评估IgY抗体作为多特异性抗人球蛋白试剂的性能。

Evaluation of IgY Antibody as a Polyspecific Coombs-Reagent.

作者信息

Calzado Esteban Justo Gutiérrez, Heredia Marlene Toledano, Duharte Jeorge Fernadez, Zarnani Amir-Hassan

机构信息

Laboratory of Antibodies and Experimental Biomodels, Center of Molecular Immunology, Santiago de Cuba, Cuba.

Department of Immunology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.

出版信息

Avicenna J Med Biotechnol. 2017 Apr-Jun;9(2):87-93.

PMID:28496948

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5410134/

Abstract

BACKGROUND

During the last twenty years, the extraction of specific egg yolk (IgY) antibodies from the immunized chickens has been accepted as a useful alternative to the immunization of mammals. The aim of the present study was immunizing the chickens with Human Umbilical Cord Serum (HUCS) and the extraction of specific anti-human globulins (IgG, C3b, and C3d) antibodies from egg yolk in order to obtain polyspecific Coombs reagent.

METHODS

The novelty of this work was the achievement of a polyclonal reagent through a very cheap alternative method in accordance with all ethical regulations required for obtaining it. Three Leghorn hens (21 weeks old) were immunized four times for a period of 66 days with 20uL of HUCS mixed with PBS/FCA or FIA each time. The extraction of IgY antibodies was performed according to the method of lipid precipitation of yolk and using water soluble fraction as the reagent material. The resulting IgY antibody was characterized by SDS-PAGE and immunoelectrophoresis and tested for the presence of hetero-agglutinins by means of direct agglutination using human erythrocytes of all blood groups treated with 0.1% papain and for indirect Coombs-test to evaluate its specificity to fractions (C3b, C3d, C4d) of human complement and human IgG, respectively.

RESULTS

Our findings show, that, the reagent obtained contains IgY and other 3 proteins (SDS-PAGE), and reacts specifically with plasma proteins, that migrate in β and ϒ regions. In immunoelectrophoresis, in addition, there is the presence of low hetero-agglutinins levels in IgY-preparation (3 lots), and the possibility to produce high amount (more than 500 ) of polyspecific Coombs-reagent in chickens is also discussed.

CONCLUSION

IgY-preparation (3 lots), and the possibility to produce high amount (more than 500 ) of polyspecific Coombs-reagent in chickens with the originality to achieve a polyclonal reagent through a very cheap alternative method in accordance with all ethical regulations required for obtaining it, was also discussed.

摘要

背景

在过去二十年中，从免疫鸡中提取特定的蛋黄（IgY）抗体已被公认为是一种替代哺乳动物免疫的有用方法。本研究的目的是用人类脐带血清（HUCS）免疫鸡，并从蛋黄中提取特异性抗人球蛋白（IgG、C3b和C3d）抗体，以获得多特异性库姆斯试剂。

方法

这项工作的新颖之处在于通过一种非常廉价的替代方法获得了一种多克隆试剂，该方法符合获取该试剂所需的所有伦理规定。三只来航鸡（21周龄）每次用20μL HUCS与PBS/FCA或FIA混合，进行四次免疫，为期66天。IgY抗体的提取采用蛋黄脂质沉淀法，以水溶性部分作为试剂材料。通过SDS-PAGE和免疫电泳对所得IgY抗体进行表征，并通过使用经0.1%木瓜蛋白酶处理的所有血型的人红细胞进行直接凝集试验检测异凝集素的存在，以及通过间接库姆斯试验分别评估其对人补体和人IgG的各组分（C3b、C3d、C4d）的特异性。