Development of criteria for immobilization of human erythrocytes of phenotypes R1R1 and R2R2 on the solid phase in determining the content of anti-D-antibodies IgG in immunoglobulin human antirhesus Rho(D) preparations by enzyme immunoassay
AbstractIntroduction. The content of anti-D-antibodies (Ab) IgG in human immunoglobulin antirhesus Rho(D) preparations is evaluated by direct competitive enzyme immunoassay (ELISA) using the International standard (IS) of anti-D immunoglobulin, commercial reagent and antigens (Ag) for the preparation of an immunosorbent. Freshly prepared and preserved D-positive erythrocytes human blood group I(0) of the R1R1 and R2R2 phenotypes obtained from at least 3 donors are Ag, which are immobilized in the solid phase in laboratory conditions. The features of their distribution in the solid phase when preparing an immunosorbent have not been studied, and the stage of reproducing the method is not standardized.
The aim of this study was to develop criteria for immobilization of human erythrocytes of the R1R1 and R2R2 phenotypes on the solid phase in determining the content of anti-D-Ab IgG in human immunoglobulin antirhesus Rho(D) preparations by direct competitive ELISA.
Material and methods. We used erythrocytes of human blood I(0) group of donors of the R1R1 and R2R2 phenotypes, IS of anti-D immunoglobulin, Standard of normal human immunoglobulin not containing anti-D-Ab IgG, biotinylated monoclonal anti-D-Ab. The immunosorbent was prepared by immobilization of pre-washed and hydrolyzed papain erythrocytes into the wells of a polystyrene tablet, quantitative assessment of their distribution in the solid phase was carried out by microscopy and computer morphometry. To establish the values of the immobilization criteria, a quantitative assessment of the anti-D-Ab IgG content in solutions of test samples with a known anti-D-Ab IgG content was carried out by direct competitive ELISA.
Results. Revealed that erythrocytes of the R1R1 and R2R2 phenotypes are not evenly distributed on the surface of the solid phase, with a tendency to aggregation depending on the phenotype. Formulas are proposed and calculated: the area of uniform distribution – relative area (Srel.), the total area of aggregated erythrocytes of the studied phenotypes (∑S), and their distribution coefficients (Kdistr.) on the solid phase. Developed criteria of immobilization of Ag on the solid phase: for erythrocytes of phenotype R1R1 Srel. must be at least 88 %, Kdistr. – no more than 0.22; for the phenotype R2R2 Srel. – at least 70 %, Kdistr. – not more than 0.23. It is shown that the results of determining the content of anti-D-Ab IgG in human immunoglobulin preparations antirhesus Rho(D) by direct competitive ELISA meet the criteria of acceptability, provided that the established criteria for immobilization of erythrocytes are met.
Conclusion. The developed criteria for the immobilization of erythrocytes of the R1R1 and R2R2 phenotypes are recommended for standardizing the stage of preparing an immunosorbent for determining the content of anti-D-Ab IgG in human immunoglobulin antirhesus Rho(D) preparations by direct competitive ELISA.
Keywords:human immunoglobulin antirhesus Rho(D); anti-D-antibodies IgG; competitive ELISA; phenotypes of erythrocytes R1R1 и R2R2; immunosorbent; criteria of immobilization
For citation: Shvedova E.V., Leshina S.A., Davydov D.S., Borisevich I.V., Kudasheva E.Yu. Development of criteria for immobilization of human erythrocytes of phenotypes R1R1 and R2R2 on the solid phase in determining the content of anti-D-antibodies IgG in immunoglobulin human antirhesus Rho(D) preparations by enzyme immunoassay. Immunologiya. 2022; 43 (2): 208–16. DOI: https://doi.org/10.33029/0206-4952-2022-43-2-208-216 (in Russian)
Funding. The study was carried out as part of a publicly funded research project No. 056-00005-21-02 and was supported by the Scientific Centre for Expert Evaluation of Medicinal Products of the Ministry of Health of the Russian Federation (R&D public accounting No. 121022000147-4).
Conflict of interests. The authors declare no conflict of interests.
Authors’ contribution. Research concept and design – Kudasheva E.Yu., Shvedova E.V., Borisevich I.V.; experimental work – Shvedova E.V., Leshina S.A.; text writing – Shvedova E.V., Kudasheva E.Yu., Davydov D.S.; final approval of the version for publication – Borisevich I.V., Kudasheva E.Yu.
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