A Fast and Straightforward Method for the Purification of Anti- Immunoglobulin G (IgG) for Coombs Wright Assay

Abstract

Introduction: Immunoglobulin G is the most abundant immunoglobulin in human blood, comprising approximately 75% of serum antibodies. Human IgG is a glycoprotein and can be an antigen for other mammals. Antibody-sensitized red blood cells (RBCs) and complement components can react with anti-human globulins resulting in their agglutination. This study aimed to prepare an antiserum against human IgG for deploying in Coombs Wright and cross-match for rare blood groups. Methods: After isolation of serum from healthy blood donors, serum proteins were precipitated using ammonium sulfate. Consequently, tangential flow filtration and ion-exchange chromatography were applied to purify IgG. SDS-PAGE and Bradford protein content assay was conducted to evaluate the quality and the concentration of the purified IgG. Rabbits were weekly injected with different amounts of the protein four times. Then, sera were obtained from the immunized mice, and total IgG was purified. Finally, the Coombs Wright test was performed on samples from brucellosis patients to validate purified IgG antibody quality. Results: Electrophoresis and Bradford assay results showed that the purified protein had considerable high purity and quantity. Protein bands of reducing and the non-reducing SDS-PAGE showed high purity of the protein along with a protein yield of 2.2 mg/L. Coombs Wright tests using the rabbit anti-human serum had a comparable result with available commercial anti-human immunoglobulin. Conclusion: The results indicated that our method for the purification of IgG was suitable for anti-human globulin preparation. This antibody can also be used in clinical diagnostic tests such as Coombs Wright, cross-match, and blood types evaluation with weak Rh or Du antigens.

Keywords

• anti-human globulin
• immunoglobulins
• coombs wright