjI893PPnAR3xa4EDhAMRWICYUkzAgT6S6UTAKRsEoGIEAABi9Uxxg/aZeAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yildiz Technical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Emrah","middleName":"Şefik","lastName":"ABAMOR","suffix":""},{"id":273578394,"identity":"4a072c91-441f-4c06-9ac7-6d58aa0d1daf","order_by":4,"name":"Melahat BAĞIROVA","email":"","orcid":"","institution":"Institute of the V. Akhundov National Scientific Research Medical Prophylactic","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Melahat","middleName":"","lastName":"BAĞIROVA","suffix":""},{"id":273578395,"identity":"8e43f905-2c3a-4c83-a68c-511f11913793","order_by":5,"name":"Adil ALLAHVERDİYEV","email":"","orcid":"","institution":"Institute of the V. Akhundov National Scientific Research Medical Prophylactic","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Adil","middleName":"","lastName":"ALLAHVERDİYEV","suffix":""}],"badges":[],"createdAt":"2024-02-18 10:16:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3966637/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3966637/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51409946,"identity":"1cc84b1a-a391-4f13-849e-e17f282c4254","added_by":"auto","created_at":"2024-02-21 04:43:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58006,"visible":true,"origin":"","legend":"\u003cp\u003eNitric oxide responses to AU-565 antigen concentration in J774 cell line (P ⩽ 0.05)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3966637/v1/a47be0199634c378b6d802de.png"},{"id":51409950,"identity":"84a9d32c-07d1-42eb-bba0-30b7a261cef1","added_by":"auto","created_at":"2024-02-21 04:43:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58839,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro immunostimulatory activity of antigen and adjuvant combinations at a concentration of 40 µg/ml in the macrophage cell culture system (P ⩽ 0.05)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3966637/v1/44744916238e1199828a10d6.png"},{"id":51409949,"identity":"b1f943a2-e831-46dc-bfa7-d61ea82f9d39","added_by":"auto","created_at":"2024-02-21 04:43:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70443,"visible":true,"origin":"","legend":"\u003cp\u003eThe cytotoxic effect of antigens and antigen-adjuvant combinations on viability in fibroblast cell culture system (P ⩽ 0.0001)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3966637/v1/21a33a98e4421e634e755b24.png"},{"id":51410385,"identity":"edee9b82-43b3-45df-942c-145b446f4321","added_by":"auto","created_at":"2024-02-21 04:51:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109280,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of serum antibody titers in mice immunized with combinations including breast cancer antiens and Complete Freund’s adjuvant or PO adjuvant\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3966637/v1/1dadd5f09e9ae8c3a0a852cf.png"},{"id":51409948,"identity":"22348cfb-728e-498d-af93-f111021f22e4","added_by":"auto","created_at":"2024-02-21 04:43:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":984146,"visible":true,"origin":"","legend":"\u003cp\u003eGradual development and colonization of hybridoma cells a)21st b)28th c)33rd d)45th day of incubation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3966637/v1/03cd8166ab21cad257c33d14.png"},{"id":51409952,"identity":"4def2b29-fb60-42b1-8632-1b696cd97fb6","added_by":"auto","created_at":"2024-02-21 04:43:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":33556,"visible":true,"origin":"","legend":"\u003cp\u003ePolyclonal antibody levels measured in supernatant samples of hybridoma cells\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3966637/v1/129b65dc42e60f5055df91fe.png"},{"id":51409951,"identity":"57e1afc8-4058-4b07-bb20-8ea58e137c38","added_by":"auto","created_at":"2024-02-21 04:43:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":53012,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic values of produced polyclonal and commercial antibodies\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3966637/v1/aadab4802b7098aaf070e92a.png"},{"id":52055769,"identity":"76c41658-38e3-48d6-a484-72ade2d0596c","added_by":"auto","created_at":"2024-03-06 02:57:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1662583,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3966637/v1/dd0ff69e-6109-4a26-ac7d-73e7b09c56f0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluating Immunoreactivity of Polyclonal Antibodies Developed against AU-565 Cell Line for Diagnosis and Immunotherapy of Breast Cancer","fulltext":[{"header":"Introductıon","content":"\u003cp\u003eCancer occurs as a result of uncontrolled division of cells. Although there are many types of cancer, the most frequently diagnosed cancer in women worldwide is breast cancer with more than 2\u0026nbsp;million new cases in 2020. Breast cancer is the second most common cause of cancer death among women in the world. At the same time, its incidence and mortality rates have been increasing steadily over the past three decades. It is estimated that 2.7\u0026nbsp;million new breast cancer cases will be detected worldwide in 2030, and deaths from breast cancer will reach 870 thousand [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Treatment methods such as surgery, chemotherapy and