Abstract
Original set of 15 clones of hybridomas producers of monoclonal antibodies (mAbs) to prostate-specific antigen (PSA) (using as a source of lymphocytes of mice of inbred strains NZB and Balb/c) was obtained. The activity in ELISA, affinity constant and titer in the culture fluid of the mAbs obtained from NZB mice are higher compared to the mAbs obtained from the Balb/c mice splenocytes. Conjugates of obtained mAbs with horseradish peroxidase were synthesized, and it allowed performing comparative epitope characterization of the resulting set of mAbs. Studied mAbs are directed to 4 epitopes of PSA molecule: 2 mAbs interacting with one of the epitopes showed cross-activity with PSA-related protein – human kallikrein 2; 2 mAbs of the same epitope specificity blocked the enzymatic activity of PSA; mAbs of the remaining two epitopes either did not affect PSA chymotrypsin activity at all, or this inhibitory effect was not significant. The obtained mAbs recognize antigenic determinants that are not screened when interacting with α1-antichymotrypsin, as well as those that are localized in the places where α1-antichymotrypsin interacts with PSA. Within each of the given two mAbs groups, there are antibodies belonging to different epitopes, which give prospects for the further use of the obtained mAbs in immunoassay and immunobiotechnology. Two alternative methods for determining of the mAbs constants of affinity (by Friguet and Scatchard) are comparable (for determination of the affinity constants of anti-PSA antibodies): coefficient of linear correlation between the affinity constants defined by different methods was 0.90.
Highlights
The Prostate-specific antigen (PSA), known as gamma-seminoprotein or callicrein-3, is a glycoprotein enzyme with a molecular weight of 34 kDa, containing 237 amino acid residues, is related to serine proteinases (EC 3.4.21.77)
Analysis of the testing results allowed dividing the obtained monoclonal antibodies (mAbs) into three groups of specificity: the 1st group contains 8 mAbs and characterize by positive signals on prostate-specific antigen (PSA) only (11B5, 12F11, 13C2, 13G4, 14C8, 21D7, 23B4, 21B7); the 2nd group contains 8 mAbs and characterize by positive signals on PSA as well as on PSA-ACT complex (17F9, 12С7, 11G5, 21F4, 27C10, 26B9, 24H7); and the 3rd group is presented by 2 mAbs, which is positive on testing of all markers (PSA, PSA-ACT complex and human kallikrein-2 (hK2))
It was established that the activity in enzyme-linked immunosorbent assay (ELISA), the affinity constant and titer in the culture liquid of the mAbs obtained from NZB mice are higher compared to the mAbs obtained from the splenocytes of Balb/c mice
Summary
The Prostate-specific antigen (PSA), known as gamma-seminoprotein or callicrein-3 (kallikrein 3, KLK3), is a glycoprotein enzyme with a molecular weight of 34 kDa, containing 237 amino acid residues, is related to serine proteinases (EC 3.4.21.77). PSA is a biomarker for PC risk assessment Serum protease inhibitors such as α1-antihymortitpsin (ACT), α2-macroglobulin (MG) and other acute-protein proteins form irreversible complexes with circulating PSA and inhibit its proteolytic activity (Fletcher, 2019; Duskova and Vesely, 2015; Haythorn and Ablin, 2011; Sydyakina et al, 2019). Study of the PSA structure and its biological activity, as well as developments in the field of immune biotechnology, sometimes require the bioreagents – antibodies that are specific to PSA. In this case, the most appropriate is the use of mAbs, since they, unlike polyclonal serums, are characterized by their exceptional specificity and homogeneity, as well as the possibility of obtaining virtually unlimited quantities. One of the important elements of hybridoma technology is the use of effective immunization schemes that would ensure obtaining a broad or narrow-line epitopic repertoire of monoclonal antibodies (mAbs) with high affinity and activity in different immunoassay methods
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