Immunochemistry-Based Diagnosis of Extrapulmonary Tuberculosis: A Strategy for Large-Scale Production of MPT64-Antibodies for Use in the MPT64 Antigen Detection Test

Abstract

Tuberculosis (TB) is a global health problem. The immunohistochemistry (IHC)-based MPT64 antigen detection test has shown promising results for diagnosing extrapulmonary TB in previous studies. However, the anti-MPT64 antibody currently used in the test is in limited supply, and reproduction of a functional antibody is a prerequisite for further large-scale use. Various antigen-adjuvant combinations and immunisation protocols were tested in mice and rabbits to generate monoclonal and polyclonal antibodies. Antibodies were screened in IHC, and the final new antibody was validated on clinical human specimens. We were not able to generate monoclonal antibodies that were functional in IHC, but we obtained multiple functional polyclonal antibodies through careful selection of antigen-adjuvant and comprehensive screening in IHC of both pre-immune sera and antisera. To overcome the limitation of batch-to-batch variability with polyclonal antibodies, the best performing individual polyclonal antibodies were pooled to one final large-volume new anti-MPT64 antibody. The sensitivity of the new antibody was in the same range as the reference antibody, while the specificity was somewhat reduced. Our results suggest that it possible to reproduce a large-volume functional polyclonal antibody with stable performance, thereby securing stable supplies and reproducibility of the MPT64 test, albeit further validation remains to be done.

Diagnosing adult and pediatric extrapulmonary tuberculosis by MPT64 antigen detection with immunohistochemistry and immunocytochemistry using reproduced polyclonal antibodies

“…In all previous studies, we used an in‐house polyclonal antibody which was available in limited amounts. With the intention for scale‐up of the test, we reproduced the primary antibody and produced a single batch of this new polyclonal antibody in large amounts [ 21 ]. Since polyclonal antibodies recognize and bind to different epitopes of the antigen in question, the performance of the reproduced primary antibodies may differ from those previously used.…”

“…Pathologists (MRP, MK, and SI) were trained in immunostaining and slide evaluation. The production of the anti‐MPT64 antibodies used in our study is described in detail by Hoel et al [ 21 ]. In brief, rabbits were immunized to produce polyclonal anti‐MPT64 antibodies, and the performance of various adjuvants–antigen combinations was assessed by IHC screening using serial dilutions to identify the optimal working dilution.…”

“…However, the antibodies evaluated in previous studies were in limited supply. As sufficient supply is a prerequisite for a test to be used in routine diagnostics, a strategy has been developed for producing larger volumes of the MPT64 polyclonal antibodies, which can facilitate the use of this test on a large scale [ 21 ]. The aim of this study was to assess the performance of the MPT64 test with the reproduced polyclonal antibodies for the diagnosis of EPTB in the high TB burden settings of Pakistan and India, and to compare the results with routine diagnostics including Xpert.…”

Detection ofMycobacterium tuberculosisin human tissue via RNAin situhybridization

“…Secreted Mtb antigens have been identified via IHC staining in Mtb-infected human lung tissue (7)(8)(9)(10)(11). Similarly, we used IHC to detect Mtb secreted antigen 85B (Ag85B) in the same pulmonary and testicular specimens used for RNAscope analysis (Figures 2, 3).…”

“…In vitro and in vivo studies have shown that several secreted Mtb antigens are present within TB lesions (6). Despite the potential of using antibodies to detect Mtb surface or secreted antigens (7)(8)(9)(10)(11), little progress has been made to improve TB diagnostics beyond ZN staining. Whereas mRNA-based diagnostic tests have been proposed for TB (12), studies comparing RNA in situ hybridization (RISH) methods with routine histochemistry and/or immunohistochemistry (IHC) for the spatial identification of Mtb are lacking, especially in paucibacillary and abacillary tissues.…”