Evaluating immunoreactivity of hybridoma-derived antibody mixtures generated against AU-565 cell line for diagnosis and immunotherapy of breast cancer

BACKGROUND: This study aimed to generate antibodies against the whole-cell lysate of the AU-565 breast cancer cell line using hybridoma technology and to assess their immunogenic properties and preliminary immunoassay-oriented reactivity. Two adjuvants, Complete Freund's Adjuvant (CFA) and Polyoxidonium (PO), were compared for their ability to enhance immune responses. Cytokine profiling was also performed to evaluate the immunomodulatory effects of antigen-adjuvant formulations. METHODS: AU-565 cell lysates were prepared by sonication and combined with CFA or PO. Immunostimulatory activity was evaluated in vitro by nitric oxide (NO) production in J774 macrophages and in vivo by antibody titers in Balb/c mice. Hybridoma cells were generated from splenocytes of immunized mice. Since single-cell cloning was not performed, antibodies were collected from pooled hybridoma cultures, representing a mixture of monoclonal antibodies recognizing multiple epitopes. Cytokine levels (IL-6, IL-12, GM-CSF, and TNF-α) were quantified by ELISA to characterize the inflammatory response pattern induced by the antigen-adjuvant formulations. RESULTS: The AU-565 antigen + CFA formulation significantly increased NO production compared with untreated control macrophages and generated the strongest antibody titers compared with the PBS control group. Cytokine profiling showed that the AU-565 antigen + CFA formulation significantly increased IL-12, GM-CSF, and TNF-α secretion compared with untreated control macrophages, indicating a pronounced pro-inflammatory response pattern in the macrophage model, while a moderate IL-6 increase relative to untreated control macrophages suggested partial support for humoral-associated signaling. The hybridoma-derived antibody mixture displayed stronger ELISA reactivity toward AU-565 and MCF-7 lysates than the commercial antibody, indicating broader relative lysate recognition under the assay conditions used. CONCLUSION: The hybridoma-derived antibody mixture showed strong immunogenicity, cytokine-associated immune stimulation, and preliminary potential for breast cancer-oriented immunoassay development; however, additional validation of specificity and analytical performance is required before diagnostic utility can be concluded. The AU-565 antigen-CFA combination not only enhanced antibody production but also promoted a favorable cytokine profile, underscoring CFA's superiority as an adjuvant in breast cancer-oriented immunogen design.