Og4C3 ELISA TEST for Filaria
This test is used to diagnose infection with wuchereria bancrofti The diagnosis is based on the use of a monoclonal Ab Og4C3 which is coated on to the surface of microtitre plates. The coating monoclonal antibody (Og4C3) has been shown to specifically recognise only Wuchereria bancrofti antigen in human sera. Principle:
1.Microtitre Plates Five U-bottom polystyrene microtitre plates pre-coated with Og4C3 monoclonal antibody.
2.Solution A (green solution) One 50 ml bottle for primary treatment of serum samples.
3.Solution B (YELLOW solution)One 50 ml bottle for secondary treatment of serum samples.
4.Antibody and conjugate diluent One 100 ml bottle (blue soloution)
5.Standard antigens (1-7) Seven dilutions of Onchocerca gibsoni
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antigen.
Each vial contains 600 ml of standard (orange cap)
6. Rabbit and-onchocerca antibody One 300 ml bottle(yelow cap)
7.Anti-Rabbit conjugate One 300 ml bottle (purple cap.) '
8.Chromogen One 60ml bottle of single component ABTS ready to use.
9.Washing buffer(X20) concentrate.
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METHOD
All steps carried out at room temperature .
Ensure that all reagents and the microtitre plates are at room temperature before use.
Quantities indicated below refer to those required for the use of ONE plate.
To prepare washing buffer, dispense one measure from the x20 dispensing bottle into 500 ml of distilled water.
1. Preparation of serum samples.
Add 50 ul of each serum sample to 50 ul of solution A (green solution) in a suitable container.(e.g. any type of 96- well microtitre plate, BUT NOT one of the coated plates supplied with this kit.)
Mix the contents of the wells by tapping the plate, and leave at room temperature for 2 minutes.
Using a multichannel pipette ,add 50ul of solution B (YELLOW solution) to each well containing the serum sample/solution A mixture.
Mix well by pipetting u p and down several times, and IMMEDIATELY transfer 50 ul of the treated serum to each of two wells of the og4C3 Ab-coated plates supplied.
Up to 40 samples in duplicate can be tested per plate (see the plate layout diagram)
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Add 50 ul of each Standard Antigen (no1- 7, Orange caps ) directly to duplicate wells of columns 11 and 12.Add the standards directly to the wells; DO NOT treat or dilute in any way.
For conjugate control (cc), add 50 ul of solution B to wells A11 and A12.
3. Place the plate in a humid container and incubate for 1.5 hours.
4. Wash the plate three times with wash buffer, invert and tap gently to remove residual droplets.
5. Prepare rabbit anti-Onchocerca antibody by adding 50 ul of rabbit anti-onchocerca antibody (YELLOE cap) to 6ml of antibody diluent (Blue solution)
Add 50 ul of the diluted rabbit antibody to all wells and incubate for one hour
8. Wash the plate three times as before.
9. Add 100 Ul of chromogen (ABTS)(do not dilute) to each well and incubate for one hour.
10.Plates can be read with a spectrophotometer at a single wavelength of 414 and 492 nm.
Blank the plate reader on wells containing conjugate control or a row of wells containing substrate in a separate blanking plate.
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INTERPRETATION OF RESULTS
A standard curve may be drawn using the optical densities of the seven standard control samples as the Y values.
The X values can be the numbers 1 to 7 corresponding to the controls. The test samples can be allocated to groups on the basis of the optical density compared with the optical density of the standard curve samples. Samples with an OD>= Standard No 2 are considered to be the positive samples.
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copy right © vcrc 2000-2006