Biology:Antibodies from lymphocyte secretions

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The antibodies from lymphocyte secretions (ALS) assay is an immunological assay to detect active diseases like tuberculosis, cholera, typhoid etc. Recently, ALS assay nods the scientific community as it is rapidly used for diagnosis of Tuberculosis. The principle is based on the secretion of antibody from in vivo activated plasma B cells found in blood circulation for a short period of time in response to TB-antigens during active TB infection rather than latent TB infection.

Procedure of ALS assay

PBMCs were separated from blood on Ficoll-Paque by differential centrifugation and were suspended in 24-well tissue culture plates culture medium. Different dilutions of PBMCs were incubated at 37 °C with 5% CO
2. Culture supernatants were collected at 24, 48, 72, and 96 h after incubation and the supernatants were test against BCG or PPD by ELISA. The ELISA titer indicate the positive or negative result.[1]

Advantages of ALS Assay in TB diagnosis

The diagnosis of TB is most complicated. The routine diagnosis for TB patient is sputum culture based. But culture need 6-8week with 10-20% false positive results.[2] A rapid serological test for diagnosis, follow-up of disease activity, and response to therapy would be useful to clinicians.[3][4] The purified protein derivative (PPD) skin test (Mantoux test) is an important tool for diagnosis of latent TB infection and disease in the developed world, but it has low predictive value in Bacillus Calmette-Guérin (BCG)–vaccinated individuals, as well as in individuals living in areas where TB is endemic. The low predictive value is a result of cross-reactivity with BCG and atypical mycobacteria, as well as false negative reactions in malnourished children.[5][6] BCG has been used as an antigen in EIAs in in vitro studies to determine disease activity, but its use was suspended because of difficulties in interpretation, problems differentiating between active or past disease, and low sensitivity and specificity.[7][8][9] The antibodies from lymphocyte secretion (ALS) assay was earlier used to detect specific antibody response after oral vaccination with a killed cholera vaccine in healthy adults without any requirement for in vitro antigen stimulation.[10]

The main advantages are:

  • High Sensitivity >93 %[11]
  • Early detection of active TB
  • This method does not require a specimen taken from the site of disease, it also may be useful in diagnosis of paucibacillary childhood TB.
  • Secreted antibody may be preserved for long time for further analysis

Pitfalls

This method cannot be applied if Mantoux test (tuberculin skin test) has been done within the last 40 days, because it can hamper the results of the ALS test. This test is used as a complementary test to other tests, e.g. chest X-ray, ESR, CRP, history of contact with active TB case, failure with conventional antibiotic treatment etc.; anti-TB therapy is not provided if only ALS test is positive. The reason is that this method is potentially an early biomarker of active infection. However, if a subject does not show any physical symptoms, the doctors cannot prescribe anti-TB treatment.

References

  1. ↑ Raqib, R., J. Rahman, A. K. Kamaluddin, S. M. Kamal, F. A. Banu, S. Ahmed, Z. Rahim, P. K. Bardhan, J. Andersson, and D. A. Sack. 2003. Rapid diagnosis of active tuberculosis by detecting antibodies from lymphocyte secretions. J. Infect. Dis. 188:364–370
  2. ↑ Kothadia SN, Deshmukh S, Saoji AM. Evaluation of direct microscopy as a screening test in the diagnosis of pulmonary tuberculosis. Indian J Pathol Microbiol 1990; 33:68–73.
  3. ↑ Al-Hajjaj MS, Gad-el-Rab MO, Al-Orainey IO, Al-Kassimi FA. Improved sensitivity for detection of tuberculosis cases by a modified Anda-TB ELISA test. Tuber Lung Dis 1999; 79:181–5.
  4. ↑ Yilmaz A, Ece F, Bayramgurler B, Akkaya E, Baran R. The value of Ca125 in the evaluation of tuberculosis activity. Respir Med 2001; 95:666–9.
  5. ↑ Huebner RE, Schein MF, Bass JB Jr. The tuberculin skin test. Clin Infect Dis 1993; 17:968–75.
  6. ↑ Donald PR. Childhood tuberculosis. Curr Opin Pulm Med 2000; 6: 187–92.
  7. ↑ Dhand R, Ganguly NK, Vaishnavi C, Gilhotra R, Malik SK. False-positive reactions with enzyme-linked immunosorbent assay of Mycobacterium tuberculosis antigens in pleural fluid. J Med Microbiol 1988; 26:241–3.
  8. ↑ Wang CR, Liu MF, Chen MY, Lin TP, Cheng CS, Chuang CY. Enzymelinked immunosorbent assay with BCG sonicate antigen for diagnostic potential of mycobacterial infection in Taiwan. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi 1989; 22:97–104
  9. ↑ Lu CZ, Qiao J, Shen T, Link H. Early diagnosis of tuberculous meningitis by detection of anti-BCG secreting cells in cerebrospinal fluid. Lancet 1990; 336:10–3.
  10. ↑ Chang HS, Sack DA. Development of a novel in vitro assay (ALS assay) for evaluation of vaccine-induced antibody secretion from circulating mucosal lymphocytes. Clin Diagn Lab Immunol 2001; 8:482–8.
  11. ↑ Rapid diagnosis of active tuberculosis by detecting antibodies from lymphocyte secretions R Raqib, J Rahman, AKM Kamaluddin, … - The Journal of …, 2003 - UChicago Press



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