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Skin Test Studies in Guinea Pigs

      The injection of whole M. leprae into guinea pigs induces a potent cell mediated response, which can be assessed by injecting small amounts of M. leprae subcellular antigen fractions (cytosol, membrane and soluble cell wall proteins) intradermally, with the subsequent measurement of the delayed type hypersensitivity response (DTH). Injections of even 1 mg quantities of these complex crude antigens generally results in a strong DTH reaction, with induration and erythema being usually between 15 to 20 mm diameters. Understanding the makeup of the individual proteins that are responsible for inducing this reaction would lead to potential development of improved diagnostic reagents for the assessment of the immune status in leprosy patients, and to develop improved skin test reagents for human use.

      In order to reduce the complexity of the crude antigens, we have used the BioRad whole gel eluter to obtain size-fractionated proteins from M. leprae cytosol. The eluter was originally developed by Dr. Peter Andersen, which he used to prepare size-fractionated proteins from the culture filtrate of M. tuberculosis. Using this technique, he was able to identify those proteins that produced the best IFN-gamma responses in sensitized mice, and eventually identified novel and immunologically important proteins, such as esat-6. The device allows one to purify substantial amounts of proteins from a preparative SDS-PAGE gel (usually loading 10-20 mg of the complex protein mixture). After electroelution, 30 fractions are obtained, each containing a relatively small set of proteins, all having a molecular weight range within 5 kDa of each other. When the set of proteins is run on a gel (see illustration below), it looks like a ladder of bands of decreasing molecular weight. The advantage of using this technique is that the proteins are isolated relatively free of SDS and can be used right away in in vitro and in vivo assays to measure parameters such as the induction of IFN-gamma in peripheral blood lymphocytes or in guinea pig DTH responses.
     

We have prepared size fractionated M. leprae cytosolic fractions and have examined their reactivity in DTH responses in guinea pigs. Most of the antigen fractions induce a potent DTH skin test response (10-17 mm diameter average) using 2-4 ug of these semi-purified antigens. We have already identified 5 of the proteins in 4 different fractions by sequencing the dominant spots from 2D gels, and so far, they are all known and immunologically relevant proteins found in M. leprae.

 


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