Hello
I have a problem with ELISA test
I try to do Ag detection ELISA for quantification my antigen
So i hyperimmunized rabbits and guinea pigs with the same antigen then designed the capture ELISA As bellow:
1- coating with Guinea pig Ab from hyperimmune serum // antigen // rabbit Ab from hyperimmune serum// antirabbit HRP conjugate// substrate then stope solution.
2- coating with Guinea pig Ab from normal serum serum // antigen // rabbit Ab from hyperimmune serum// antirabbit HRP conjugate// substrate then stope solution.
3- coating with Guinea pig Ab from hyperimmune serum // PBS // rabbit Ab from hyperimmune serum// antirabbit HRP conjugate// substrate then stope solution.
4- coating with Guinea pig Ab from normal serum serum //PBS // rabbit Ab from hyperimmune serum// antirabbit HRP conjugate// substrate then stope solution.
5- coating with Guinea pig Ab from hyperimmune serum // PBS // PBS// antirabbit HRP conjugate// substrate then stope solution.
6- coating with Guinea pig Ab from normal serum serum //PBS // PBS// antirabbit HRP conjugate// substrate then stope solution.
As a result in the cases 2. 3. 4 i have got OD less but very close to the OD of the case 1, while 5 and 6 were equal the blank's OD.
My question, is it possible to have such ant species reaction between rabbits and guinea pig sera / or Ig G. and how can i solve this reaction
Thanks in advance for your comments
This is hard to interpret without the absorbance values for 1,2,3 & 4 but I am assuming that they are all high compared with the blanks 5 & 6.
So you have binding of HRP conjugate in the absence of antigen. And you also have no signal in the absence of the rabbit IgG detector, which rules out cross-reaction of the anti-rabbit HRP conjugate with GP IgG on the plate. Thus the rabbit IgG must be binding non-specifically to your plate (to the GP IgG or the plastic or the blocking agent). A lack of blocking would give rise to a high signal in 5&6, thus there is most likely a failure to remove non-specifically bound rabbit IgG in the wash steps.
I would add rabbit igG to a plate of your immobilised GP IgG and try different wash solutions in triplicate wells. You'll also need a few wells without rabbit IgG to define the background. Once you have low background with rabbit IgG added to the plate you can repeat your original expt with antigen.
Hello Nick
Thank you for the recommendations
Sorry for not including the OD results.
please you can find bello results of one of the experiments we have performed
in fact we tried to neutralize the capture antibodies with negative GP serum as as result we got OD value less than 0.1
Please note. GP HYP is just for the one plate which was actually coated with Guinea pig hyperimmune sera, i couldn't manage the explanations on the graphic in better way
I will follow your recommendations
The other possibility is that the rabbit hyper-immune serum contains antibodies that cross reacting with guinea pig Ig. This is not an uncommon finding. One way to eliminate this is by adding purified guinea pig IgG to the rabbit hyper-immune serum and filtering it [I use Acrodisc 13mm syringe filters with 0.2 um HT Tuffyn membranes] prior to adding to the plate. The guinea pig IgG will bind to any anti-guinea pig antibodies in your rabbit hyper-immune serum and those immune complexes will be retained by the filter. I used to do this routinely to block anti-mouse Ig cross reactive antibodies in a commercial goat anti-rat Ig product for FACS.
You will have to do some test runs to determine that correct amount of guinea pig IgG to add.