Hi Yves, yes you can forget the blocking. It's only really required for commercial assays which need to be stored at room temperature for guaranteed longer times (6 month up to three years). In this case you need some protein to stabilize the secondary structure of the antibody on the solid phase. Also some sugar is required (1 ... 3% mannose and/or glucose) to bind some water. In ELISA plates which are used within a short time, coating without blocking is sufficient. In more than 400 ELISA I validated, I couldn't improve any of the important ELISA quality parameters. For sure not the detection limit, but also not the imprecision. The detergent in the washing buffer has the major function to bind with it's hydrophobic end to the polystyrol rings at the solid phase. It happens with an very high affinity in an very short time. Just an simple experiment: Put an serial dilution of your detergent (start with 0.1% Tween 20) on a naked plate just for some minutes .... and try to bind your antigen/antibody on the solid phase in a second step after washing with PBS.

The blocking will have no impact on the immune reaction. If you have a "low affinity" antibody (especially monoclonal antibody), you have to have longer incubation times, the detection limit depends very much on the affinity (law of mass action), the upper detection limit as well. The additional problem with monoclonals is their multirecativity (also in commercial antibodies). You should check them by using different antigens on the solid phase. ssDNA or other repetitive antigens like PRP or cytokeratins, collage are very often recognized by those antibodies (Kießig, ST, Jahn, S, Hiepe, F, Volk, H-D, Fietze, E, Zugehör, M, Porstmann, T, v.Baehr, R (1990) Multireactive human monoclonal antibodies. Allerg Immunol 36:163-177).

About washing: I've forgot to add "three times" in total 5 minutes. Washing in ELISA is nothing else than to dilute as much as possible. It's done by removing as much as possible and filling as much as possible volume for washing (e.i. 10 µL residual volume of the sample, 250 µL for washing = 1:25, second washing 1:625, third washing 1:15625). Some shaking could be also helpful, 4 times washing as well.

Dear rafael,

first of all,I think your protocol has some problem,

- your coating condition and its time is good . but your blocking time is very long and unusual .60 min or 120 min in 37 C with BSA 1% or gelatin 1% usually give good result.

on the other hand,in your protocol reaction time for primary antibody is so long,because antibody - antigen reaction is reversible and will equilibrate in some minute(based on antibody affinity) and 60 min or 90 min for most antibodies are enough

after that, what is exactly your problem?Do you have problem with false positive or background color?your test sensitivity is low?

do you have low OD for positive control?

I have low OD in a positive control that is coated with a protein , but the positive control where it is coated with a small peptide is OK. I'm wondering if, as the small peptide is more stable, it could last longer than the protein.

Hello everybody!

I am totally new at ELISA and I was wondering if I have to do setup before running all the samples?

I mean how would I know the appropriate dilution of primary antibody?

Also, I use serum as sample so, does it need to be diluted or use it directly?

I really appreciate it if you help me out!

Thanks in advance!

Mostafa

Thank you very much Yves for your kind answer.

Do I have to work triplicate? so a kit with 96 stripes can be used just for less than 25 samples :-( beside setting up the serum dilution, right?

on the sheet it recommended a distinct dilution for primary AB. so It is just serum dilution to find.

Would it be Ok if I work duplicate? would the data be still significant?

I would like to get to know any thing I could about ELISA, so if It is ok with you, please help me out with these subjects:

When do I have false positive or background color?

When does the test sensitivity is low?

why low OD may happen for positive control?

and if any trouble may happen please add yourself, you know, because ELISA is very an expensive test for us , so I can not afford to let anything goes wrong.

Thank you very muchhhhhh.

I ll look forward to your answer.

Mostafa

Hi Mostafa, if you mean technical triplicate (assaying the same sample three times) instead of biological triplicate (three different samples belonging to the same treatment), then I'd say a duplicate is enough. In my experience and my personal opinion, I find more useful to assay six samples from the same group (not even in duplicates) and not two samples in triplicates. I've done mostly ELISA of conditioned media of cancer cell lines, and some of mice sera.

Hope it helps.

Kind regards,

Mariano

Thank you very much my friends for sharing your experiences with me.

I wish you all luck in your research.

Sincerely

Mostafa

Hi Rafael,

the low of mass action is also valid for ELISA. If you have an specific antibody (in your sample) and a normal affinity (higher than 10Exp10 mol/L) that the immune reaction depends only on the diffusion of the analyte to the solid phase. That's normally done within 10 - 15 min. But normally incubation times are adjusted to 60 or 90 min. That's only due to have sufficient time to fill and empty the plate and to avoid (incubation-)time depended errors.

Immune reaction running always better at room temperature. There is no reason the incubate the immune reactions longer than 90 min. Only in very rare cases it's can help to increase the sensitivity (decrease the detection limit), but this will increase all unspecific reaction as well.

and something else to safe time: Forget the blocking. You are coating the plates overnight, thats fine, remove the liquid, store the plates frozen until use. Before you start the incubation with your analyte, just wash the plates three times with the washing buffer (e.i. PBS, 0.1% Tween 20 v/v). The detergent will bind to any free hydrophobic free binding site at your plate/solid phase.

Summary:

1. Coating overnight at 4 °C

2. washing 5 min

3. Incubation: 60 - 90 min at 25 °C

4. washing 5 min

5. second incubation 60 - 90 min

6. washing 5 min

7. substrate reaction 10 - 20 min

8. stop

9. measure

Just one last remark: The higher the affinity of your antibodies, the shorter the incubation steps can be. Now you need only a method the fill your plate in very short times (e.i. by using multichannel pipettes).

Hi Yves, yes you can forget the blocking. It's only really required for commercial assays which need to be stored at room temperature for guaranteed longer times (6 month up to three years). In this case you need some protein to stabilize the secondary structure of the antibody on the solid phase. Also some sugar is required (1 ... 3% mannose and/or glucose) to bind some water. In ELISA plates which are used within a short time, coating without blocking is sufficient. In more than 400 ELISA I validated, I couldn't improve any of the important ELISA quality parameters. For sure not the detection limit, but also not the imprecision. The detergent in the washing buffer has the major function to bind with it's hydrophobic end to the polystyrol rings at the solid phase. It happens with an very high affinity in an very short time. Just an simple experiment: Put an serial dilution of your detergent (start with 0.1% Tween 20) on a naked plate just for some minutes .... and try to bind your antigen/antibody on the solid phase in a second step after washing with PBS.

The blocking will have no impact on the immune reaction. If you have a "low affinity" antibody (especially monoclonal antibody), you have to have longer incubation times, the detection limit depends very much on the affinity (law of mass action), the upper detection limit as well. The additional problem with monoclonals is their multirecativity (also in commercial antibodies). You should check them by using different antigens on the solid phase. ssDNA or other repetitive antigens like PRP or cytokeratins, collage are very often recognized by those antibodies (Kießig, ST, Jahn, S, Hiepe, F, Volk, H-D, Fietze, E, Zugehör, M, Porstmann, T, v.Baehr, R (1990) Multireactive human monoclonal antibodies. Allerg Immunol 36:163-177).

About washing: I've forgot to add "three times" in total 5 minutes. Washing in ELISA is nothing else than to dilute as much as possible. It's done by removing as much as possible and filling as much as possible volume for washing (e.i. 10 µL residual volume of the sample, 250 µL for washing = 1:25, second washing 1:625, third washing 1:15625). Some shaking could be also helpful, 4 times washing as well.