Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

Biochemical evidence for structurally distinct H-2Dd antigens differing in serological properties

H-2D^d antigens, as defined by the private H-2.4 determinant, exist as two immunochemically distinct populations in H-2^a and H-2^dm2 splenocytes and in the transformed cell line, RADA1(H-2^a). The two populations are distinguishable by the anti-H-2.28 serum, k/r anti-h2, which is directed, in part, against the H-2.28 family of public determinants encoded by the D end of the b haplotype. Sequential precipitates of lentil-lectin-purified glycoprotein extracts metabolically labeled with radioactive amino acids reveal that approximately one-quarter to one-third of the H-2D^d antigens, designated H-2D^d (b28⁺), react with this antiserum, whereas two-thirds to three-quarters, designated H-2D^d(b28^–), do not. Paired-label tryptic peptide maps in this and a previous study indicate that H-2D^d(b28⁺) and H-2D^d(b28^–) are closely related structurally and are more likely to represent modified forms of the same gene product rather than products of different genes, although the existence of closely related genetic loci is not rigorously excluded. Together, H-2D^d(b28⁺) and H-2D^d(b28^–) have a radioactivity level seven times higher than H-2L^d, which also reacts with the anti-H-2.28 serum but which lacks the H-2.4 determinant. As yet unresolved, however, is the question of whether the observed quantitative differences between these three antigens reflect actual molar differences at the cellular level, or whether the variation is the result of metabolic or compositional factors. In any case, a complex serological and structural relationship is found to exist between antigens encoded by the D/L end of the MHC.     

Advances in immunoproteomics for serological characterization of microbial antigens

We propose a multi-dimensional strategy, associating immunodetection to a protein fractionating two-dimensional liquid chromatography tool, for serological characterization of microbial antigens. The originality of such immunoproteomic approaches resides in their application in large-scale studies for rapid serotyping of micro-organisms, evaluation of immunomes and could be proposed in the development and monitoring of vaccines.     

Lymphocyte antigens in Norwegian goats: serological and genetic studies

Altogether, 292 goat alloantisera were screened for antilymphocyte reactivity in a two-step dye exclusion microcytotoxicity test. Fifteen different lymphocyte antigen specificities were characterized by cluster analysis and absorption studies. The specificities were designated N1-N15 (N for Norwegian). Lymphocytes from 247 Norwegian dairy goats were tested. Each animal displayed from none to four of the characterized specificities. Lysostrip testing and family studies indicated that the specificities N1-N14 were coded for by multiple alleles belonging to at least two closely linked loci. It is suggested that these loci are part of the caprine major histocompatibility complex. Family studies gave strong evidence that the specificity N15 was not coded for by genes located in the same region as the other 14 specificities. Absorption studies showed that this specificity was located on both lymphocytes and erythrocytes.     

Rapid test for the serological separation of staphylococci from micrococci

A simple test for the serological separation of staphylococci from micrococci is described, which is based on the quite different cell wall peptidoglycan structures of these two genera. Antisera to (pentaglycyl-epsilon-amino-n-hexanoic acid)20-albumin agglutinated without exception all staphylococci and gave no positive reaction with micrococci or other bacterial cells. To obtain a good reaction, it was necessary to extract the cells with hot trichloroacetic acid for 30 min. Antisera to (tri-L-alanyl-epsilon-amino-n-hexanoic acid)22-albumin reacted strongly with micrococci containing oligo-L-alanine bridges in their peptidoglycan, but did not agglutinate staphylococci or other bacteria lacking alanine interpeptide bridges.     

Erythrocyte antigens in Norwegian goats: serological and genetic studies.

Goat alloantisera and bovine blood typing reagents were used to characterize eight erythrocyte antigen specificities in Norwegian goats by cluster analysis, absorption and family studies. Most of the goat sera were produced by injecting dams once or twice with blood cells or blood from their own kids. The characterized specificities were designated N1-N8. The two specificities N5 and N8 were recognized both by goat alloantisera and by reagents against the bovine factors E'1 and E'2 (N5) and I (N8), which are allelic factors in the bovine B-system. In goat families, the two specificities also behaved as alleles. Consequently, the locus or gene system coding for these specificities was called the B-system of goats. The six other erythrocyte antigens were provisionally assigned to six separate loci. In addition, a bovine anti-sheep R factor reagent reacted with cells from 3.3% of the goats tested, whereas a monoclonal antibody against the Forssman antigen reacted with all the goats tested.     

