Immunoanalytical methods are frequently employed in the detection of hazardous small molecular weight compounds. However, antibody development for these molecules is a challenge, because they are haptens and cannot induce a humoral immune response in experimental animals. Immunogenic forms of haptens are usually prepared by conjugating them to a protein carrier which serves as an immune stimulator. However, the carrier is usually considered merely as a bulk mass, and its biological activity is ignored. Here, we induced an endocytic receptor, transferrin receptor, by selecting its ligand as a carrier protein to enhance antibody production. We conjugated aflatoxin, a potent carcinogenic food contaminant, to transferrin and evaluated its potential to stimulate antibody production with respect to ovalbumin conjugates. Transferrin conjugates induced aflatoxin-specific immune responses in the second immunization, while ovalbumin conjugates reached similar antibody titers after 5 injections. Monoclonal antibodies were successfully developed with mice immunized with either of the conjugates.
Antibodies are the basic components of immunoanalytical systems used for detection of a wide range of analytes. Although there are some ground rules for antibody selection, analyte- and assay-specific criteria are the ones that determine the ultimate success of the immunoassays. In this study, we introduced an effective antibody selection procedure for the development of immunoaffinity columns for aflatoxins. The designed scheme puts emphasis on solvent- and matrix-related characterization steps and was used to comparatively evaluate eight monoclonal antibodies. The selected antibody was tolerant to 40% methanol, 20% acetonitrile, 30% acetone and 40% ethanol and did not interact with corn, red pepper or hazelnut extracts. Immunoaffinity columns developed with the selected antibody were validated by 15 independent aflatoxin analysis laboratories.
Hepatitis B is an infection of the liver caused by the Hepatitis B virus (HBV). HBV is one of the major causes of acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma, and it is a serious global public health problem. It is estimated that 350 million individuals worldwide are infected with the virus, which causes 620,000 deaths each year. People most at risk include antibody who has unprotected sexual intercourse, drug users who share needles and syringes, health care workers in contact with potentially contaminated blood or body fluids, anyone in intimate contact with the infected person. Therefore, the development of economic and accurate detection systems and potential alternative antiviral approaches for HBV detection could be important for fighting viral hepatitis. Hepatitis B diagnosis has been based on the detection of serologic markers. Testing for these markers helps to determine the presence of past or ongoing HBV infection, the acute, chronic or subclinical carrier state of the disease, response to therapy, and/or the immune status of the patient. Hepatitis B virus surface antigen (HBsAg) is the first serological marker to appear in the circulation, well before clinical symptoms, and is the viral component usually found in the highest concentration in the serum of HBV-infected patients. The presence of anti-HBs in serum indicates previous exposure to HBV and long-lasting acquired immunity. Low serum titres of anti-HBs, however, it can signal a lack of immunity to future HBV infection. In this study, diagnostic systems based on the use of monoclonal antibody (MAb) and polyclonal antibodies (PAb) were developed and conjugated enzyme and biotin for early and sensitive diagnosis of Hepatitis B which is still one of the important infectious diseases in Turkey. Sandwich ELISA kit systems were generated by using both 2G3 MAb (as capturing agent) and 2G3-HRP or 2G3-biotin conjugates (as detecting agents). Homemade sandwich ELISA tests were compared with the other conventional sandwich ELISA tests by using hepatitis B positive and negative infection serum. They were shown that our system gave reliable results. When the homemade HBsAg ELISA system were compared with the other commercial kit by using 280 patients' sera, it was shown that our system corresponded with the results of negative and positive samples at ratio of 96 %. When the homemade anti-HBsAg ELISA system was compared with the commercial kit by using 173 patients' sera, it was shown that our system corresponded with the results of negative and positive samples at ratio of 91 %. In subsequent studies by the final tests of homemade ELISA kit, it was observed that Biotin-labeled kits responded very close results with the 97% conformity level when compared with commercial kits.
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