Using 5% skimmed milk with 0.5% tween as a coating buffer is probably is not a very good choice. The milk contains a lot of proteins that might be immunopositive by your rabbit antiserum (as a Primary Antibody). I would use something like PBS (pH 7.0-7.5) as coating buffer for your antigen.

You can avoid background by taking another reading at 570nm and subtract this reading from your actual reading at 450nm or so.

Further you should be conscious during washing the plate, give 1-5 mins gap between each wash and soak the plate properly before proceeding for the next step of the experiment. there should not be any bubble or remnant of wash buffer after soaking.   

Using 5% skimmed milk with 0.5% tween as a coating buffer is probably is not a very good choice. The milk contains a lot of proteins that might be immunopositive by your rabbit antiserum (as a Primary Antibody). I would use something like PBS (pH 7.0-7.5) as coating buffer for your antigen.

@ Viktor, yes the possibity that, skimmed milk may have reactive proteins to primary antibody of rabbit. However, use of BSA also, gave same higher reading.. We to speculate the role of blocking solution in this higher reading, we are still standardising tween concentrations with primary antibody... Hope it works better..

I would advise you to make sure the washing is thorough and no milk precipitate is on the plate after each wash. but most importantly if the milk is not a problem, check your coating buffer, pH  must is an important aspect. Also I would like to suggest getting rid of skimmed milk in your coating buffer.

High background means dark color development or high (OD) readings. Possible causes are insufficient washing, contamination wash buffer, and salt concentration in incubation/wash buffer. So wash your plate properly and shock properly there should not be any bubble in plate. Prepare fresh buffer, read plate immediately after adding stop solution.

Good Luck!!

You did not say how you make solutions of antibodies! At start make primary and secondary antibodies solutions in milk, let these stand in a refrigerator for 30 min and make hard spin down at least at 10.000 g 15 min. Use supernatant - carefully. If this does not work, do not use Tween in coating and you may consider carbonate buffer if proteins are hydrophobic.  At higher pH this will enhance hydrophobic binding to the plate. As substitute for milk try gelatin, but concentration must be found  by titration on the plate. Sometimes it works grate, but also could be even worse! That is life in a lab. Try to find high grade gelatin (it dissolves in saline itself and without heating) and start with 0.1-0.2% and go up. Tween in antibodies and washing solutions should not exceed 0.5% and preferably should be 0.05%.  Use only high grade Tween that YOU have opened to make stock solution, make sure you do not have any kind of microbial contamination from water downstream.

1. What plates are you using?

2. What is the antigen used to coat the plates?  Has this been titred?

3. What is the coating buffer used?  Coating time & temperature?

4. What blocking step is used.  Time, temperature, & blocking solution?

5. What diluent are you using for the dilution of the rabbit serums & conjugate (times & temperatures)

6. What washing buffer are you using, how many washes and are what times between washes?

7. Are you using OPD, TMP or other substrate, time & temperature?

8.  how are you reading your plates, single or dual wavelength and what wavelengths are you using?

Hi Vimarsh, to start, there are some things that I don't understand. The buffer you are reporting with skimmed milk and tween is the coating or the blocking buffer??? If it is the coating buffer, i think the use of tween is incorrect. Nor Tween or proteins should be present in coating buffer. The use of tween as a blocking agent is not as effective as protein blockers (milk, BSA, OVA, gelatin, among others). The interactions between Tween and the plate are not strong enough and irreversible, and any stronger interaction will displace it. The Tween concentration recommended for the washing buffer is of 0.05%. Other aspect is the incubation times, in this kind of ELISAs is recommended 1 or 2 hours. The anti-rabbit you are using is conjugated to what??? Because if you are using a biotin-streptavidin detection system, the milk interferes. How many wash steps you are using??? How you are preparing your antibodies???

@ All, Antigen coating with Carbonate Coating Buffer

PBS 1X with tween (0.05%) + 5% Skimmed Milk Powder- block 1 hour

1 x pbs wash

1:125, 1:250 1:500 and 1:1000 dilutions in PBS 1X with tween (0.05%) : Primary Antibody (Rabbit sera)

1 hour @ 25 degrre temp

3 times PBST wash + 3 times PBS wash

1:15,000 Sec Ab (As per manufacturere Gudance) Goat Anti rabbit igG

3x PBST wash + 3x PBS wash

1x TMB /H2o2

20 min dark

1N H2SO4 stop.

reading @ 450 with 620 ref. 

@ Richard S Green

1. Nunc 96 Flat Base

2. Conjugated Peptide Coated. Titer, No idea.

3. Carbonate buffer: coating overnight @ 4 degrees

4. Blocking with 5% Skimmed Milk Powder in PBST (0.05%)

5. PBST (0.05%) Antibody Dilution (25 degrees), 1:125, 250, 500 and 1000

6. PBST (0.05%) and PBS 1x (Three times after 1Ab and 2Ab addition)

7. TMB/H2O2 : 1x 20 min reaction time @ 25 degree temp

8. Biorad Elisa reader with perfect Caliberation. @450 with ref of 620 nm.

Any suggestions sir?

There are some issues I still don't understand... for example... why you use tween in the blocking buffer??? detergents interfere the blocking... you already have high concentrations of milk that assures the blocking of the plate sites... I recommend the PBS-T for all the wash steps... i don't understand the extra washes with only PBS... why??? if you are trying to remove the background with wash steps, try 4 or 6 wash steps with PBS-T after the conjugate step... what is the incubation time with the conjugate??? how do you prepare it??? try PBS-T + 1% milk... why you read in 2 wavelength if you are stopping with sulfuric??? what do you consider as background readings??? what you are using as a blank of the assay???

1.  Plates can cause great differences in backgrounds. Nunc Maxisorp vs Polysorp may show a difference, try Costar.  Typically when developing an assay, I would try 3 or 4 brands each with a high and medium bind.  You should find the best plate and then have more confidence in your assay.

2. Carbonate buffer may not be the best coating buffer and PBS may work better.

3.  High antigen concentrations can cause high background and should be titred.

4.  What are your blanks like?  Does your conjugate bind to the skim milk powder and give high blanks.  The milk powder concentration could also be a problem.  Milk powder is cheap but sometimes you may need to try other proteins e.g. ovalbumen or IgG free BSA

5.  Plate washing.  with some assays it is necessary to fill the plate with the wash solution and let it stand for 1 - 3 minutes before emptying.  I would recommend 5 or 6 washes all with tween.

6.  The source of the conjugate can make a difference, different manufacturers and grades.  See if you can beg a few microlitres of different anti rabbit off collegues and compare them.