After washing three times with TBST and once with TBS, TMB) was added and the membranes were let stand for 3 mins while the color developed. The reaction was stopped by rinsing the membranes with distilled water. The antigen-blotted membranes were prepared as described above and incubated for 1 hr in Block Ace at room temperature. Before incubating with membranes, 15 mL of urine samples were preincubated with 7 μL of E. coli lysate with shaking to block nonspecific binding for 1 hr at room temperature. Next, the membranes were incubated with the primary antibody for 1 hr at room temperature with shaking. Other procedures were the

same as for the serum assay. Statistically significant differences GSK2126458 molecular weight was determined by the Mann-Whitney’s U-test. Differences with P < 0.05 were considered significant. Affinity purification detected MPB64 as a His-Tag fusion RG-7388 nmr protein, mostly in the insoluble fraction, with a molecular mass of about 30 kDa. We speculate that recombinant MPB64 is sequestered into inclusion bodies by E. coli and thus rendered insoluble (Fig. 1). We examined the reactivity of MPB64 protein by western blotting using pooled serum from five patients with active TB and serum from healthy individuals as a control. All of the fractions of pooled patient serum that were tested, including the sonicated soluble

fraction, the soluble fraction after freezing and thawing, and the sonicated insoluble fraction, showed a specific band at about 30 kDa (Fig. 2a and b). In contrast, serum from healthy individuals showed no such bands (Fig. 2c). These findings confirmed that MPB64 is specifically present in the serum of patients with active TB and is detected by an MPB64-specific IgG antibody. In order to determine the amounts of antigen, we blotted several amounts (300 ng to 18.3 pg/dot, each ¼ dilutions) of proteins to membranes in duplicate. The results of these dot-blot assays are shown in Figure 3a: we detected signals from 300 ng to 4.7 ng Dynein of antigen in the patients’ pooled serum. However, we did not detect signals from 18.8 ng in healthy subjects. Based on these results, we decided that the optimal amount of purified

MPB64 protein for detecting the specific reaction for this assay is 18.8 ng. When we examined serum and urine samples for M. tuberculosis by dot-blot assay using purified MPB64 antigen, we rated the reaction as “2 (++)” if we observed a strong signal, “negative” for no signal, and “1 (+)” for a weak signal. Figure 3 shows examples of each type of assay result. Relevant clinical data and the results of dot-blot assay using MPB64 antigen for a representative patient are shown in Figure 4. The patient had many bacterial cells on culture and an increased ESR on admission. After 2 months of hospitalization, when their TB was considered to be in the active phase, the number of colonies had increased about twofold and the ESR from 50 to 100 mm.