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The use of immunofluorescence (IF) technique to detect and evaluate expression levels and localization of cellular proteins and other antigens of interest through the antibodies in their cellular or tissue context has become a standard approach among researchers. Optimizing primary antibody concentrations/dilutions is an essential step in the fluorescent antibody staining protocol. The steps in IF staining are similar to those of the immunohistochemistry (IHC) technique. The use of IHC technique to determine the optimal working dilutions of primary antibodies for IF staining of formalin-fixed paraffin-embedded (FFPE) tissues sections can minimize time wasting and cumbersome approach of using direct IF single labeling using variable dilutions of both primary and secondary antibodies. We used IHC staining technique to determine the working dilutions of the respective primary antibodies by staining 3-µm sections of recommended positive FFPE tissue sections using 3 different dilutions of the primary antibodies and an isotype control (used at the highest concentration). Digital images of sections stained were reviewed in ImageScope by a Consultant Pathologist for positivity, intensity, and histologic distribution. We adopted the IHC predetermined optimal dilutions of primary antibodies to CD4, CD8, CD16, CD21, CD56, CD68, CD163, FOXP3, and PD1 to carry out IF staining of FFPE tissue sections. This approach has helped to remove the complexities associated with grappling with 2 unknown to optimize for both the primary and secondary antibodies using IF technique.

We compared the performance of tuberculin skin test (TST), Quantiferon-TB Gold in-tube (QFT-GIT), and T-SPOT.TBin diagnosing latent tuberculosis (LTBI) among childhood TB contacts in a TB endemic setting with high BCG coverage. We evaluated the performance of interferon gamma release assays (IGRAs) and TST when combined in an algorithm.

ABSTRACTOverlapping peptides of Mycobacterium tuberculosisantigens ESAT-6 and CFP-10 offer increased specificity over the purified protein derivative skin test when they were used in an ex vivo enzyme-linked immunospot (ELISPOT) assay for gamma interferon detection for the diagnosis of M. tuberculosisinfection from recent exposure. We assessed whether equivalent results could be obtained for a fusion protein of the two antigens and whether a combined readout would offer increased sensitivity in The Gambia. We studied the ELISPOT assay results for 488 household contacts of 88 sputum smear-positive tuberculosis (TB) cases. The proportions of subjects positive by each test and by the tests combined were assessed across an exposure gradient, defined according to sleeping proximity to a TB case. Eighty-eight (18%) subjects were positive for CFP-10 peptides, 148 (30%) were positive for ESAT-6 peptides, 161 (33%) were positive for both peptides, and 168 (34%) were positive for the fusion protein; 188 (39%) subjects had either a positive result for a peptide or a positive result for the fusion protein. There was reasonable agreement between the peptide and the protein results (kappa statistic = 0.78) and no significant discordance (P= 0.38). There was a strong correlation between the fusion protein and combined peptide spot counts (r= 0.9), and responses to the peptide and the proteins all increased significantly according to M. tuberculosisexposure. The proportion of subjects positive for either the pool of peptides or the fusion protein offered maximum sensitivity, being significantly higher than the proportion of subjects positive for ESAT-6 peptides alone (P= 0.007). A fusion protein of ESAT-6 and CFP-10 is equivalent to overlapping peptides for the diagnosis of latent M. tuberculosisinfection. Use of a combination of peptides and fusion protein offers improved sensitivity.