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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.
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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.
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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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