Hi Linda,

blocking is in most cases not necessary. Don't waist casein or other proteins protein like that. Use Tween (or Triton X-100) in the incubation buffer. This will block any free binding site due to the hydrophobic interaction with the polystyrole rings. You need blocking for plates only, if you would like to store them for longer periods at room temperature. The problem with the blocking is, that the blocking protein will replace (slowly) the coating antibody. This will decrease your OD's for the positive controls!!

Don't wash the plates after coating. Same phenomenon. The detergent will replace the coating protein, slowly but surly. Store the plates frozen and wash them immediately only before using!!!!

To optimize the solid phase antibody concentration go for a checkerboard titration. Stat with 0.1 µg/mL antibody concentration, 2 columns, double the concentration for the next two columns ...

Use two samples, one highly positive, one negative, dilute them from one factor less then you are currently using up to the 8 steps for the different rows.

Incubate better for 90 min. Use if available multichannel pipettes to avoid, that the first sample will incubate much longer than the last due to the time shift during pipetting.

Do all the other incubation steps (you can do them all with 100 µL including stopping or if you need to save material, the ELISA will work also as a 50 µL assay for all steps.

In the evaluation compare per solid phase concentration the Positive/Negative-Ratio and search for the optimum under the condition, that the maximal OD is not higher than 2.0. Due to the Lambert-Beer-Low, measurements higher than 2.0 getting more and more imprecise. (OD is the reciprocal value of the transmission!).

Put the graph of your evaluation here and I will comment on this.

check your refrence cotrolls(-ve and +ve) if ok increase the concetration of your caoting Ag and decrease the concentration of the detection Ab

What kind is your microplate? Which species are your ABS from, which species are your samples from?

1. make sure that the couting is fine. You need about 2 - 10 µg/L of your antigen/antibody in a carbonate buffer pH 9.6. Store at 4 °C overnight. Remove next morning the fluid as complete as possible. Store the plates frozen until use.

2. Use a washing buffer PBS 0,1% tween 20 (0.05% is also fine), wash always three times with 200 µL/well, wait at each washing step 1 min.

3. Use an incubation buffer (it's your washing buffer [including the detergent tween 20] + protein (BSA, HSA, ...) 3% is sufficient. Make sure that your sample dilution is sufficient. Take a look at the expected concentrations, ELISA's are very sensitive (lower detection limit is at 1ng/mL) and the measurement range is about two orders of magnitude. Add some phenol red to see where you are pipetting .... The missing detergent in your incubation buffer could be one of the reasons, but in this case, the blank is normally still very low.

4. incubate for 90 min (only longer if you need really much lower detection limits)

5. wash (3 times)

6. Use your incubation buffer to dilute the labeled antibody (a concentration of 1 µg/mL is sufficient). Don't look for dilutions, look for concentrations.

7. wash (3 times)

8. use a absolutely clean tray for your substrate solution. (minimal contaminations by the enzyme leads to a major background reaction). This could be one of the reasons for your background including the high blank!!!

9. Stop the reaction (depending on the substrate: TMB best with H2SO4, 1 mol/L)

use Room temperature incubations, dilute (more) your conjugate in TBS-T + bsa 1% , works for me! :)

There are many reasons that contribute to the high background signals.

1. Your diluent or blank may be contaminated

2. The quality of BSA used is not the quality required in an ELISA

3. You may have to dilute further your conjugated antibody.

Mr Stephan has given a very useful advice

Follow the protocol given by Stephan Kiessig step by step. best of luck.

Dear Linda,

I had once the same problem. I was able to solve it by using different containers for the detection antibody and the detection reagent. In our reaction container (for a multichannel pipette) the HRP binds extremely strong (plastic kind?) so we decided to use one container for the HRP dilutions and another for the TMB reagent (detection reagent). I hope this helps you. Have a nice day, kind regards...

Thanks for all the reactions!

- As a microplate I use greiner medium binding ELISA plate.

- The species of my antibodies are: mouse polyclonal as a capture antibody, which I incubate overnight at 4°C. For a second antibody I use a rabbit polyclonal and as a detection antibody with HRP I use a goat aniti-rabbit poly-HRP..

according to the manufacturer there shoudn't be any crosslinking between these species.

- My samples are human serum.

- For coating buffer i use carbonate/bicarbonate pH 9,6. After the incubation overnight i wash the plate with an automatic washer, 4 times with 300µl, 1xPBS with 0,05%Tween. Then the block, 5%BSA 1xPBS 200µl incubated for 1 hour at room temperature. Washing step. Then the standards and samples which i dilute in 1%BSA 1xPBS, 100µl incubated for 1 hour at room temperature. Washing step. Then the second antibody diluted in 1%BSA 1xPBS, 100µl incubated for 1 hour at room temperature. Washing step. Then the detection antibody, again diluted in 1%BSA 1xPBS, 100µl incubated for 1 hour at room temperature. Washing step. 100µl TMB incubated for 20 minutes at room temperature in the dark. After the color is formed I stop the reaction with H2SO4 50µl and I meassure by 450-650nm.

- I always use clean disposable containers for each step so no contiminations are possible. And also clean disposable pippeting tips.

In my next experiments i'm using different concentrations for the antibodies so maybe that will help. And also i'm going to check using different blocking buffers (caseine, protifar..)

I will also check if it helps to put Tween in my incubation buffers :)

I increase the number of washs to 5 after conjugate, and I also use Casein 0.5% intead of BSA, is a very good blocking agent; see the pdf files:

http://www.nuncbrand.com/files/en-577.pdf (about casein)

http://www.labobaza.pl/download/productFile/15350czynnik_blokujacy_elisa.pdf (about blocking and detergent)

Thankyou! today I diluted my antibodies further but it didn't decrease the blanc as much as I had hoped.

