I am working on serum sample of rat mouse. rotavirus given to rat through mouth and after 42 days collected serum and stool then done indirect ELISA on negative, positive and test samples. negative sample is mouse eaten buffer.
in indirect ELISA by IgG or IgA as a secondary antibody , negative control and pre samples have more OD than to test or positive controls and all pre samples have high OD .
in all my samples even negative control after period of experiment had increase in OD.
i was used skimmed milk, FBS, Casein and tween as a blocker but did not alter my results.
WHATS PROBLEM
WHO I can reduce OD of negative control and pre serums
If it is totally a commercial ELISA system then you can try the ELISA again changing the negative sample or diluting the samples more.
If It's your own protocol I guess you need to check a few things one by one-
1. Check your blocker using BSA or other protein without tween
2. Check your wash buffer's efficiency
3. Try again changing the dilution of secondary antibody
4. Check your stop solution's efficiency
It sometimes happen, due do different reasons. So, don't worry. Hope you overcome this..!!
Dear Somaye,
have you tried PBST with BSA (1% m/v should be ok) as diluent and blocking buffer?
Otherwise, please post as much details from your experiment as possible, esp. buffer compositions.
Ideally before initiating the experiment you should have done the titre check of the pre immune sera by indirect ELISA and then proceeded with your treatment. Only those rat which have low titre should have been taken for the experiment.
yes i try BSA 1% IN PBST results in this condition similar another blocker.
i try 1:500 1:3000 1:10000 secondary antibody but alone in 1:500 we see OD.
If you have to use the secondary 1:500, something is wrong. It's most likely background noise that you are seeing. Usually, secondaries are used 1:10000 or more dilute (1ul in 10ml, because it's convenient). Normally, I'm using them 1:50k or more dilute (we pre-dilute secondaries in Stabilzyme 1:5 or 1:10 when opening the vial, so they will last for ages).
Is the peroxidase of the secondary working? Make sure, there is no sodium azide in any buffer that gets in contact with the peroxidase.
Then, do you have a positive control to include in your assay? Do you get any signal with the virus preparation you're feeding the mice?
We use 1:10000 &1:3000 secondare Antibody(HRP conjugate) but we dont have OD
We dont have sodium azide we have just PBS buffer in stool sample and serum samples
We have positive and negative control
Negative have more OD than positive sample
In positive control we have little response
Are your reagents ok?
To test the peroxidase conjugate and the reagents, start with 1ul of the HRP conjugate, dilute into 11ul PBST and make some serial dilutions 1ul+10ul. Use a working secondary as control.
Then, add 10ul of chromogen solution to 1ul of each dilution. You should see colour development instantly. If not, get new chromogen or try to add a little hydrogen peroxide (e.g. prepare a ml of a 1% solution and add 1ul to each of the test solutions you have prepared in the last step).
If the chromogen should already be blue when it comes out of the bottle, it has become bad, no chance to reactivate it, get a new one.
We use IgG and IgA HRP secondary antibody but in 2 situation we get similar results
the results that i was said prevoiusly
we use this reagent for rabbit serum and we dont have OD
But by negative rat serum get OD