radiotherapy are involved in the treatment of breast cancer. With the development of immunotherapy and targeted drug treatment approaches, more effective results could be obtained in breast cancer treatments [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Cancer immunotherapy, as an alternative treatment method, has developed significantly in recent years. Unlike other therapeutic concepts, immunotherapy primarily aims at preventing the metastatic spread of the disease and improving the life quality of affected individuals. Moreover, by introducing cancer cell antigens to B and T lymphocytes, immunotherapy can induce immunity on individuals before the onset of the disease, and provide a faster response than current treatments. Furthermore, therapeutic vaccination is also accepted as a part of immunotherapy since administration of antibodies can recognize and destroy cancer cells. However, no effective and reliable vaccine formulation has been created against breast cancer so far. Another important limitation of breast cancer therapy is the inadequacy of present diagnostic methods [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mammography and ultrasound are the techniques that are frequently applied for breast cancer diagnosis. Acquisition of false positive and false negative results is the major limitation of mammography applications. Moreover, suspicious individuals are exposed to high radiation during diagnosis with mammography. On the other side, some small solid tumors can miss from ultrasound screening despite the fact that this technique does not require radiation. In addition, sensitivity of ultrasound is not high as mammograpghy for screening of breast cancer cells[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Finally, some breast cancer types are not diagnosed by screening and invasive technique such as biopsy is the only option that would be successful to provide accurate diagnosis in that cases[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Owing to limitations of current diagnostic methods, it is very important to develop new diagnostic kits for the accurate and rapid identification of breast cancer.\u003c/p\u003e \u003cp\u003ePolyclonal and monoclonal antibodies which are produced by hybridoma technology have been used in the diagnosis of many diseases in recent years. The biggest advantage of hybridoma technology-based diagnostic kits is that they demonstrate high specificity to capture special antigens[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition design of the most appropriate protocol to produce hybridoma technology-based monoclonal antibodies is so important. The most suitable adjuvant should be selected to improve immunogenicities of used antigens during immunization of animals because this directly influences the specificity of produced antibodies to capture special antigens[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the diagnostic values of hybridoma-based antibodies that have been created to date was not adequate enough so they have failed to be used for preparation of diagnostic kits against breast cancer. We assume that diagnostic performances of produced mono or polyclonal antibodies could be advanced by administration of a strong adjuvant in immunization. In the scope of this study, we explored the assistant performances of one microorganism based adjuvant such as Complete Freund's and one polymeric adjuvant called as Polyoxidonium (PO) in order to check their capacities to improve immunogenicities of whole breast cancer cell antigens. In this study, the importance and effects of adjuvants in hybridoma technology were also investigated. Although in some studies it was stated that Complete Freund's adjuvant is more toxic and not active enough compared to other adjuvants there are also several studies in which Complete Freund's adjuvant (CFA) was used and positive results were obtained in stimulation of humoral and cellular immune responses [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, until now, studies examining the antibody production of hybridomas created using breast cancer cell antigens in conjunction with Complete Freund's adjuvant and the determination of the diagnostic valence of the obtained antibodies are quite insufficient. There is also no study in the literature that invesitigates diagnostic and immunogenic features of antibodies acquired by hybridoma technology that are produced against breast cancer cell antigens supported by PO as an adjuvant. Hence the main goal of the present study was to generate hybridoma-based polyclonal antibodies against AU-565 breast cancer cell line antigens accompanied by CFA or PO as an adjuvant and explore the comparative efficacies of these two adjuvants over diagnostic values of produed polyclonal antibodies. The immunogenic features of antigens in combinations with CFA or PO was also compared in current study.