The blanc was a little lower when i diluted my capture antibody (mouse polyclonal) 2000x times in stead off 1000x times.

Tomorrow I will check the use of different blocking buffers and also increase my blocking incubation time. (from 1 hour to 2 hours)

in the microplate, where did you put the blank wells? you can use the iner wells and leave the outerwells (close to the outside of the microplate ) and fill those with water.

the conjugate you should USE tween, and you only use 1% BSA, you need tween in the conjugate!

I always use this formula for conjugate: BSA 0.1-1% or gelatine 0.1-0.5% +PBS-T or TBS-T , but always with tween 20, 0.05%.

or some of your capture/ 1st antibodies react to BSA! if you have a polyclonal. In this case you have to change the blocking agent, or test other bloking agents.

Hi Linda,

blocking is in most cases not necessary. Don't waist casein or other proteins protein like that. Use Tween (or Triton X-100) in the incubation buffer. This will block any free binding site due to the hydrophobic interaction with the polystyrole rings. You need blocking for plates only, if you would like to store them for longer periods at room temperature. The problem with the blocking is, that the blocking protein will replace (slowly) the coating antibody. This will decrease your OD's for the positive controls!!

Don't wash the plates after coating. Same phenomenon. The detergent will replace the coating protein, slowly but surly. Store the plates frozen and wash them immediately only before using!!!!

To optimize the solid phase antibody concentration go for a checkerboard titration. Stat with 0.1 µg/mL antibody concentration, 2 columns, double the concentration for the next two columns ...

Use two samples, one highly positive, one negative, dilute them from one factor less then you are currently using up to the 8 steps for the different rows.

Incubate better for 90 min. Use if available multichannel pipettes to avoid, that the first sample will incubate much longer than the last due to the time shift during pipetting.

Do all the other incubation steps (you can do them all with 100 µL including stopping or if you need to save material, the ELISA will work also as a 50 µL assay for all steps.

In the evaluation compare per solid phase concentration the Positive/Negative-Ratio and search for the optimum under the condition, that the maximal OD is not higher than 2.0. Due to the Lambert-Beer-Low, measurements higher than 2.0 getting more and more imprecise. (OD is the reciprocal value of the transmission!).

Put the graph of your evaluation here and I will comment on this.

Yesterday I tested the different blocking buffers and increased the blocking time to two hours on my standards. I also tested putting 0,05%Tween in my incubation buffers.. The blocking buffers I tested were:

- BSA 5% for 1 hour (how i normally do it)

- BSA 5% for two hours

- Casiene for two hours

- Protivar 5% for two hours

- Protivar 3% for two hours

And i had a row which i used 1%BSA 1xPBS 0,05%Tween as incubation buffer to see the difference between what i normally do.

My results show that 5%BSA for 1 hour is still the best.. 5%BSA for two hours decreased my signal a little.

Casiene & protifar decreased my signal very much.

Tween in the incubation buffer decreased my blanc a little and increased my signal a little so that was good.

I don't know what to do to decrease my blanc further.. and still the weird results from my samples (which were almost the same value as my blanc no mather wich dilution i used)

Thankyou Stephan for your suggestion, I will think about it and see if I can test it :)

Any other suggestions or tips are still very welcome!

you didn't mention in what buffer you used to dilute the conjugate!!

you can use now these new conditions and try to test your conjugate diluitions but in TBS-T + BSA 1%,

Sorry, normally i diluted all the antibodies, so also my detection poly-HRP antibody in 1%BSA 1xPBS.

Yesterday i used 1%BSA 1xPBS + 0,05% Tween. That decreased my blanc a little and it increased my signal from the standards a little so i think i will use Tween in my incubation buffers from now on :).

my last option to reduce the blank backgound would be to reduce the conjugate concentration from 1:3000. to 1:4000 in TBS-T + BSA 1%.

I did that yesterday and it did decrease the blank.. I compared results from: 1:3000, 1:4000 and 1:5000, but my signal from the standards from 1:4000 and 1:5000 decreased almost half the value of which i got from 1:3000.

Tomorrow I'm going to use a couple of different buffers (TBS, HEPES & SSC) en different ELISA plates (Greiner high binding, Corning medium binding & Corning high binding). See if there's any difference between that and what i normally do: PBS buffer and Greiner medium binding plate.

Hi!

I already did some experiment like that, and I used the Costar (now Corning) flat High binder at room temperature , but with a lot of blocking, in my case casein, and for a low backgound I use the Sterilin flat microplate, I hope this help!

Hi Linda,

high binding plates causing often high backgrounds, despite of blocking. Use normal ELISA plates. The high binding plates are only required for very small proteins, peptides, very hydrophilic proteins etc.

Stephan- Could you please provide reference (source) for normal ELISA plates. 

Hi, 

I am getting similar problem as Linda's in my Antisoy trypsin inhibitor ELISA but I managed to reduce the background by changing the blocking solution from PBS-BSA to StartlingBlock blocking buffer but I can't get my standard curve right and my initial concentration of the Soy antigen is 1mg/mL diluted 10-1 to 10-6 and I am using a crude soy product could this be a reason? any thought??

Would anyone please explain why Stephan's suggestion is getting downvotes?

 Hi Vineeta,

the plates from any manufacturer works (Greiner, Nunc, ...) whatever you want. For commercial assays, I used often Nunc-Plates.