3. Stick Elisa (Indirect)


Detection of tuberculous antibodies


A. Principle:

M. tuberculosis ES-31 antigen is immobilized on cellulose acetate membrane sticks. The test sample is then incubated and the bound antibody is detected using enzyme labelled second antibody. The immune reaction is revealed using suitable substrate.


B. Materials :

a.Cellulose acetate membrane sticks.

b.0.25 M SPB        (Appendix -I)

c.0.05 M SPB        (Appendix -I)

d.0.01 M SPB        (Appendix -I)

e.PBS/T       (Appendix -I)

f.3% Gelatin in 0.05 M SPB.

g.Anti-human IgG-penicillinase conjugate.

h.Penicillinase substrate (Appendix - I)

i.M.tb ES-31 antigen (Appendix -II)

j.Test sera.


C. Method :


1.Coat 1ng/5ml M.tb ES-31 antigen to CAM sticks and block with 3% gelatin for 2 hr at 370C. 

2.Wash sticks 3 times with PBS/T (as described earlier).

3.Incubate antigen coated sticks in 500 ml volume of diluted (1: 600 in PBS/T) positive and  negative  sera  for  1 hr  at  370C  in  duplicates in small plastic vials. (5 ml to 3 ml PBS/T). 

4.Wash  sticks 5  times  with PBS/T  ( for  washing add PBS/T, shake gently & discard after 3 min.)

5.Incubate each stick in 500 ml of optimally diluted anti-human IgG Penicillinase conjugate at 370C for 30 min. 

6.Wash sticks 9 times with PBS/T (as described earlier).

7.Change sticks to labelled glass tubes.

8.Add 500 ml of starch-iodine-penicillin 'V' substrate to each tube   and incubate at 370 C.

9.Note the time of decolorization for each tube.

10.Serum sample showing complete decolorization at least 4 minutes earlier than the negative  control serum is considered positive for tuberculous antibody. Serum sample showing complete decolorization at least 2-3 minutes earlier than the negative control serum is considered Borderline Positive for tuberculous antibody and the test needs to be repeated.




1.Blood samples collected on filter paper can also be used as test samples. Collect 20 ml of blood on Whatman No.3 filter paper and allow to dry at room temperature. It was observed that 20 ml of blood covers a circle of diameter of about 0.9 cm. Hence alternatively blood can be collected directly on filter paper in such a way that it covers a circle of diameter of more than 0.9 cm and after drying a circle of 0.9 cm diameter is cut for elution. Cut the filter paper with dried blood samples into small pieces and elute into 0.5 ml of 0.05 M, PBS, pH 7.2 in a tube by shaking at 370C for 2 hr and leave the tubes at 40C for overnight. After centrifugation at 600 g for 10 min, separate the supernatant and store at -200C with 0.01% Sodium azide as preservative. [Undiluted filter paper eluate is assigned an equivalent serum dilution of 1: 50, assuming serum content of blood as 50%.]


2.After antigen coating and blocking with gelatin, the CAM sticks may be dipped in 5% sucrose solution and incubated at 370C for 30 minutes for long time preservation. The stick needs to be washed thrice with PBS/T before use.




1.Nair ER,  Kumar S,  Reddy MVR  and  Harinath BC. Mycobacterium tuberculosis  H37 Ra ESAS-7 an excretory - secretary antigen fraction of immunodiagnostic potential in pulmonary tuberculosis; Indian J Clin. Biochem (1998) 13(2),98-105. 

2.Nair ER, Banerjee S, Kumar S, and Harinath BC. Isolation and characterization of a 31 kDa  mycobacterial  antigen from  tuberculosis  sera and  its identification with in vitro released culture filtrate antigen of M.tb H37 Ra bacilli; Scand J Infect  Dis. (2000) 32 :551-556.