Development and evaluation of a novel multiple-antigen ELISA for serodiagnosis of tuberculosis

State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, No. 220 Handan Road, 200433 Shanghai, PR China.
Tuberculosis (Edinburgh, Scotland) (Impact Factor: 2.71). 07/2009; 89(4):278-84. DOI: 10.1016/
Source: PubMed


In this study, we describe the development and evaluation of a novel multiple-antigen ELISA for rapid diagnosis and screening of active tuberculosis (TB). The humoral immune responses of 136 active TB patients and 57 healthy subjects against antigens Rv3425, 38kDa and lipoarabinomannan (LAM) from Mycobacterium tuberculosis H37Rv were examined by ELISA. Three essential results were obtained. (i) Rv3425 antigen is a potential candidate for serodiagnosis of active TB. Of 136 active TB patients, Rv3425 antigen provided a sensitivity of 31.6%, lower than that of LAM antigen, but higher than that of 38kDa antigen, with an overall specificity of 100%. (ii) For 62 smear-negative pulmonary TB patients and 15 extra-pulmonary TB patients, the multiple-antigen test provided a sensitivity of 43.5% and 26.7%, respectively, representing an improvement over acid-fast bacilli (AFB) smear-based diagnosis. (iii) Compared with the single-antigen ELISA and the two available commercial kits, the multiple-antigen test offered the highest accuracy (71.0%). In conclusion, the multiple-antigen ELSIA test based on Rv3425, 38kDa, and LAM antigens is a potentially useful tool for the serodiagnosis and screening of active TB. Combinations of Rv3425 with other mycobacterial antigens may also be worthy of further investigation.

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Available from: Jun-Wei Zhao
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    • "Our hypothesis relies on the heterogeneous humoral response to different antigens as the cause of the increased specificity of the assay (Zhang et al. 2009), in agreement with current guidelines for the diagnosis of infections involving an antibody response, thus suggesting the importance of having multiple antigens to define the antibody profile related to early diagnosis and follow-up of the specific illness (Steller et al. 2005; Sartain et al. 2006). Although further work is now needed to establish "
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    ABSTRACT: Unlabelled: Several serological diagnostics rely on enzyme-linked immunosorbent assay (ELISA) to detect bacterial infections. However, for some pathogens, including Bartonella henselae, diagnosis still depends on manually intensive, time-consuming assays including micro-immunofluorescence, Western blotting or indirect immunofluorescence. For such pathogens, there is obviously still a need to identify antigens to establish a reliable, fast and high-throughput assay (Dupon et al. ). We evaluated two B. henselae proteins to develop a novel serological ELISA: a well-known antigen, the 17-kDa protein, and GroEL, identified during this study by a proteomic approach. When serum IgG were tested, the specificity and sensitivity were 76 and 65·7% for 17-kDa, respectively, and 82 and 42·9% for GroEL, respectively. IgM were found to be more sensitive and specific for both proteins: 17-kDa protein, specificity 86·2% and sensitivity 75%; GroEL, specificity 97·7% and sensitivity 45·3%. IgM antibodies were also measured in lymphoma patients and patients with Mycobacterium tuberculosis infection to assess the usefulness of our ELISA to distinguish them from B. henselae infected patients. The resulting specificities were 89·1 and 93·5% for 17-kDa protein and GroEL, respectively. Combining the results from the two tests, we obtained a sensitivity of 82·8% and a specificity of 83·9%. Our work described and validated a proteomic approach suitable to identify immunogenic proteins useful for developing a serological test of B. henselae infection. Significance and impact of the study: A reliable serological assay for the diagnosis of Cat Scratch Disease (CSD) - a pathological condition caused by Bartonella henselae infection - has not yet been developed. Such an assay would be extremely useful to discriminate between CSD and other pathologies with similar symptoms but different aetiologies, for example lymphoma or tuberculosis. We investigate the use of two B. henselae proteins - GroEL and 17-kDa - to develop a serological-based ELISA, showing promising results with the potential for further development as an effective tool for the differential diagnosing of B. henselae infection.
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    • "We have not found LAM to be a particularly good antigen for the study and diagnosis of TB in badgers using the ELISA format (unpublished work). The evaluation of LAM as a serodiagnostic antigen in human TB is also not encouraging, although sensitivity is improved if combined with other antigens (Zhang et al., 2009; Beyene et al., 2010). Assessment of different antigens, singly and in combination with animals in a single study, would be valuable. "
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    • "Briefly, each serum sample was considered positive if the mean absorbance was greater than the cut-off point. For the multiple-antigen ELISA results, single antigen ELISA data was re-evaluated and the serum tested was determined to be positive according to the following criteria: (1) any 2 or more antigens specifically react with serum with the absorbance greater than the cut-off value calculated from the mean absorbance (A405nm) plus 2 standard deviations of healthy controls or (2) any antigen that specifically reacts with serum when the cut-off value was calculated from the mean absorbance (A405nm) plus 3 standard deviations of healthy controls [25]. "
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