MODULATING EFFECT OF POLYCLONAL ANTIBODIES AGAINST AFLATOXIN B₁ MACROMOLECULAR COMPLEXES ON AFLATOXIN INDUCED HEPATOCARCINOGENESIS

Abstract

Polyclonal immunoglobulin G antibodies were raised in adult male albino rabbits against aflatoxin B₁-bovine serum albumin, aflatoxin B₁-histone H₁ and aflatoxin B₁-microsomal proteins complexes after a single intradermal multiple site injection of water in oil emulsion of the complexes. Two different combinations of the complexes namely a mixture of aflatoxin B₁-bovine serum albumin and aflatoxin B₁-histone H₁ complexes and a mixture of the three aflatoxin-protein complexes were also used in raising antibodies. The antisera obtained from the rabbits immunized with each immunogen showed the presence of large amounts of antibodies as from the third week after immunization. The antibody content of the different antisera increased gradually from the third week until maximum production occurred in the eighth or ninth week after immunization. This was followed by a gradual decrease till the termination of bleeding in the twelfth week after immunization. Binding studies on the interaction of the purified immunoglobulin G antibodies with each of the antigenic complexes using equilibrium dialysis showed that the number of binding sites for aflatoxin B₁ on the antibody molecule varies from 0.78 to 1.43 for AFB -BSA antisera, 0.86 to 1.52 for AFB₁-H₁ antisera, 0.77 to 1.65 for AFB₁ -MP antisera, 0.65 to 1.60 for antisera to AFB₁-BSA combined with AFB₁-H₁ and 0.80 to 1.53 for antisera to a combination of AFB₁-BSA, AFB₁-H₁ and AFB₁-MP. The classical first association constant binding of aflatoxin B₁ to the antibodies also varied between 19.83 and 44.49 x 10³M⁻¹ for AFB₁ -BSA, 19.07 and 49.92 x 10³M⁻¹ for AFB₁-H₁, 19.13 and 33.83 x 10³M⁻¹ for AFB₁-MP, 12.86 and 46.69 x 10³M⁻¹ for AFB₁-BSA + AFB₁-H₁ + AFB₁-MP complexes. The free energy change for the binding ranges from -5.86 and -6.34 Kcal/mol for AFB₁-BSA, -5.84 and -6.41 Kcal/mol for AFB₁ -H₁, and -6.18 Kcal/mol for AFB₁-MP, -5.60 and -6.18 Kcal/mol for AFB₁-BSA+ AFB₁-H₁ and -5.82 and -6.23 Kcal/mol for AFB₁-BSA+AFB₁-H₁+AFB₁-MP complexes. Hapten inhibition reaction between the immunogens, their respective immunoglobulin antibodies and aflatoxin B₁ shows that aflatoxin B₁ inhibit the binding of the antibodies to their specific complexes. These findings indicate that the antibodies against the complexes are specific for aflatoxin B₁ and there is little difference in their effectiveness for aflatoxin B₁. Injection of antitoxin B₁ (7mg/kg body weight) into wistar rats caused 65% mortality and the development of highly diffused bile duct hyperplasia, enlarged hepatocyte nuclei, necrosis and vacuolation of hepatocytes in the surviving rats. Increased levels of serum alkaline phosphatase, gamma glutamyl transferase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase were also observed in these rats. The observed hepatic lesions are however greatly reduced in rats that are preimmunized with AFB₁-BSA and AFB₁-H₁ complexes before being challenged with the same dose of aflatoxin B₁. These findings suggest that the polyclonal antibodies may be of use as immunodetective devices for quantitation of aflatoxin in body fluids such as serum, plasma, milk and urine. They could also be used for immunointerception of the toxin via passive immunization. Furthermore the antibodies may have great potentials in vaccine development for immunoprophylaxis against aflatoxin B₁ in man and animals.