Hello,

Ensure that coating concencentration of antigen is ok, and blocking of plate is ok. PH of your  buffer could be a responsible. Ensure PH of different buffer is ok.

what conjugate dilution are you using?

Probable reason would be your blocking and conjugate dilution. Try increasing the conjugate dilution and ensure that this background is not due to improper blocking.

Cheers

I checked both 1:2000 and 1:4000 dilutions which is given by manufacture.

Increase the dilutions upto 10000. Probably it will work

Are you blocking with goat serum? if not, you should.

@ Sushant, but manufacture has given only 1:2000 and 1:4000 dilutions. 

@Gemilson, I am blocking with skimmed milk.

Manufacturer recommends dilution but we have to see the cutoff value at which positive is positive and negative is negative.

High amount of conjugate binds non specifically with the polystyrene plate (since there are no antibodies in case of negative sera or if blocking is improper) and gives a false background.

Manufacturers can only give guidelines, but each experiment needs its own optimization. All the above comments are correct. You need to check your blocking conditions and make sure you dilute the antibody far enough to minimize the background. Based on the description of your assay, it seems that goat serum should be in your blocking buffer (in addition to skimmed milk and Tween-20). Good luck!

Thanks Sushant. I agree with you. I'll check and will back to you soon.

@ Jan, I use only 5% skimmed milk in PBS for blocking. sorry, I did a mistake in my question. 2ry antibody should be corrected as goat anti-human IgG.

Bhagya, you should probably first check if the background comes (partly) from the primary in your serum samples or (also) from the secondary antibody. You can check this by comparing a view samples with some micro-wells omitting the sera. It would also be good to compare pathogen-coated micro-wells with mock-pathogen-coated microwells from unconditioned culture media in the same experiment.  If any of the micro-wells without the control serum samples still give background, you know the secondary antibody is not good enough. You may need a new secondary affinity purified IgG with minimal cross-reactivity to serum proteins for the species of pathogen you are working with. This may solve part of the problem, because you see difference between control sera and patient sera. I would be curious how this difference holds up after improving on your goat-anti human IgG secondary.

See http://everestbiotech.com/non-specific-binding-background-and-noise/

Also interesting to read would be http://everestbiotech.com/common-trust-commercial-antibodies/ and the links therein.

You can try  increasing the number of washes between the incubations, you can try leaving the washing buffer for 1-2 minutes during washes, just let the "soak". You can try lowering the conjugate concentration but this will affect the ODs. Also make sure you use the fresh aliquote of your conjugate. 

Three thing you have to consider to minimize background while optimizing indigenous ELISA. After coating antigen you have to block plate with high concentration of inert protein like 0.3 to 1% gelatin (boom less than 90) or milk protein. Thoroughly wash plate after each step (five to seven times) with washing buffer containing 0.05% Tween-20. Dilute (twice or thrice time more as suggested by manufacturers) enzyme conjugated secondary antibody in dilution buffer containing  high concentration of inert protein like BSA up to 5%. I hope this may solve your problem.

With best wishes

Assay development is an art; many variables need to be looked at. It may appear easy to handle variables once at a time, but when interactions between variables exist (and they do exist), then optimization may turn into a nightmare.

I do respect all the answers above. However, one has to look at the raw data to sort out the origin of background / false reactivities. You cannot optimize an assay in one or two rounds of experiments. I recommend factorial DOE (Design of Experiments) to have some a rational design.

You need to describe in more details your assay to obtain specific answers.

I would take into consideration all the indications above and consider to include the same amount of IgG per well. In addition I would take into account the nature of the mix used to coat the plate. Perhaps you should purify a Little bit more your antigen.

Good luck

Luisa Villar

I have checked several commercial products of 2ndary antibody, but none of them worked perfectly. All of them had non-specific binding more or less. As an alternate, we developed the indirect competition ELISA in which the pathogen is firstly incubated with dilutions of serum and then detected by HRPO-labeled anti-pathogen mAb. The lineality and specificity of this competition assay is fine, but as you know other classes of pathogen-specific Ig's are also counted. It is difficult to establish a direct IgG assay.

There are several things to optimize:

The best parameter for the optimizations are:

1. Negative control (N) has to be at an OD below 0.1

2. Positive control (P) has to be at an OD above 1.0

3. Look for the optimal P/N-ratio

First search for an optimal solid phase. You should use a checkerboard titration. Normally an optimal concentration for solid phase coating is at 2 … 10 µg/mL in a 0.1 mol/L carbonate buffer pH 9.6. For each concentration two columns of your ELISA plate. The first for a serial dilution of your positive sample and the second for the serial dilution of your negative sample.

Than you need to optimize your incubation buffer. I’ve got always the best experience using a modified PBS with increase salt concentrations. The doubled salt (NaCl) will reduce unspecific binding and therefore the background. The inert protein (Gelafusal) is important. Cross reactions should be excluded, constant protein concentration should be given during the incubation despite of different dilutions. Gelafusal is a drug, used as plasma expander, available via pharmacy and best defined as all other drugs. Gelafusal is also free of any animal antibodies. Tween is a detergent reducing unspecific hydrophobic interactions with other proteins and the solid phase. Anyone has this in his dilution / incubation buffer and that why any blocking is nonsense and will increase background reaction and costs only.

So you will need about 4 days to get your assay optimized.

1. You can increase the concentration of you Blocking reagent

2. You can try a different Blocking reagent

3. You can increase your washing force and time

4. You can let the plate stay for 1-2 minutes after every wash to soak the washing buffer

5. You can try with a fresh Conjugate.

6. You can try decreasing the concentration of conjugate

What is your positive control here?