What do you mean by "unable to get proper result"?  You will get some level of absorbance in negative samples, which you use to set the background threshold for your reaction.  If you are seeing contamination, then you need to throw away ALL of your reagents (except the antibody stock solution), wash ALL of your equipment and sterilize your work area.  Good luck!

As with many of these questions, not enough detail. What is the lysis buffer, the sample and  primary and secondary antibodies.  Have you titred your primary and secondary antibodies.  Why is the volume of TMB much less than the coating volumes.  What volume did you use for your antibodies etc.  And finally, give some results.  What are they for results the blank, sample and negative control?

I agree with both answers given so far: there is not enough information for a correct answer. Yet as we started with similar problems, I can give here some few proposals, which could be of help. I think your main task is to reduce the background noise. For this reason the first steps as well as the washing procedures are important. Often the right plastic material is also essential; so we used e.g. MaxiSorp Immunoplates from Nunc, which worked well. Furthermore, in our laboratory the samples were adsorbed for 2 hours at room temperature followed by an incubation in 5 % Tween 20 in PBS at 4° overnight (the latter buffer was also used for all the washing steps). Another point, which you should have in mind is the concentration of the first antibody: when you have to use it in such high concentrations you can get trouble of false positive results; usually an ELISA should be sensitive enough to use antibody dilutions of not less than 1:100. Good Luck!

To write a productive recommendation I need to understand the system and its goal. You mention two antibodies, but don´t say against what. In any case, a 30 fold dilution use to be a very high concentration for the primary antibody and it could produce cross reaction. On the other hand, if you really need such a high concentration, probably your antibody is not pure enough.

Give more details and receive a more precise answer.

I would like to thank you all for answering the question. I used mouse antibody as primary and goat antimouse as secondary. I tried once again the ratio of primary antibody as 1:30, 1:100 and 1:200. Last one  gave a reading of 0.9. I did not use sample this time, it was just a blank(coating buffer+lysis buffer). I used TMB 100ul sorry i wrote it wrong in the question. Basically I am not getting a considerable difference in the reading of blank and sample. Absorbance is nearly same.

Is the goat antimouse reacting with your milk blocking?  There can be cross reactivity between cow, sheep and goat.  suggest that you coat your plate, block and wash as normal, skip mouse primary Ab, just add goat antimouse, incubate, wash and develop.  you should need to try other blocking proteins from non-ruminant source e.g. ovalbumen or change the secondary antibody.  Some assays require the wash step to be filled with wash buffer and left to soak before emptying for the next wash, sometimes 1 - 3 minutes between washes and soaking cannot be always replaced with more washes

I read your procedure once again and once again I understand nothing. If I would to prepare a sandwich type ELISA, my procedure would be the following:

You say that you first coats the plate with buffer and sample. That means that any protein in your sample could be attached to the plate. Later you add primary antibody (I suppose it binds the sample) and latter the secondary one (I suppose it binds the primary one but I don´t not why. I don´t believe that this offers a great amplification of the signal. If the secondary antibody binds the sample your procedure has no sense to me).

It is very important to know what the primary and the secondary antibodies bind.

If the primary and secondary antibodies bind the sample, the procedure is roughly  the one I detailed above (you can mix some steps).

Totally agree with Gerardo plus give results

Dear Pamari,

we  are working with plant pathogens and for some of those we also use the indirect plate trapped antigen ELISA that you describe here.

I think, that the problem you are describing here arises from non proper binding of the antigen to the plate. Use carbonate binding buffer pH 9,6 1:1 together with your lysis buffer to bind the antigen to the plate and proceed with the usual protocol (of course titrate your antigen- 1st and 2nd antibody reaction and always use freshly prepared buffers and blocking agents). If this does not help, try high binding plates and a stronger blocking agent. What is your lysis buffer composed of?

You can make the carbonate buffer easily by yourself: Coating buffer (pH 9.6) 1.59 g sodium carbonate (Na2CO3) 2.93 g sodium bicarbonate (NaHCO3) 0.20 g sodium azide (NaN3) Dissolve in 900 ml H2O, adjust pH to 9.6 with HCl and make up to 1 l. 

I completely concur with Joachim. Without knowing what capture antibody you are using, and not knowing what antigen is being captured, it sounds like the most crucial issue here is during the plate coating. Typically, you want a buffer near pH 9 - 10 range. The reason for this is that proteins will covalently stick to the ELISA plate [be it polystyrene or PVC] via tyrosine residues, and you want to be near the pKA of the phenolic group for the reaction to occur.

A second issue may arise during blocking. Casein or dried milk are often used because they are a cheap source of non-specific protein, but in at least on case I know of, casein or dried milk as block actually interfered with the ELISA, because there are many bacteria in the dried milk or casein preps, and they bound up all the antibody for the test antigen [phosphoryl choline], so BSA [ELISA grade is preferable. 1% is sufficient, but I often used 3% to be sure.

In the primary antibody step, a 1:30 dilution seems very low to me, unless it is a fairly dilute prep. In this step, I typically use 0.1% BSA; [I use the same concentration BSA in the secondary step].

I haven't used TMB - is that an Alkaline Phosphatase substrate, or a Horseradish peroxidase substrate? I'm guessing it may be a HRP substrate, since to kill AP ELISAs you use NaOH, not H2SO4. If it's HRP based, the quality of your peroxide is a keep factor. I found I needed to get fresh bottles monthly when I was doing lots of HRP ELISAs.

Finally, 30 minutes seem like a long time for an HRP based ELISA. Typically, mine plates were ready to stop within minutes of adding substrate. Anything more than 4 plates, and I would have to stop adding substrate, kill the first 4 plates, and then continue with the next 4, etc.

Dear Pari,

what you are performing is an antigen coated plate (ACP) also called plate trapped antigen (PTA) indirect ELISA, and you are not using a capture antibody, right? to attach the antigen tightly to the plate you should use a high pH coating buffer. The lysis buffer with the antigen may also hinder the proper binding of the antigen to the plate. If so you can try out deionized water with 0,5 to 1% PVP as antioxidant. When using such high concentrations of primary antibodies asyou do, you will get high background. Use the proposed coating buffer for coating the plate with your antigen of choice, then block with 0,1 % of BSA (as proposed by Gary). Dilute your primary antibody to 1:500 or 1000 and after washing add the secondary (conjugated) antibody. Then do the development with your substrate. In plant virology we normally use p-nitro phenyl phosphate (p-NPP) as substrate. But it should work as well with HRP- substrates. Please let us know if this works.

Thank you all for helping me to get through. I am working on inhibition of acetylcholinesterase using blueberry extract. I corrected few steps as 

1. I used 5%milk for blocking.

2. Primary antibody dilution 1:300

Thank you all for your valuable suggestion. I was doing the extraction process so could not perform ELISA. I would be starting again from next week and definitely post the outcome. I apologize for the late reply. Thank You.