Increasing, the Specificity of Polyclonal Antibodies to Human and Mouse β2-Microglobulin as an Alternative to the Use of Monoclonal Antibodies in Immunological Analysis

Abstract

Highly specific reagents, i.e., proteins and antibodies to them, are the necessary components of systems for verifying the effectiveness of transgenic/knockout animal biomodels. In particular, the identification of   mouse and human β2-microglobulin in the protein fractions of organs and tissues of transgenic and β2m knockout mice of several HLA lines, which have been created in recent years at the Scientific Center of  Biomedical Technologies of the FMBA of Russia, is the most important stage of their certification. At  the first stage of our research,  E. coli  producing strains of recombinant mouse and human β2-microglobulin (mβ2mg and hβ2mg) were obtained, the proteins were isolated and purified. At the next stage of the work, affine sorbents with immobilized mβ2mg and hβ2mg were obtained. To increase the species specificity of the serum, “rabbit-anti-hβ2mg” were depleted against the recombinant protein mβ2mg, and, conversely, “rabbit-anti-mβ2mg” were depleted against the recombinant protein hβ2mg. Highly specific antibodies were purified from depleted sera using affinity sorbents. Using dot- and western-blotting methods оn the example of depleted and affinity-purified rabbit-anti-hβ2mg antibodies, a significant increase in  their specificity relative to hβ2mg was shown.