A Novel Sensitive Targeting the

Immunoassay 5-Methylthio-d-Xylofuranose-Lipoarabinomannan Epitope Meets the WHO's Performance Target for Tuberculosis Diagnosis. ABSTRACT The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufﬁcient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to puriﬁed LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (conﬁdence interval [CI], 80% to 97%) and speciﬁcity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio- D -xylofuranose (MTX)-dependent epitope in LAM that is speciﬁc to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and speciﬁc novel LAM point-of-care assay with the aim to meet the WHO’s performance target for TB diagnosis. cells Supernatants indirect immunizing low cross-reactivity BSA. The sequences of heavy and light chains of B cells the activity cloned into separate and cotransfected into HEK293 cells. The transfected supernatants were tested by indirect ELISA to identify the antibodies 27D2) reactivity to puriﬁed heat-killed M. a of detection using rapid speciation assays the Capilia TB test; TAUNS, Japan), or molecular The in this study using the Alere LF-LAM test run according the was read two different who com- pared the test line with the card provided by the and results. assay but showed poor speciﬁcity (63%), with some TB-negative samples giving signals as high as 10-fold above the blank signal. While we were able to develop a LAM assay using the FIND 28/A194-01 pair that provided excellent analytical sensitivity, our results suggest that the performance of urinary LAM assays using only antibodies targeting non-TB speciﬁc arabinan epitopes may ultimately be limited by cross-reactivity with urinary LAM from other sources, such as non-TB mycobacteria (NTMs) or related organisms of the Actinomycetales order. In particular, the Ara6 structures are not unique to M. tuberculosis LAM, and our cross-reactivity studies conﬁrmed that the FIND 28/A194-01 pair cross-reacts with the nonmycobacterial actinomycetes Nocardia , Gordonia , Rhodococcus , and Tsukamurella , which are all known to produce LAM with Ara6 structures (38, 39). It is likely that CS-35 and the polyclonal antibodies used in the Alere LF-LAM test and previous commercial ELISAs have similar limitations in speciﬁcity. An attempt to improve an older commercial ELISA, the Clearview TB test, by concentrating urine prior to analysis revealed that the sensitivity could be signiﬁcantly improved but also revealed a corresponding decrease in speciﬁcity (35). Similarly, the need to reduce false-positive results from the Alere LF-LAM test led the manufacturer to revise the reference card of the test toward a higher assay cutoff in 2014, which increased the speciﬁcity but decreased the sensitivity. Furthermore, the cross-reactivity of the Alere LF-LAM test to mouth-residing Actinomyces and Nocardia is likely the reason that the assay is not speciﬁc enough for LAM detection in sputum (40). We also found that both the ECL assay using the FIND 28/A194-01 pair and the Alere LF-LAM test were susceptible to cross-reactivity from environmental