Rapid test for the serological separation of staphylococci from micrococci.

A simple test for the serological separation of staphylococci from micrococci is described, which is based on the quite different cell wall peptidoglycan structures of these two genera. Antisera to (pentaglycyl-epsilon-amino-n-hexanoic acid)20-albumin agglutinated without exception all staphylococci and gave no positive reaction with micrococci or other bacterial cells. To obtain a good reaction, it was necessary to extract the cells with hot trichloroacetic acid for 30 min. Antisera to (tri-L-alanyl-epsilon-amino-n-hexanoic acid)22-albumin reacted strongly with micrococci containing oligo-L-alanine bridges in their peptidoglycan, but did not agglutinate staphylococci or other bacteria lacking alanine interpeptide bridges.     

Immunodiffusion studies on the antigens of Histoplasma capsulatum: comparison of mycelial histoplasmin with the yeast phase reagent Histolyn-CYL

Immunodiffusion assays were performed to determine if H and M antigens associated with mycelial histoplasmin were present in the yeast phase reagent, Histolyn-CYL (H-CYL). Neither H nor M antigens could be detected in H-CYL. However, when H-CYL was reacted against human histoplasmal sera a positive reaction was evidenced in all instances in which a response was elicited with a reference mycelial immunodiffusion reagent.     

Chemical and biological properties of mycobactins isolated from various mycobacteria

Nine different strains of mycobacteria grown on media deficient in iron all produced mycobactins. Most strains produced one mycobactin in great preponderance. Mycobacteria from clearly distinct taxonomic groups gave mycobactins differing in the structure of their nuclei. One group of taxonomically related mycobacteria produced mycobactins having the same nucleus but with different distributions of side chains within the homologous mixtures. Simple methods are described for identifying mycobactins on a small scale; these may be of value in classifying mycobacteria. Structures are proposed for mycobactin A from Mycobacterium aurum, mycobactin R from M. terrae, mycobactin F, produced together with mycobactin H by M. fortuitum, and mycobactins M and N from M. marinum. The first three of these differ from known mycobactins in details of substitution and configuration of asymmetric centres in the nucleus. Mycobactins M and N are substantially different, having only small acyl groups (acetyl and propionyl respectively) at the hydroxamic acid centre of the mycobactic acid moiety. Both are homologous mixtures having long-chain saturated 3-hydroxy-2-methyl acid fragments in the cobactin moiety. All mycobactins so far isolated promote almost maximal growth of M. johnei at 30ng./ml. in liquid medium. The activity of some mycobactins extends to much lower concentrations, mycobactin S showing significant growth promotion at 0.3ng./ml. Mycobactin M or N in combination with mycobactins having a long side chain in the mycobactic acid moiety exerts a mutually antagonistic effect on the growth of M. johnei, the mixture giving less growth than either mycobactin separately. Mycobactin M also decreases the growth of M. kansasii and M. tuberculosis on liquid media. These antagonistic effects are probably caused by a lengthening of the lag phase.     

The effects of deoxycholate and sodium dodecyl sulphate on the serological reactivity of antigens isolated from six Bacteroides reference strains

The detergents sodium dodecyl sulphate (SDS) and sodium deoxycholate (NaD) are frequently used as solvents for macromolecular polysaccharide complexes in immunochemical and serological techniques. The influence of the disaggregating surfactants on the serological reactivity of endotoxins isolated from six serotype specific reference strains of the Bacteroides fragilis group was investigated by comparing haemagglutinating and precipitating reactivities of antigen solutions in phosphate buffered saline (PBS), NaD and SDS. All antigens were phenol/water extracted endotoxins. Solutions of antigens isolated from serotypes A, B, C and D in PBS exhibited mainly serotype specificity and a few well known low-titer cross reactions; solutions in NaD showed additional cross reactivity, which was enhanced by solubilization of the antigens in SDS. In immunoelectrophoresis endotoxins isolated from serotypes A and C and dissolved in NaD or SDS showed additional precipitation lines compared to solutions of the same antigens in PBS. These changes in the serological reactivity are of relevance for investigations where the serological specificity of antigens is in question.