Evaluation of IgY Antibody as a Polyspecific Coombs-Reagent

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 ml/egg) of polyspecific Coombs-reagent in chickens is also discussed.

Conclusion: IgY-preparation (3 lots), and the possibility to produce high amount (more than 500 ml/egg) 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.

Keywords: Chicken; Egg yolk antibody; Immunization; Immunoglobulins.

Figure 1.

Standard concentrations of globulins in human normal serum.

Figure 2.

Concentrations of globulins in thirteen samples of umbilical cord sera. See no presence of IgA and IgM in any sample. There is very high C3 (complement fraction) concentration in three of them (in red).

Figure 3.

Comparison of concentrations for globulins between human normal sera (left) and chosen umbilical cord sera samples (right).

Figure 4.

SDS-PAGE (no reducing conditions). 1. Specific IgY-Preparation against chosen human umbilical cord sera by WSF method (50 μl of preparation, aprox. 50 μg). 2. Standard of human IgG (20 μg). 3. Standard of mouse monoclonal IgG (10 μg). 4. Standard of chicken IgY from Sigma (10 μg). Compare lanes 1 and 4. The position of upper band in lane 1 correspond to band of pure IgY (180 kDa approx.) in lane 4. See the bands in lanes 2 and 3 underneath 180Kd that correspond to mammals IgG monomers (150 kDa approx).

Figure 5.

Immunoelectrophoresis. (A) Total human serum; (B) IgY-Preparation against chosen umbilical cord sera; (C) Commercial human IgG. Globulins-β (Globulins Beta) represents two specific arcs of precipitation toward β region (C3, C4) of migration of total plasma globulins and globulin-Y(Globulins Gamma) represent specific arc of precipitation that migrates toward Y region (human IgG) of total plasma globulins (Top part of the figure). Lower of the figure represents specific arc of precipitation toward human IgG to compare sense of migration in immunoelectro-phoresis.

Figure 6.

Specific activity of original IgY-preparation and the presence of hetero-agglutinins levels.

Figure 7.

Specific activity of IgY-preparation after fivefold dilution of reagent (left) and demonstration of no presence of hetero-agglutinins in it (right).