Optimization and intensification of synthesis protein-protein and protein-hapten conjugates for highly sensitive immunoassays

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O. Yu. Galkin
N. P. Metalnikova
Yu. P. Gorgo


It is known a lot of methods of conjugation of antibodies with enzymes that can produce conjugates with high antigen-binding and enzymatic activity. A variety of functional groups of enzyme and antibody molecules is used for immunoenzymatic conjugates synthesis (for example, amino, sulfhydryl, and aldehyde groups). Enhancement of diagnostic kits can be realized by improving of immunoenzymatic conjugates, and methods for its synthesis and purification. Intensification and optimization of the protein conjugates synthesis is possible by increasing the influence of macrokinetic component of the conjugation reaction. Hardware and tool implementation of this principle is the replacement of the adsorbent which is used in conjugation with an inert filter material and circulation the reaction mixture through filter material. The optimal conditions for the synthesis of the conjugates have been determinate. It has been proved the possibility of the protein conjugates synthesis for a significantly smaller (10 min) period of time compared to traditional methods. Temperature is significantly less effect on the reaction rate of conjugation in the case of the optimized method, when macrokinetic factors influence on its course more intensively. It has been shown that proteins and antibody conjugates with biotin and peroxidase conjugates prepared by an optimized method are more active, and they can be used to develop highly sensitive immunoassays.

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