\u003c/p\u003e"},{"header":"Material and method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eAU-565 breast cancer cell line culture used for antigen production was grown in RPMI-1640 medium containing 10% FBS, 1% Pen-step. For cytotoxicity and nitric oxide studies, L929 Fibroblast cell line culture was grown in RPMI-1640 medium containing 10% FBS, 1% Pen-step, and J774 macrophage cell line culture was grown in RPMI-1640 medium containing 10% FBS, 1% Pen-step. P3-X63-Ag8.653 myeloma cell line used for fusion was cultured. P3-X63-Ag8.653 myeloma cell line was grown in with RPMI-1640 containing 20% FBS and 1% Pen-Strep and treated with 20 \u0026micro;g 8-azaguanine/mL prior to the hybridoma protocol [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Breast Cancer Antigens\u003c/h2\u003e \u003cp\u003eSonication procedure was applied to obtain AU-565 breast cancer antigens. The cell pellet was attached to the sonicator probe. The sonicator was set to 40 seconds and 3 Cycles. The power potentiometer of the sonicator generator was set to 40%. Sonication was repeated 5 times. The resulting lysate was centrifuged at 10,000 rpm for 3 minutes. The supernatant was taken and measured in UV-Vis spectrophotometer. To determine the protein content inside the lysate, the Warburg-Christian method was used with a UV-vis spectrometer under 280 and 260 nm wavelengths [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNO ve MTT Analysis\u003c/h2\u003e \u003cp\u003eThe amount of nitric oxide (NO) produced by the cells was determined by the Griess method in order to determine the immunostimulatory activities of the vaccine formulations prepared at different concentrations by combining the antigens obtained by sonication method alone or in combination with different adjuvants in macrophage cell culture systems. Antigen prepared at seven different concentrations (10 \u0026micro;g/ml, 20 \u0026micro;g/ml, 40 \u0026micro;g/ml, 80 \u0026micro;g/ml, 100 \u0026micro;g/ml120 \u0026micro;g/ml, 160 \u0026micro;g/ml) after incubation of 1*10\u003csup\u003e5\u003c/sup\u003e cells/ml macrophage cells in an oven at 37⁰C containing 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours, and antigen\u0026thinsp;+\u0026thinsp;adjuvant (Complete Freund's (40 \u0026micro;g/ml) and PO (100 \u0026micro;g/ml) combinations were added. Supernatants were collected after 48 hours of incubation. Griess reagent was added to the supernatants and reacted. For Griess reagent, 2.5 ml of phosphoric acid, 0.1 g of N-(1-Naphthyl) Ethylenediamine and 1 g of Sulfanilamide were prepared. It was added to 100 ml of distilled water. Then, 50 \u0026micro;l of the culture medium was taken and added to the 96-well plates for NO measurement. Griess reagent was added to 96-well plates. It was incubated for 10 minutes at room temperature. Absorbance values were measured at 540 nm on an ELISA Reader[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor cell viability analysis, antigen prepared in seven different concentrations (10 \u0026micro;g/ml, 20 \u0026micro;g/ml, 40 \u0026micro;g/ml, 80 \u0026micro;g/ml, 100 \u0026micro;g/ml, 120 \u0026micro;g/ml, 160 \u0026micro;g/ml) and antigen\u0026thinsp;+\u0026thinsp;adjuvant (Complete Freund's (40 \u0026micro;g/ml) and PO (100 \u0026micro;g/ml) were used. Then MTT reactant was applied to the fibroblast cells incubated for 48 hours. Cell viability rates were evaluated with MTT salt (5 mg/ml stock solution) containing 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltatrazilium bromide. 10 \u0026micro;l of MTT solution was added to each well containing sample in 96-well plates. Cells in the well plate were incubated for 3 hours at 37\u0026deg;C in the dark. After the incubation, the liquids containing the MTT solution were aspirated and removed. 100 \u0026micro;l of dimethylsulfoxide (DMSO) was added to each well. Afterwards, the well plates were kept in the dark at room temperature for 30 minutes. Cell viability analysis was performed by measuring at a wavelength of 570 nm [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Animals\u003c/h2\u003e \u003cp\u003e Vaccination procedures and blood collection procedures to be applied in animals were carried out at Bezm-i Alem University Experimental Animals Research Center after the approval of Istanbul Bezm-i Alem University Experimental Animals Ethics Committee (Ethical number: 2018.05).\u003c/p\u003e \u003cp\u003eExperimental animal studies were determined by statistical calculation and started with 3 female Balb/c mice (6 weeks old) in each group. 3 groups were created. Approximately 0.2 mL of blood samples were taken from each group for pre-vaccination tail titer testing. The first group was determined as the PO group. In the first group, a mixture of 100 \u0026micro;g sonicated breast cancer antigen and 250 \u0026micro;g PO adjuvant was formed and intraperitoneally administered with a volume of 0.2 mL (27). The second group was determined as Complete Freund's group. In the second group, a 1:1 mixture of 100 \u0026micro;g sonicated breast cancer antigen and 100 \u0026micro;g Complete Freund's complete adjuvant was formed and administered intraperitoneally with a volume of 0.2 mL[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The third group is the control group. Mice were vaccinated in one-week period until antibody response was approximately 10 times higher than control. Blood samples were isolated from animals before each vaccination. Except for the control group, only breast cancer antigen was used in the last booster dose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell Fusion and Cloning\u003c/h2\u003e \u003cp\u003e10 week old