To obtain purified H and M
antigens suitable as primary standards in the serological diagnosis of
histoplasmosis by
agar gel double-diffusion tests, H and M reactive components of
histoplasmin were fractionated by column chromatography by using
Sephadex G-100,
Sephadex G-200, and
diethylaminoethyl cellulose. Six fractions from
diethylaminoethyl cellulose were reactive in
agar gel double-diffusion,
complement fixation, and capillary precipitin tests. When examined by electrophoresis on acrylamide gel, one M
antigen (fraction 2, molecular weight greater than 200,000) and two H
antigens (fractions 5 and 6, molecular weight greater than 200,000) each gave essentially a single
protein band. In
agar gel double-diffusion and
complement fixation tests with sera from patients with proven cases of
histoplasmosis,
blastomycosis, or
coccidioidomycosis, these two fractions of
H antigen and the one of M
antigen reacted only with sera from proven or suspect cases of
histoplasmosis and showed reactivity with those sera known to contain only the anti-H or anti-M antibody, respectively. Fraction 2 (M
antigen) and fractions 5 and 6 (H
antigens) had
carbohydrate-to-
protein ratios of 1.60, 0.77 and 0.78, respectively. Both
antigens contained
galactose,
glucose,
mannose, and
hexosamine, with
mannose being the predominant
sugar. Fraction 2 was characterized by a high
proline and
glucose content, whereas fractions 5 and 6 contained higher concentrations of
galactose,
mannose,
glycine, and
alanine. Each of these products appeared to separate into two active fractions, one of a molecular weight greater than 200,000 and one of a molecular weight less than 35,000. The M
antigen component of fraction 2 was still characterized by a high
proline content, whereas the
H antigen components of fractions 5 and 6 had a high content of
glutamic acid,
serine,
glycine, and
proline.