4. Plate Peroxidase Elisa ( Inhibition )



Detection of filarial antigen

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.