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

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. Antibodysensitized red blood cells (RBCs) and complement components can react with antihuman globulins resulting in their agglutination. This study aimed to prepare an antiserum against human IgG for deploying in Coombs Wright and crossmatch 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 ionexchange chromatography were applied to purify IgG. SDSPAGE 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 nonreducing SDSPAGE showed high purity of the protein along with a protein yield of 2.2 mg/L. Coombs Wright tests using the rabbit antihuman serum had a comparable result with available commercial antihuman immunoglobulin. Conclusion: The results indicated that our method for the purification of IgG was suitable for antihuman globulin preparation. This antibody can also be used in clinical diagnostic tests such as Coombs Wright, crossmatch, and blood types evaluation with weak Rh or Du antigens.