Balb/c mice from the control group were sacrificed to collect peritoneal macrophages as the feeder layer for hybridoma cells prior to cell fusion. Peritoneal macrophages were harvested in RPMI-1460 medium. One of the Balb/C mice with ELISA titers 10 times higher than the control was sacrificed. Spleen cells of the sacrificed mouse were collected and fused with myeloma cell (10:1) by adding PEG1500 (Sigma-Aldrich\u0026reg;) in RPMI-1640 containing 20% FBS mixture. The fused cells were centrifuged at 1250 rpm for 10 minutes. After centrifugation, cells were resuspended in RPMI-1640 medium supplemented with 20% FBS and HAT (hypoxantine-aminopterin-thymidine). The diluted fused cell solution was seeded into a 96-well plate. The plate was incubated for 14 days at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e (28). On the 14th day, the media were taken from the medium in which the cells were found, and the antibody level was determined. At the following days HAT medium was replaced with HT (hypoxanthine-thymidine) medium and the cells were incubated. Antibody measurements were performed by taking samples from the supernatant of hybridomas at certain intervals [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eBreast cancer antigen produced by sonication method was prepared in 0.05 M carbonate coating solution (pH: 9.6). 100 \u0026micro;L of antigen solution was added to 96 well plates, with 10 \u0026micro;g/ml antigen in each well. The prepared well plate was incubated at 4\u0026deg;C overnight. After incubation, the well plate was washed three times with phosphate buffered saline (PBS) containing 0.05% Tween-20. Each well was coated with 2% powdered milk and incubated for 1 hour at 37\u0026deg;C. Serum samples were diluted 1:50 with 2% PBS/Tween 20/Milk. After rinsing prepared serum samples were added to the wells and incubated for 1 hour at 37\u0026deg;C. Washing was done after incubation. The alkaline phosphatase conjugated IgG antibody was diluted 1:1000 with PBS/Tween20. 100 \u0026micro;L of diluted antibodies were included to each well and the well plate was incubated at 37\u0026deg;C for 1 hour. Following to washing step, 10 mg of substrate (p Nitrophenyl Phosphate, Sigma-Aldrich) was dissolved in 200 mL of prepared substrate buffer (0.02 g ZnCl2; 0.04 g MgCl2; 1.5 g Glycine, pH: 10.4). Prepared substrate solution was added to each well as 100 \u0026micro;L. The well plate was incubated for 30 minutes at room temperature in the dark. At the end of these procedures, the well plate was measured at 405 nm in the ELISA Reader [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 9 software was used for statistical analyses. The data was analyzed by one-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s multiple comparison tests.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eDetermination of NO Activity\u003c/h2\u003e\n \u003cp\u003eIt is well known that nitric oxide (NO) is an important component of immunity and has two main roles in the immune response: destroying pathogens and regulating immune pathways. NO is the key molecule in modulating Th1 and Th2 responses. Low NO concentrations can stimulate Th2, while high NO concentrations can promote Th1 immune response. In addition, Acosta et al. showed the correlation between the increased NO response of vaccinated animals and the level of protection against related pathogens [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, we investigated the effect of obtained vaccine formulations on concentrations of NO released by J774 macrophages to assess in vitro immunogenicity of vaccine formulations. Secreted NO level is an important tool for determining the probable stimulatory effects of vaccine formulations. In different studies it was established that vaccine candidates have ability to increase NO activity at a high rate[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Initially enhanced concentrations of sonicated antigens were applied on macrophages in order to detect the most appropriate antigen dosage that stimulated NO response. The results indicate that the highest NO level was detected when macrophages exposed to 40 \u0026micro;g/ml antigen concentration. At this concentration, there was a 1.2-fold increase compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). According to outputs of this assay, it was decided to use 40 \u0026micro;g/ml concentration of sonicated antigens for further investigations.\u003c/p\u003e\n \u003cp\u003eAdjuvants are substances that act to improve the immunogenicity of antigens for leading to high immune responses after vaccine administrations[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Here, we calculated the in vitro immunogenic features of antigen-adjuvant combinations in terms of secreted NO levels from treated macrophages. Antigens were combined with Freund\u0026rsquo;s and PO adjuvants and stimulatory activities of combinations were compared with sole antigens. It was exerted that produced NO amounts were significantly high in the groups where macrophages were treated with antigen\u0026thinsp;+\u0026thinsp;adjuvant combinations rather than macrophages exposed to pure antigens alone. In the study, the NO activity of antigen\u0026thinsp;+\u0026thinsp;adjuvant combinations is higher than pure antigens. Approximately 1.47-fold increase was recorded in the group treated with combination of antigen and Complete Freund\u0026apos;s adjuvant, while association of antigens with PO adjuvant stimulated 1.3-fold advancement at secreted NO levels in contrast to use of antigens alone (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eMTT assay was conducted for establishment of biocompability ratios of prepared vaccine formulations. Viability degrees of treated macrophages were evaluated and compared for pure antigen and antigen-adjuvant combinations. