ELISA negativ control? - (Nov/21/2005 )
hello everybody,
we're working in our lab on ELISA but now we have a silly situation and we would like to ask for some advices.
Ok, we are working with two different recombinant proteins which we're coating on the plates. Our negative control is HIV-protein at a concentration of 1µg/ml. the two recombinant proteins were coated at different concentrations corresponding to previous tests with specific antibody. After coating with the antigen we incubated the plates with serum from patients with hematological malignancies. Following washingsteps,secondary antibody incubation etc. are still the same for both assays. The problem is, that for one protein the negative control (HIV) is really negativ whereas the positive serum gaves higher results as expected but for the other protein the result for HIV is nearly the result for the serum... 
anyone an idea???? please, please, please 
or any idea for another "good" negative control??
thanks a lot in advance for your help!!!
we're working in our lab on ELISA but now we have a silly situation and we would like to ask for some advices.
Ok, we are working with two different recombinant proteins which we're coating on the plates. Our negative control is HIV-protein at a concentration of 1µg/ml. the two recombinant proteins were coated at different concentrations corresponding to previous tests with specific antibody. After coating with the antigen we incubated the plates with serum from patients with hematological malignancies. Following washingsteps,secondary antibody incubation etc. are still the same for both assays. The problem is, that for one protein the negative control (HIV) is really negativ whereas the positive serum gaves higher results as expected but for the other protein the result for HIV is nearly the result for the serum...
anyone an idea???? please, please, please
or any idea for another "good" negative control??
thanks a lot in advance for your help!!!
It looks like your negative assay contain a mistake somewhere
1/ Might be your coating which is not efficient due to the nature of your rec protein (properties, buffer, contaminants and so on)
2/How do you treat your derum before the assay ? Some reactive Ab's might be dead !
3/What do you mean for good negative contrôl ? You say that the signal was the same for HIV and your protein ! Do you mean that the results are too high ?
4/ If it's a positive signal in your negative contrôl just make sure that nothing in this serm should be positive Has they been typed for HIV ? How do you know that they should be negative !
5/ Might comes from the HIV protein tht you use is there anything common with other type of viral antigen more common ?
Try to be more precise that we could hepl you better !
Pesji
hello everybody,
we're working in our lab on ELISA but now we have a silly situation and we would like to ask for some advices.
Ok, we are working with two different recombinant proteins which we're coating on the plates. Our negative control is HIV-protein at a concentration of 1µg/ml. the two recombinant proteins were coated at different concentrations corresponding to previous tests with specific antibody. After coating with the antigen we incubated the plates with serum from patients with hematological malignancies. Following washingsteps,secondary antibody incubation etc. are still the same for both assays. The problem is, that for one protein the negative control (HIV) is really negativ whereas the positive serum gaves higher results as expected but for the other protein the result for HIV is nearly the result for the serum...
anyone an idea???? please, please, please
or any idea for another "good" negative control??
thanks a lot in advance for your help!!!
It looks like your negative assay contain a mistake somewhere
1/ Might be your coating which is not efficient due to the nature of your rec protein (properties, buffer, contaminants and so on)
2/How do you treat your derum before the assay ? Some reactive Ab's might be dead !
3/What do you mean for good negative contrôl ? You say that the signal was the same for HIV and your protein ! Do you mean that the results are too high ?
4/ If it's a positive signal in your negative contrôl just make sure that nothing in this serm should be positive Has they been typed for HIV ? How do you know that they should be negative !
5/ Might comes from the HIV protein tht you use is there anything common with other type of viral antigen more common ?
Try to be more precise that we could hepl you better !
Pesji