PRODUCTION OF POLYCLONAL ANTI-HUMAN HEMOGLOBIN A, C, F AND S ANTIBODY IN RABBIT

Abstract

Polyclonal antibodies represent a group of mixture of antibodies produced by different B-lymphocytes in response to the same antigen. Thus, different antibodies in the group recognize different parts of the antigen. Anti-human haemoglobin was developed in rabbit using haemoglobin isolated from normal adult human red blood cells as the immunogen. Purified haemoglobin A, C and S from adult human red blood cells and F from cord blood was used. Blood samples were collected into EDTA bottles from individuals with known haemoglobin genotype AA, AS, AC and SS, and from neonates ( day old babies ). The various samples genotype were confirmed by cellulose acetate paper electrophoresis. Based on the relative mobility of haemoglobin A, C, F and S after alkaline electrophoresis, various bands were cut and eluted with phosphate saline buffer. Hemoglobin concentration was determined spectrophotometrically. The eluate concentration was adjusted to 1mg/ml . 1ml hemoglobin was mixed with 10μl ,of adjuvant ( PPD Tuberculin mammalian, Tween 80 0.005%, Phenol 0.25%, sigma) and inoculated intramuscularly (IM) into the 6 month old New Zealand white rabbits. The eighteen animals used were divided into; two as control, eight inoculated with adjuvant(two animals for each hemoglobin A, C, F and S) and eight inoculated without adjuvant. The second and the third inoculations were performed on days 14 and 28 without adjuvant, and fourth inoculation was done on day 42. Seven days after the final inoculation blood samples were taken from the rabbit into plain bottles and serum was separated. Immunized rabbit serum was assayed for total protein, albumin, globulin and serum electrophoresis. Some other portions of the sample were used for antigen/antibody testing which include capillary precipitin test, agar diffusion test and agar plate test. Evidence of precipitin formation was first noted in serum obtained from animals with adjuvant within 2 hours while evidence of precipitin formation was delayed for up to six hours in serum obtained from animals without adjuvant. With undiluted anti hemoglobin sera, positive precipitin reactions were obtained with dilutions of antigen of as high as 1:1,000 (1 μg/dl haemoglobin). Positive precipitin rings were also observed with dilution of anti sera as high as 1:40 in all the reactions. Precipitin formation between serum obtained from immunized Rabbits and hemolysed red cell was an evidence of polyclonal antibody production.