The detection of anti‐cord factor (trehalose 6,6′‐dimycolate) IgG antibody in active (smear‐and/or culture‐positive) and inactive (smear‐ and culture‐negative) tuberculosis patients is a useful serodiagnostic tool that can be used for early clinical diagnosis of the disease. We estimated the titers of anticord factor IgG antibody in the sera of tuberculosis patients, and compared them with those of Mycobacterium avium‐infected patients. Most of the serum samples obtained from the tuberculosis patients were highly reactive against M. tuberculosis (MTB) cord factor isolated from M. tuberculosis H37Rv, a human‐type mycobacterial strain, whereas they were less reactive against M. avium (MAC) cord factor. Similarly, most of the serum samples of the MAC‐infected patients were highly reactive against MAC cord factor and less reactive against MTB cord factor. These results suggest that anti‐cord factor IgG antibody recognizes the mycolic acid subclasses as an epitope which comprises cord factor, since MTB and MAC cord factor differ in mycolic acid subclasses and molecular species composition. To clarify the exact antigenic epitope in cord factor and to find out a more sensitive and specific diagnostic test antigen, we examined the reactivity of patients' sera to glycolipids containing trehalose (cord factor and sulfolipid) obtained from various mycobacterial species. Furthermore, the reactivity of human antisera to various mycolic acid subclasses (α‐, methoxy and keto mycolic acids) of MTB cord factor was compared. We found that anti‐cord factor IgG antibody in the sera of human tuberculosis patients most strikingly recognized methoxy mycolic acid in the cord factor of M. tuberculosis, whereas it recognized α‐ and keto mycolic acids weakly. Pre‐absorption studies of antibody with MTB cord factor or methoxy mycolic acid methyl ester showed that anti‐cord factor antibody was absorbed partially, but consistently. This is the first report describing that the specific subclass of mycolic acid from mycobacteria is antigenic in the humoral immune system of human tuberculosis infection.
An ELISA with cord factor (trehalose-6,6P-dimycolate) is useful for the serodiagnosis of tuberculosis. To clarify the exact antigenic epitope in cord factor, recognized by a rabbit anti-cord factor IgG antibody, and to ascertain the most sensitive and specific diagnostic test antigen, rabbits were immunized with two kinds of cord factors isolated from Mycobacterium tuberculosis or Mycobacterium avium and the reactivities of the sera were tested against cord factors or the component mycolic acid methyl esters by ELISA. The serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against M. tuberculosis cord factor, but less reactive against M. avium cord factor. In contrast, the serum from rabbits immunized with M. avium cord factor was highly reactive against M. avium cord factor but less reactive against M. tuberculosis cord factor. Moreover, the serum from rabbits immunized with M. tuberculosis cord factor reacted against mycolic acid methyl esters, especially methoxy mycolic acid methyl ester. On the other hand, the serum from rabbits immunized with M. tuberculosis cord factor was less reactive against trehalose-6-monomycolate and not reactive against sulfolipid (2,3,6,6P-tetraacyl trehalose 2P-sulfate). From these results, it was concluded that the anti-cord factor IgG antibody, produced experimentally in rabbits, recognized the differences in the cord factor structures, i.e. the hydrophobic moiety rather than the carbohydrate moiety. It was also noted that the serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against methoxy mycolic acid as an epitope. This paper is the first to describe how the anti-cord factor IgG antibody can recognize the mycolic acid subclasses, which differ according to the species of mycobacteria. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Abbreviations: FAB/MS, first atom bombardment mass spectrometry; MAC-TDM, cord factor (trehalose-6,6P-dimycolate) isolated from Mycobacterium avium; Mt-TDM, cord factor (trehalose-6,6P-dimycolate) isolated from Mycobacterium tuberculosis; PBS-T, phosphate-buffered saline containing 0.05% (v/v) Tween-20; SL, sulfolipid (2,3,6,6P-tetraacyl trehalose 2P-sulfate); TB, tuberculosis; TLC, thin layer chromatography; TMM, trehalose-6-monomycolate; w/o/w, water in oil in water
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