Figure 1.
Two formats of sandwich ELISA.
Format 1: Monoclonal antibody (MAb) based, where both the capture and detection antibody are MAb's. Format 2: Bispecific Monoclonal antibody (bsMAb) based, where the capture antibody is a MAb but the detection antibody is a bsMAb.
Table 1.
Different processing conditions for simulated serum samples.
Figure 2.
SDS-PAGE and Western blot of BSA-Hex and unconjugated BSA.
A: Coomassie stained SDS-PAGE gel showing BSA-Hex, which contains 15.2 sugar residues per carrier, and unconjugated BSA, running at 81.4 kDa and 66.3 kDa, respectively. Lane M, standard molecular marker; lane 1, BSA-Hex; lane 2, BSA. B: Probe of BSA-Hex and BSA after transfer to nitrocellulose with MAb CS-35 in a Western blot showing reactivity only with BSA-Hex.
Figure 3.
Optimization of different assay parameters.
A: Optimisation of the capture antibody (CAb), where the concentration 8 µg/ml was found to be optimum. B: The optimisation of detection antibody (DAb), where the concentration 4 µg/ml was found to be optimum. C: The optimisation of conjugate (st-HRPO), where the dilution 1∶8,000 was found to be optimum.
Figure 4.
Immunoswab in different matrices.
A: The immunoswab performed after spiking the BSA-Hex antigen in serum and urine and the limit of detection (LOD) was found to be 0.5 and 5.0 ng/ml in respective matrices (encircled in red). B: The immunoswab performed after spiking the bacterial LAM isolated from M. tuberculosis H37Rv, in serum and the LOD was found to be 0.5 ng/ml.
Table 2.
Specificity study of the assay.
Figure 5.
The effect of chemical treatment on the antigen detection level in the simulated and clinical samples.
A: The effect of different chemical treatments (as mentioned along x-axis) on antigen detection level (in simulated samples). The three different antibody concentrations are mentioned at the top right hand side. Each condition (under each antibody concentration) was tested in triplicates. B: The antigen level detected with the clinical samples. The clear bar shows the result without (w/o) any pre-treatment while the pre-treated samples are shown via the dark bar. The first four samples were negative and rest were positive clinical samples. Each sample was tested in duplicate.
Figure 6.
The clinical samples were analysed in the microtitre plate. The line in the middle showed the cut-off point that was chosen to be mean+2 SD (standard deviation). The green bars (with alphanumeric sample number) showed the negative samples, all of which lied below the line. The red bars (with numerical sample number) showed the positive samples, nine of which were above the line.