New immunoassay systems based on recombinant human lactoferrin

D A Semenov,O V Sviridov,I I Vashkevich

doi:10.29235/1561-8323-2021-65-3-290-302

D A Semenov, O V Sviridov + Show 1 more

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https://doi.org/10.29235/1561-8323-2021-65-3-290-302

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Abstract

In this work, soluble and solid phase immunoreagents, including recombinant human lactoferrin (rhLF), a complex of rhLF with europium ions, rabbit antiserum to rhLF, anti-rhLF immunoglobulin purified by antigen-affinity chromatography and the conjugates of this immunoglobulin with an Eu3+ chelate or horseradish peroxidase have been obtained by a combination of biochemical and synthetic methods using rhLF as an initial compound. Biospecific interactions of the reagents in four immunochemical systems were assessed by measuring the enzyme activity or time-resolved fluorescence. The study resulted in the development of fast and precise immunoassays for biologically active rhLF in transgenic goat milk and in protein fractions obtained in the course of pure rhLF manufacture, as well as in pharmaceutical preparations and food additives.

Highlights

Soluble and solid phase immunoreagents, including recombinant human lactoferrin, a complex of rhLF with europium ions, rabbit antiserum to rhLF, anti-rhLF immunoglobulin purified by antigen-affinity chromatography and the conjugates of this immunoglobulin with an Eu3+ chelate or horseradish peroxidase have been obtained by a combination of biochemical and synthetic methods using rhLF as an initial compound
Biospecific interactions of the reagents in four immunochemical systems were assessed by measuring the enzyme activity or time-resolved fluorescence
The study resulted in the development of fast and precise immunoassays for biologically active rhLF in transgenic goat milk and in protein fractions obtained in the course of pure rhLF manufacture, as well as in pharmaceutical preparations and food additives

Summary

Introduction

Soluble and solid phase immunoreagents, including recombinant human lactoferrin (rhLF), a complex of rhLF with europium ions, rabbit antiserum to rhLF, anti-rhLF immunoglobulin purified by antigen-affinity chromatography and the conjugates of this immunoglobulin with an Eu3+ chelate or horseradish peroxidase have been obtained by a combination of biochemical and synthetic methods using rhLF as an initial compound. При работе с системой прямого одностадийного ИФА, в отличие от описанной выше методики непрямого ИФА, в лунки микропланшета, функционализированного адсорбцией рчЛФ, последовательно прибавляли 0,05 мл пробы, содержащей 0 или от 0,4 до 32,4 мг/л рчЛФ, и 0,05 мл раствора с концентрацией 4,0 мкг/л конъюгата анти-рчЛФ ПАт-ПХ, и после инкубации в течение 45 мин при комнатной температуре без встряхивания, лунки промывали и сразу же выполняли этапы ферментативной детекции.

Results

Conclusion