Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity

Autor(es)

de Lima Fontes, Marina ^[1] ; Neves, Franciny Mara de Lima ^[1] ; Santos, Kelvin Sousa ^[1] ; Fusco-Almeida, Ana Marisa ^[1] ; Giannini, Maria Jose Soares Mendes ^[1] ; Felisbino, Sergio Luis ^[2] ; Deffune, Elenice ^[3] ; Moroz, Andrei ^[1]

Afiliação do(s) autor(es)

^[1] Sao Paulo State Univ UNESP, Sch Pharmaceut Sci, Monoclonal Antibody Lab, Dept Clin Anal, Araraquara, SP - Brazil

^[2] Sao Paulo State Univ UNESP, Inst Biosci, Extracellular Matrix Lab, Dept Morphol, Botucatu, SP - Brazil

^[3] Sao Paulo State Univ UNESP, Sch Med, Tissue Engn Lab, Dept Urol, Botucatu, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: FRONTIERS IN IMMUNOLOGY; v. 12, DEC 7 2021.
• Citações Web of Science: 0

Resumo

Monoclonal antibodies (mAbs) have been a valuable tool to elucidate several biological processes, such as stem cell differentiation and cancer, and contributed to virtually all areas of biomedical sciences. Yet, it remains a challenge to obtain mAbs specific to poorly expressed epitopes, or to epitopes that are actually involved in important biological phenomena, such as cell differentiation and metastasis. Drug-induced subtractive immunization, and recently the multiple tolerization subtractive immunization (MTSI) technique, reported by our group, have the potential to level up the field, as they direct the host ` s immune response towards these epitopes. However, due to cyclophosphamide (CY) treatment, high mice mortality can be observed, and only a few data are available on how these techniques affect the immune system of mice. Tolerogen and immunogen cells, RWPE-1 and PC-3 cells, respectively, were individually seeded at 2 x 10(4) cells/cm(2), and then adjusted to 2 x 10(6) cells per mouse before immunization, which was conducted in a subtractive approach (MTSI) with CY. Immunosuppression of mice was recorded via total white blood counting, as well the reactivity of circulating polyclonal antibodies (pAbs). General parameters, including weight, physical appearance, and behavior on mice subjected to three different concentrations of CY were recorded. mAbs were obtained using classical hybridoma techniques, using the spleen of immunized mice. After purification, antibodies were characterized by Western blotting, and Indirect immunofluorescence. In conclusion, all CY dosage were efficient in creating an immunosuppression state, but only the 100 mg/kg body weight was feasible, as the others resulted in extensive mice mortality. pAbs obtained in the peripheral blood of mice showed more reactivity towards tumor cells. MAbs 2-7A50 and 2-5C11 recognized antigens from tumor cells, but not from their non-tumor counterparts, as shown in western blotting and immunofluorescence assays. MTSI technique was successful in generating mAbs that recognize tumor-specific antigens. (AU)

• Processo FAPESP: 17/20404-0 - Desenvolvimento de imunosensor para detecção de células prostáticas tumorais circulantes utilizando anticorpos monoclonais
• Beneficiário: Marina de Lima Fontes
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 18/19083-7 - Imunosensores para detecção de células prostáticas tumorais circulantes utilizando anticorpos monoclonais murinos
• Beneficiário: Andrei Moroz
• Modalidade de apoio: Auxílio à Pesquisa - Regular