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows the cell viability results of J774 macrophage cells exposed to different vaccine formulations. As can be seen, the antigens did not exhibit significant toxicity against macrophages at investigated concentrations. In antigen-adjuvant combinations, the 40 \u0026micro;g/ml antigen-40 \u0026micro;g/ml adjuvant combination with the highest NO response gave the ideal toxicity response. Therefore, further immunization studies were designed with the vaccine formulation including 40 \u0026micro;g/ml antigen-40 \u0026micro;g/ml adjuvant since this mixture is superior in regards to stimulating in vitro NO responses and being non-toxic for macrophages. Overall, biocompatibility results of the current paper are consistent with our previous study in which antigen and antigen-adjuvant combinations were non-toxic against L929 cell lines [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eEvaluation of Antibody Titers after Immunization\u003c/h2\u003e\n \u003cp\u003eFor detection of in vivo immunostimulatory performances of selected formulations, Balb/c mice were vaccinated with antigen-adjuvant mixtures at concentrations, described above. Antibody responses against sonicated breast cancer antigens remarkably enhanced in immunized mice compared to the control when they were applied together with Complete Freund\u0026apos;s and PO adjuvants. Nevertheless the most significant increment was recorded in the group immunized with the combination including sonicated antigens and Complete Freund\u0026apos;s adjuvant. As it is clearly seen in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, initial ELISA values measured in sera from control groups were between 0.05 and 0.06. In negative controls, these values were measured between 0.04 and 0.05. After the 5th vaccination, antibody levels have been started to raise in Complete Freund\u0026apos;s adjuvant group. Following to 10th vaccination with antigen-Complete Freund adjuvant combination, measured specific antibody levels against breast cancer antigens were about 10-times higher than the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The massive improvement detected in serum samples of this group were considered as sufficient for further hybridoma applications. On the other hand, only a 2-fold increase in antibody levels was observed after the 11th vaccination of antigens accompanied by PO adjuvant (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). These outputs reflect that Complete Freund\u0026rsquo;s adjuvant were more vigorous to induce the immunogenic features of breast cancer antigens in comparison to PO adjuvant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eMorphological Screening of Hybridoma Cells\u003c/h2\u003e\n \u003cp\u003eFollowing to determination of high murine antibody titers in response to vaccination with antigen and Complete Freund\u0026rsquo;s adjuvant combinations, immunized mice in this group were sacrificed and their spleens were collected safely for further hybridoma applications. Subsequently, splenocytes isolated from the organs of immunized mice were fused with myeloma cells and appearances of hybridoma cells were monitored for 55 days. Figure 5 indicates the images that were taken from the flasks where hybridoma process was conducted in various time slots. As it is obviously observed from the images, first hybridoma colonies were established at 21st day of the culture and the number of colonies notably augmented in the upcoming days. Rapid and excessive growth of colonies in HAT medium within examined time intervals was the demonstration of successfully maintenance of hybridoma procedure (Fig. 5).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eAntibody Levels Produced by Hybridoma Cell\u003c/h2\u003e\n \u003cp\u003eThe antibody levels in hybridoma cells that were produced against breast cancer antigens were assessed with ELISA methodology conducted on supernatants isolated from the culture at 14., 21., 33., 45. and 55. days of fusion. It was discovered that the produced antibody amounts in hybridoma cells accelerated until the 55th day of incubation. The antibody level of the hybridoma culture exhibited approximately 1.25-fold raise at day 55 compared to day 14 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, nearly 2-times increment was recorded in antibody levels generated by hybridoma cells at the 55th day of incubation in comparsion to control. These outcomes are the signs implying that concentrations of antibodies secreted from the hybridomas outstandingly improve at upcoming days of incubation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eComparison of produced polyclonal and commercial antibodies and their