Use of an antigen-capture assay for characterisation of monoclonal antibodies to mycobacterial lipoarabinomannan.

Monoclonal antibodies directed to six separate antigen molecules of Mycobacterium leprae have been tested in an antigen-capture assay based on combined use of polyclonal ("capture") and monoclonal ("detector") antibody reagents. This approach provides a potentially versatile, sensitive and specific assay for detection and relative quantitation of M. leprae antigens. Characterisation of monoclonal antibodies to mycobacterial lipoarabinomannan (LAM-B) by the antigen-capture assay indicates that some of the antigenic determinants present on LAM-B from M. leprae may be either absent altogether or present at much lower concentrations on the corresponding LAM-B structure form M. tuberculosis.

A Mycobacterium leprae-specific human T cell epitope cross-reactive with an HLA-DR2 peptide.

Mycobacterium leprae induces T cell reactivity and protective immunity in the majority of exposed individuals, but the minority that develop leprosy exhibit various types of immunopathology. Thus, the definition of epitopes on M. leprae antigens that are recognized by T cells from different individuals might result in the development of an effective vaccine against leprosy. A sequence from the 65-kD protein of this organism was recognized by two HLA-DR2-restricted, M. leprae-specific helper T cell clones that were derived from a tuberculoid leprosy patient. Synthetic peptides were used to define this epitope as Leu-Gln-Ala-Ala-Pro-Ala-Leu-Asp-Lys-Leu. A similar peptide that was derived from the third hypervariable region of the HLA-DR2 chain, Glu-Gln-Ala-Arg-Ala-Ala-Val-Asp-Thr-Tyr, also activated the same clones. The unexpected cross-reactivity of this M. leprae-specific DR2-restricted T cell epitope with a DR2 peptide may have to be considered in the design of subunit vaccines against leprosy.

Exact definition of species-specific and cross-reactive epitopes of the 65-kilodalton protein of Mycobacterium leprae using synthetic peptides.

With the use of solid phase synthesis of peptides corresponding to major and minor peaks in a Hopp-Woods hydrophilicity plot, the epitopes for 10 of 14 known different mAb to the Mycobacterium leprae 65-kDa protein, a prominent T and B cell Ag of this bacillus, have been located in the primary structure. Five epitopes have been precisely mapped by using the synthetic peptides in inhibition ELISA experiments, and five others have been located on peptides of 22 amino acids or less in length. The epitope of an important species-specific antibody, IIIE9, which may be useful for seriodiagnosis of leprosy, appears to be distinguished from the epitope of the antibody IVD2, widely cross-reactive among mycobacteria, not by its sequence, but only by its critical residues. All epitopes studied appear continuous insofar as can be determined by this approach.

Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan.

Each of more than 30 monoclonal antibodies that had been raised against Mycobacterium leprae and previously classified as reactive with carbohydrate was shown to be directed against lipoarabinomannan, a prominent, highly pervasive, myo-inositol-phosphate-containing, cross-reactive antigen within the leprosy bacillus. Some of the antibodies preferentially bound to the lipopolysaccharide of M. leprae rather than to that of Mycobacterium tuberculosis, suggesting the presence of distinguishing structural features. The presence of alkali-labile inositol 1-phosphate in the lipopolysaccharide from M. tuberculosis and its apparent absence from the M. leprae product may account for the difference.

Characterization of antibody-reactive epitopes on the 65-kilodalton protein of Mycobacterium leprae.

Twenty-three monoclonal antibodies (MAbs) prepared in seven different laboratories were studied, all of which recognized the 65-kilodalton (kDa) protein of Mycobacterium leprae as determined by Western blotting or gel radioimmunoassay or both. Fourteen of the MAbs recognized different epitopes, as evaluated by cross-competition studies using radiolabeled MAb and unlabeled inhibitors; the species specificity of these epitopes was defined by nitrocellulose dot blot immunoassays with bacterial sonic extract antigen preparations from 23 species of mycobacteria. Each of the 14 distinct MAbs recognized a 65-kDa protein produced by a lysogenized Escherichia coli Y1089 host containing cloned rDNA which included the gene for the M. leprae 65-kDa protein. Of the 14 distinct MAbs, 1 recognized an epitope found only on M. leprae, and the others recognized epitopes present on as few as 8 or as many as all 23 of the mycobacterial species studied. Identification of these distinct 65-kDa protein epitopes and use of the MAbs which recognize them should assist future structural studies of this protein and characterization of the T-cell reactive and serodiagnostically useful portions of the molecule.

ELISA detection of IgM antibodies against phenolic glycolipid-I in the management of leprosy: a comparison between laboratories.

IgM antibodies to the phenolic glycolipid-I (PGL-I) antigen of Mycobacterium leprae were detected by different ELISA techniques in three laboratories (in New York, Colorado, Seattle, U.S.A.). The agreement on seropositivity and overall correlation between techniques was excellent. A positive linear correlation between the bacterial index (BI) and anti-PGL-I IgM, previously reported by the New York laboratory, was detected by all techniques. The role of erythema nodosum leprosum in decreasing the relationship of BI versus anti-PGL-I IgM was seen by the New York laboratory with sera diluted 1:20 and ABTS substrate solution and by the Colorado laboratory but not by New York with sera at 1:300 and OPD substrate or by the Seattle laboratory.

