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.
Note:
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.
References:
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.