diagnostic valence\u003c/h2\u003e\n \u003cp\u003eDiagnostic valences of produced polyclonal antibodies and commercially obtained antibodies against different breast cancer antigens were compared using ELISA technique. It was determined that the produced polyclonal antibody exhibited 2.9-times higher diagnostic value against AU-565 breast cancer antigens compared to commercial antibodies (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). It was also established that the produced polyclonal antibody elicited 2.3-times greater diagnostic value against MCF-7 breast cancer antigens in contrast to commercial antibodies (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). These ouputs exert that produced polyclonal antibodies are effective to capture the antigens of both MCF-7 and AU-565 cell lines.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMany treatment approaches are currently being developed against breast cancer. However, it is believed that immunization of the body against breast cancer could be more powerful for prevention of this highly deadful disease[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For this purpose, vaccine formulations that can create immunity against many types of breast cancer have been tried to be developed, so far. Whole tumor cell lysates, immunogenic synthetic peptides derived from tumor antigens, DNA-based and dendritic cell-based vaccines are the most frequently used tools for stimulation of immunity against breast cancer[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Despite of many advantages, there are some limitations of biotechnological cancer vaccine candidates [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For instance, most peptide vaccines are only influential over cancer types that are positive for human leukocyte antigen (HLA) and therefore patients who do not express common HLA classes cannot be treated with the vaccine [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Hence, there is a huge necessity for development of a comprehensive vaccine that could cover all breast cancer subtypes in general. Accordingly, using whole cell lysates including entire immunogenic cancer antigens is one of the most ideal vaccine approach to combat breast cancer. Vaccination with whole cell cancer lysates could trigger the formation of specific antibodies against various antigens and substantial acquired immunity can be provided against breast cancer. In previous studies, experimental animals were exposed to peptides of breast cancer proteins in conjuction with Incomplete Freund adjuvant (IFA) and successful outcomes were achieved [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The choice of adjuvant is very substantial in the emergence of humoral and cellular immune response. Although many adjuvants could activate either Th1 or Th2 response, Complete Freund's adjuvant (CFA) is an efficient instrument for stimulation of both Th1 and Th2 cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, Complete Freund adjuvant has some disadvantages due to its toxic effect. In recent years, the use of more effective, non-toxic adjuvants have attracted attention in vaccine studies. For this purpose, many targeted vaccine delivery vehicles have been designed and polymeric adjuvants capable of regional degradation have also started to be used in vaccine applications. In one of the vaccine study against Leishmaniasis, it was reported that PO adjuvant was highly effective in regards to enhancement of immunogenic features of antigens used together while demonstrating no toxicity and therefore this adjuvant was suggested to be utilized in vaccine researches [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Accordingly, this study is the first in the literature to compare the effectiveness of Complete Freund's adjuvant with PO adjuvant as an alternative in production of polyclonal antibodies by hybridoma technology against breast cancer. We initially evaluated the secreted nitric oxide amounts from macrophages treated with antigen and adjuvant combinations. This assay was followed with the calculation of in vivo antibody levels in mice immunized with different vaccine formulations prepared with two distinct adjuvants and their immunostimulatory performances were compared. The vaccine formulation with higher scores was selected and examined for further studies including detection of diagnostic values of produced polyclonal antibodies. According to the results obtained, it was discovered that whole cancer cell antigens prepared with sonication method readily achieved the induction of antibody production in immunized mice independent from type of utilized adjuvants. However, we calculated that the vaccine formulation prepared with Complete Freund's adjuvant lead to 5-times higher antibody production than the vaccine formulation prepared with PO adjuvant. Therefore, in further examinations involving creations of polyclonal antibodies with hybridoma technology, it was planned to consider the vaccine formulations produced with antigens and Complete Freund's adjuvant. The emerged hybridoma cells were discovered to release notable levels of polyclonal antibodies and produced antibody concentration in supernatant was ascertained to increase daily until 55th day of incubation.