Immunochemical characterization of a protein associated with Mycobacterium leprae cell wall.

A panel of nine monoclonal antibodies to Mycobacterium leprae were used to characterize a protein antigen of the bacillus. Two monoclonal antibodies (IVD8 and IIIE9) were specific for M. leprae and reacted with an epitope (CWPa) present on a protein molecule associated with the cell wall fraction of M. leprae. This protein, designated cell wall-associated protein (CWP), lost its immunoreactivity upon treatment with trypsin and had an apparent molecular weight of 65,000, though additional lower-molecular-weight forms of the protein were observed by immunoblotting. Four other cross-reactive epitopes (CWPb, CWPc, CWPd, and CWPe) were defined on the same molecule using seven independent monoclonal antibodies. Therefore, M. leprae possesses a trypsin-sensitive, heat-stable protein associated with the cell wall which contains at least one species-specific and four cross-reactive antigenic determinants.

Monoclonal antibodies to a 28,000 mol. wt protein antigen of Mycobacterium leprae.

Monoclonal antibodies (MoAb) have been used to analyse a protein antigen from Mycobacterium leprae with a subunit mol. wt of 28,000 daltons. Three different patterns of species specificity were observed with two antibodies being specific for M. leprae, two partially specific, and one broadly cross-reactive amongst all mycobacteria. Competitive binding and sandwich assays demonstrated that the specific and partially specific antibodies recognized closely related regions of the molecule while the cross-reactive antibody recognized a spatially separate epitope on the same polypeptide chain. Identification of specific and cross-reactive epitopes on a single antigenic molecule may be of considerable importance for understanding the functioning of the cell-mediated immune system during leprosy infection and the use of MoAb for such analyses is discussed.

Biochemical alterations in the serum of armadillos (Dasypus novemcinctus) infected with Mycobacterium leprae. A preliminary report.

Armadillos (Dasypus novemcinctus) were inoculated with Mycobacterium leprae isolated from lepromas taken from untreated lepromatous patients or from the spleen of an armadillo previously infected with human M. leprae. The effect of the infection on the serum levels of lactic dehydrogenase (LDH), alkaline phosphatase (AlkP), glutamate-oxalacetate (GOT) and glutamate-pyruvate (GPT) transaminases was investigated. In general, there was a good correlation between positive evidences of infection and alterations in the levels of LDH, GOT, and GPT. Although elevations in LDH levels were more striking, elevations in GOT and GPT levels were more consistent with the disease. When an absolute increase in the total LDH activity was not observed in a M. leprae-infected animal, an increase in the level of LDH isozyme V was still clearly evident. Serum levels of alkaline phosphatase were not affected by the disease. The ratio GOT/GPT (greater than 1.0) in the infected animals reflected and supported the chronic nature of the disease and the liver involvement. The enzymatic alterations are not, however, specific for leprosy.

Use of a polysulfone membrane support for immunochemical analysis of a glycolipid from Mycobacterium leprae.

Polysulfone membranes have been used as a solid support for chromatography and immunoblotting of phenolic glycolipid I from Mycobacterium leprae. These membranes have an advantage over other supports such as nitrocellulose and silica gel in that very little non-specific background binding of antibodies occurs and assays can readily be carried out with IgM antibodies from human sera. An example of use of the polysulfone chromatography system for detection of phenolic glycolipid I in sera from leprosy patients is described.

Production and characterization of a murine monoclonal antibody recognizing a shared mycobacterial polysaccharide.

An IgM monoclonal antibody specific for mycobacterial arabinomannan was produced by the fusion of splenocytes from BALB/c mice immunized with purified arabinomannan with NSI/1 myeloma cells. Specificity was demonstrated by gel-radioimmunoassay, and by inhibition of binding using the purified polysaccharide. The monoclonal antibody recognized the arabinomannans from all 18 species of mycobacteria tested, including Mycobacterium leprae. This antibody expands the number of defined mycobacterial antigens against which monoclonal antibodies have been produced, and has potential application in studies concerning the pathogenesis of mycobacterial disease.

Antibodies to mycobacterial arabinomannan in leprosy: correlation with reactional states and variation during treatment.

An enzyme-linked immunosorbent assay was used to measure antibody to mycobacterial arabinomannan in serial serum specimens obtained over the initial 12-31 months of therapy from nine patients with leprosy. The antibody level in pretreatment sera was directly proportional to the quantity of Mycobacterium leprae present in each patient as assessed by six-site scrapings (r = 0.75). The three patients with the lowest antibody levels (OD 0.1-0.3) had uncomplicated courses and their levels declined slowly with treatment. Three patients with intermediate antibody levels (OD 0.7-1.1) each experienced a reversal reaction during therapy; serial antibody titers in all three followed a triphasic pattern over the course of the reaction. The two patients who developed erythema nodosum leprosum during therapy had extremely high levels of antibody initially (OD greater than 1.5), which fell slowly with time and which were unaffected by the reactional state. The pretreatment antibody level to arabinomannan reflects the amount of M. leprae present and may have predictive value for the development of reactional states.