Article Figures & Data
Figures
- FIG. 1.
(A to C) Reactivity of LL/BL, TT/BT, and NEC sera to selected full-length recombinant proteins by ELISA using protein A. (A) ML0405; (B) ML2331; (C) ML2055. Results are shown as mean ODs ± standard deviations. Closed circles, LL/BL sera (n = 5); closed squares, TT/BT sera (n = 6); open squares, normal sera (n = 6).
- FIG. 2.
Reactivity of identified full-length recombinant proteins to pooled LL/BL sera by Western blot. Lanes 1 and 4, ML2331; lanes 2 and 5, ML0405; lanes 3 and 6, ML2055. Lanes 1 to 3 developed with anti-His monoclonal antibody (BD Biosciences). Lanes 4 to 6 developed with pooled LL patient sera used for expression cloning (n = 5). Positions of molecular mass markers are indicated in kilodaltons. The experiment was repeated three times, and a typical representation of the results is shown.
- FIG. 3.
(A to C) Reactivity of individual antigens to LL sera, pulmonary TB sera, and NEC serum to recombinant proteins measured by ELISA using protein A. LL (n = 24), pulmonary TB (n = 10), and NEC (normal) (n = 8) sera were tested. An arbitrary cutoff for reactivity of an OD of 0.2 is indicated by a horizontal line.
- FIG. 4.
IgM and IgG reactivity of six LL index cases, household contacts, and normal sera to (A) NDOHSA, (B) ML0405, (C) ML2331, and (D) ML2055, measured by ELISA. Serum dilution was 1:200. Number of index case serum samples was 6, the number of household contact (HC) serum samples was 28, and the number of NEC (normal) serum samples was 5. Each index case had a minimum of four household contacts included in this study. An arbitrary cutoff for reactivity of an OD of 0.2 is indicated by a horizontal line.
- FIG. 5.
Potential utility of ML0405 and ML2331 in the serological diagnosis of BL patients. (A) ELISA of BL patients with a BI between 3 and 4.17 (n = 6) and LL patients with a BI of >5 at a serum dilution of 1:800. At this serum dilution, all household contact and NEC sera used in this study were tested at an OD of <0.2 (data not shown). At a lower BI, reactivity to NDOHSA (IgM) was reduced, and in one case (BI, 3.0), serum reactivity was markedly higher in response to ML0405 (IgG). (B) ELISA at the same serum dilution using combinations of antigens. In the patients with the lowest BI, a measured combination of all three antigens (IgGAM) improved signal compared to NDOHSA alone (IgM). An arbitrary cutoff for reactivity of an OD of 0.2 is indicated by a horizontal line.
Tables
- TABLE 1.
Antigens of M. leprae identified by expression cloning using pooled LL/BL patient serad
Antigen No. of clonesa Sizes (kDa) TB homologue (% identity) Description Mean ELISA OD450 ± SDb LL/BL NEC ML0050/ML0049 2 10, 9 Rv3874 (40.0) Secreted; CFP-10, ESAT-6 0.52 ± 0.87c 0.06 ± 0.01 Rv3875 (36.3) ML0091 4 23.7 Rv3810 (52.7) Potentially secreted ND ND ML0317 2 56.9 Rv0440 (94.8) GroEL2, 60-kDa chaperonin 2 ND ND ML0405 2 40.7 Rv3616c (62.7) Previously unknown 2.31 ± 0.45 0.13 ± 0.02 ML0568 5 18.3 Rv1435c (38.9) Previously unknown 0.34 ± 0.22 0.13 ± 0.04 ML1213 3 80.1 Rv1565c (76.5) Previously unknown 0.51 ± 0.21 0.23 ± 0.04 ML2028 3 34.8 Rv1886c (88) Secreted; Ag85b 2.29 ± 1.26 0.52 ± 0.21 ML2055 6 29.5 Rv1860 (66.8) Membrane protein 1.87 ± 1.19 0.19 ± 0.05 ML2655 2 36.3 Rv0129 (82.0) Secreted; Ag85c 2.82 ± 0.81 1.88 ± 0.28 ML0097 1 35.4 Rv3804c (82.0) Secreted; Ag85a ND ND ML1812 1 50.5 Rv1477 (78.8) Previously unknown ND ND ML2331 2 26.5 Rv3717 (82.0) Previously unknown 2.09 ± 1.07 0.11 ± 0.06 ML2496 3 24.2 Rv0351 (68.6) DnaK, 70-kDa heat shock protein ND ND ↵a Number of different clones identified in the screen encoding the full or partial sequence of the same antigen.
↵b OD450 ± standard deviation in ELISA developed with protein A at a serum dilution of 1:200 (minimum of n = 6 for each measurement). ND, not done.
↵c ELISA results with ML0050-ML0049 fusion protein.
↵d n = 5.