\u003c/p\u003e \u003cp\u003eThe generated polyclonal antibodies are supposed to be used for immunotherapy of breast cancer since they have ability to conjuct specifically with tumor cells. Besides, it can be also thought that antibodies obtained with hybridoma technology can be used for diagnostic purposes against breast cancer. In the diagnosis of breast cancer, the mostly applied methods are mammography, ultrasound and biopsy [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, these techniques require either surgical intervention or long time to obtain accurate results. On contrary, antibody-mediated serological diagnostic kits enable the detection of breast cancer antigens with a more rapid, reliable and sensitive way [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, the biggest advantage of diagnostic kits involving polyclonal antibodies produced with hybridoma technology may be that they are specialized to capture the breast cancer-specific antigenic molecule in the sample, not the antibody. This approach can help to overcome the serious disadvantage of alternative serological kits whose working principle is relying on the arrest of antibodies in blood sample since tumor specific antibodies are rarely present in blood of breast cancer patients. Currently, diagnostic kits coated with antibodies are being developed against breast cancer, but no effective results have been obtained so far. The fundamental reason for low sensitivity of newly formed kits is the application of monoclonal antibodies during production. Although monoclonal antibodies indicate high potential in diagnosis and treatment of various cancers, their action is interfered with specifically binding to a single epitope region of tumor antigens, resulting in low avidity results compared to polyclonal antibodies[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Accordingly, the use of polyclonal antibodies in development of diagnotic kits could be considered more advantageous in contrast to monoclonal antibodies. At the final step of our study, the avidity properties of polyclonal antibodies derived from hybridomas that were developed against sonicated breast cancer cell antigens were explored and their performances were also compared with commercially available monoclonal antibodies. Eventually it was established that polyclonal antibodies produced from hybridomas demonstrated superior ability to interact with breast cancer antigens acquired from AU-565 cells in contrast to commercial antibodies. Interestingly, we also decipher that currently generated polyclonal antibodies also indicated robust immunoreactivity to antigens obtained from MCF-7 cells while commercial antibodies did not induce such an effect. These outputs reveal that diagnostic value of currently produced polyclonal antibodies were enormously higher than conventional antibodies and they could be used for diagnosis of breast cancer cases originated from different sources. Furthermore it is predicted that these newly generated polyclonal antibodies can readily detect metastic forms of breast cancer as well as its non-metastatic types and offer a great potential to be used in diagnostic kits which will recognize many antigenic epitopes of various cancer cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eConsequently, sonicated breast cancer lysates obtained from AU-565 cell line were combined with two separate adjuvants in order to produce innovative vaccine candidates within the scope of this research. The in vitro immunostimulatory efficacies of formulations were proven since they greatly ameliorated NO levels secreted from macrophage cells. The in vivo immunostimulatory activities of vaccine candidates were determined by evaluation of antibody titers upon immunization. According to the results combination including breast cancer antigens and Complete Freund's adjuvant was more influential in terms of stimulating humoral response and increasing antibody levels compared to combination formulized with PO adjuvant. Polyclonal antibodies were produced from splenocytes of mice immunized with breast cancer antigens and Complete Freund's adjuvant with hybridoma technology and improved amounts of antibodies were assessed within hybridoma cell culture upon long-term incubation. Finally, it was demonstrated that isolated polyclonal antibodies elicited strong interaction with antigens that were obtained from both AU-565 and MCF-7 cell lines. In conclusion, immunostimulatory activities of breast cancer antigens that were acquired with sonication method applied on AU-565 cell line were indicated for the first time in this study. It was also revealed that efficacy of antigens could be advanced by the use of adjuvants. Although Complete Freund's adjuvant was more powerful in regards to influencing B lymphocytes to create high titers of antibodies, PO adjuvant also induce the macrophages to secrete great amounts of NO. Hence, we foreseen that both formulation could be further used as immunotherapeutic vaccine candidate against breast cancer, however combination involving Complete Freund's adjuvant would be more promising. Moreover, obtained polyclonal antibodies could be further utilized for development of reliable and sensitive diagnostic kits for identification of different breast cancer types since they exerted strong immune reaction with breast cancer antigens with distinct behaviors. So, we predict that gathered outputs in this study will further make a huge contribution to researches related to development of immunotherapy approaches and diagnostic kits against breast cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declared that they had no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, M. I. and E.S.A.; methodology, software, formal analysis and investigation, M. I., A. D., A.P.Z.; writing\u0026mdash;original draft preparation, M. I. and E.S.A.; review and editing, A. A., M. B, and E.S.A.; visualization, supervision, project administration, M. I. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was supported by the Yildiz Technical University Scientific Research Project Coordination Department (Project No: FBA-2018-3359).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eŁukasiewicz S, Czeczelewski M, Forma A, Baj J, Sitarz R, Stanisławek A. Breast Cancer-Epidemiology, Risk Factors, Classification, Prognostic Markers, and Current Treatment Strategies-An Updated Review. Cancers (Basel). 2021;13(17).\u003c/li\u003e\n\u003cli\u003eMoo TA, Sanford R, Dang C, Morrow M. Overview of Breast Cancer Therapy. PET Clin. 2018;13(3):339-54.\u003c/li\u003e\n\u003cli\u003eSimonian M, Haji Ghaffari M, Negahdari B. Immunotherapy for Breast Cancer Treatment. Iran Biomed J. 2021;25(3):140-56.\u003c/li\u003e\n\u003cli\u003eReeves RA, Kaufman T. Mammography. StatPearls. Treasure Island (FL) ineligible companies. 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Methods Mol Med. 2000;40:23-34.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, polyclonal antibodies, hybridoma, diagnosis, immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-3966637/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3966637/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreast cancer is the most commonly diagnosed cancer type in women and approximately 700 thousand people around the world lose their lives due to breast cancer every year. Mammography and ultrasound are the techniques that are frequently applied for the diagnosis of breast cancer. However they involve several limitations such as low sensitivity and exposing to high radiation. Additionally, false negative and false positive results could be obtained in conventional diagnostic methods for breast cancer. So it is crucial to generate new diagnostic kits which enable rapid and accurate detection of breast cancer. Antibodies created using hybridoma technology can be considered in the diagnostic kits since they are important tools to bind cancer cell antigens. Although monoclonal antibodies are usually utilized in antibody-mediated diagnostic kits and they possess high specificity in diagnosis and treatment, they conversely indicate low avidity to tumor antigens in comparison to polyclonal antibodies because they can only bind to a single epitope region. Therefore, polyclonal antibodies display a pivotal role in recognition of many epitopes of breast cancer cells. The major aim of this study is to create polyclonal antibodies against whole cell lysate of AU-565 cell line by hybridoma technology and examine their diagnostic value by comparing with conventional antibodies. The acquired tumor cell antigens were supplemented with two distinct adjuvants Complete Freund\u0026rsquo;s Adjuvant (CFA) and Polyoxidonium (PO) while preparing the formulations for immunization. Thus we also evaluated in vivo immunogenic properties of antigen-adjuvant combinations and compared immunostimulatory efficacies of CFA and PO over prepared antigens. The outputs revealed that whole cell antigens reinforced with CFA demonstrated robust immunostimulatory activities, in vivo by enhancing the produced antibody levels in mice excessively. Polyclonal antibodies that were obtained from spleens of mice immunized with AU-565 cell antigens and CFA combinations were highly effective to capture the antigens that were isolated from different breast cancer cell line. It was detected that obtained polyclonal antibodies exhibited stronger immune reactions with breast cancer antigens when compared with conventional antibodies. Consequently, considerable immunostimulatory performance of AU-565 cell antigens and CFA combination was shown as a vaccine candidate and high diagnostic value of polyclonal antibodies produced in response to vaccination with mentioned formulation was established for the first time in the present study.\u003c/p\u003e","manuscriptTitle":"Evaluating Immunoreactivity of Polyclonal Antibodies Developed against AU-565 Cell Line for Diagnosis and Immunotherapy of Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-21 04:43:23","doi":"10.21203/rs.3.rs-3966637/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"63cc26e9-6ca3-4b85-84cc-a787fc855c39","owner":[],"postedDate":"February 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-06T02:49:05+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-21 04:43:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3966637","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3966637","identity":"rs-3966637","version":["v1"]},"buildId":"cTy_lsJlmDsVRNrSptgXS","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}