A. Principle:
The rabbit anti Bm mf ' S ' antibody is used for the detection of
filarial antigen present in the sera samples. The wells in Nunc microtitre
plate are sensitized with the antibody and further incubated with test sera.
The filarial antigen is detected by incubating the wells with Bm mf ES antigen
peroxidase conjugate.
B. Materials :
a.Nunc microtitre plates
b.0.06 M carbonate buffer pH 9.6
(Appendix-I)
c.1% BSA in carbonate buffer pH 9.6
d.0.05 M PBS pH 7.2
(Appendix-I)
e.Bm mf ES antigen peroxidase conjugate
(prepared by periodate method as described earlier).
f.PBS/T (Appendix-I)
g.Rabbit anti Bm mf ' S '
antibodies (Appendix-II)
h.OPD substrate (Appendix-I)
i.ELISA reader
j.Test sera.
k.Negative control & Positive control sera
C. Method :
1.Prepare optimal dilution of anti Bm mf 'S' antibody ( 50 mg/ml ) in 0.06 M carbonate buffer, pH 9.6.
2.Dispense 100 ml of diluted anti Bm mf ' S ' antibodies in each well & incubate in humid chamber
at 370 C for 3 hr.
3.Wash the wells once with PBS/T (for washing add PBS/T in each well
shake gently & discard after 1
min).
4.Add 200 ml
of 1% BSA in each well incubate
at 370C for 2 hr. (sera dilutions may
be carried out at this stage.
Dilute 5 ml of each serum to 500 ml in PBS/T).
5.Remove BSA solution & wash the wells twice with PBS/T.
6.Dispense 100 ml of optimally diluted sera 1:100 in PBS/T to each well
in duplicates & incubate in humid
chamber at 370C for 2 hr (including positive & negative controls).
7.Remove sera from plate & wash 5 times with PBS/T as described
above.
8.Dispense 100 ml of optimally diluted Bm mf ES antigen peroxidase
conjugate in PBS/T to each well & incubate in humid chamber at 370C for 2
hr.
9.Remove conjugate solution from plate & wash 5 times with PBS/T.
10.Add 100 ml of
freshly prepared OPD
substrate to each
well & incubate at room temperature (250 C) for10-15 min in dark.
11.Stop reaction by adding 1N HCl.
12.Read the OD at 492 nm using
ELISA reader
13.Serum sample showing lower O.D. (by a difference of 0.2 compared to
negative control) is considered as positive.
Note :
1.Sensitized plate may be covered and stored at 40C until use.
2.Sera samples have to be treated with acid & heat as follows and
used in inhibition ELISA to detect
immunecomplexed antigen
Acid & heat treatment of sera
a.Add 20 ml test sera to 100 ml of PBS (0.05 M; pH 7.2).
b.Add 100 ml of glycine HCl buffer (0.1 M; pH 2.8, Appendix-I) and incubate at 650C for 15 minutes with constant gentle
shaking.
c.Allow to cool to room temperature and add 100 ml of PBS (2 M; pH 8.0,
Appendix-I) for neutralization.
d.Dilute the sample to a final dilution of 1:100 by adding 1:7 ml
PBS/T.