1. Coating: 1 - 10 µg/mL in a 0.1 mol/L carbonate buffer pH 9.6, overnicht at 4 °C

2. Remove your coating, store the plates upside down frozen until use. Do not block the plates, it's a kind of superstition

3. Washing 3 times: PBS (0.01 mol/L Phosphate pH 7.2, 0,3 mol/L NaCl, the increases NaCl will reduce unspecific binding) + 0.1% Tween 20. This detergent will block any free binding site

4. Incubation with sample, standards and controls in PBS 0.1% Tween + 3% BSA (of hydrolysed gelatine which shows less problems), some phenol red will show you where you are pipetting. 90 min, room temperature

5. washing 3 times

6. inconation with your labelled antibody in PBS, Tween, BSA Phenol red (normal concentration is at 1 µg/mL), 90 min, room temperature

7. washing 3 times

8 substrate reaction (TMB, H2O2) ready to use: http://www.seramun.com/products/substrates/horseradish-peroxidase.html. incubate for 20 min

9. stop: 1 mol/L H2SO4

10 measurement at 490 nm.

When you start, you need a sample where your analyte is in. Start with a chekcer board titration: antibody concentration at the solid phase between 1 µg/mL an max 10 µg/mL. Antigen concentration should be below 10 µg/mL at the first dilution. Dilute by factor 3 to get a larger range in the first experiments.

Leave the 90 min for incubation, this avoid time gradients over the plates, Make sure that all buffers are at room temperature to avoid temperature gradients.

If possible, use a chromogenic substrate, so you can follow the reaction visually. Other substrates didn't improve the test performance (detection limit). The detection limit depends (according to the law of mass action) only on the affinity of the first antibody. 

https://www.piercenet.com/files/TR0065-ELISA-guide.pdf

Dear Mam

I am having HRP-linked Antibody and ;-Actin (13E5) Rabbit mAb and Pierce ECL  Substrate that is a peroxidase substrate for enhanced chemiluminescence (ECL)

will it work for elisa systme? ijsut need stop silution and blocking solution additionally/ right

could you please add up if any thig more is required just want to be sure before i start up the standarization

1. Coating: 1 - 10 µg/mL in a 0.1 mol/L carbonate buffer pH 9.6, overnicht at 4 °C

2. Remove your coating, store the plates upside down frozen until use. Do not block the plates, it's a kind of superstition

3. Washing 3 times: PBS (0.01 mol/L Phosphate pH 7.2, 0,3 mol/L NaCl, the increases NaCl will reduce unspecific binding) + 0.1% Tween 20. This detergent will block any free binding site

4. Incubation with sample, standards and controls in PBS 0.1% Tween + 3% BSA (of hydrolysed gelatine which shows less problems), some phenol red will show you where you are pipetting. 90 min, room temperature

5. washing 3 times

6. inconation with your labelled antibody in PBS, Tween, BSA Phenol red (normal concentration is at 1 µg/mL), 90 min, room temperature

7. washing 3 times

8 substrate reaction (TMB, H2O2) ready to use: http://www.seramun.com/products/substrates/horseradish-peroxidase.html. incubate for 20 min

9. stop: 1 mol/L H2SO4

10 measurement at 490 nm.

When you start, you need a sample where your analyte is in. Start with a chekcer board titration: antibody concentration at the solid phase between 1 µg/mL an max 10 µg/mL. Antigen concentration should be below 10 µg/mL at the first dilution. Dilute by factor 3 to get a larger range in the first experiments.

Leave the 90 min for incubation, this avoid time gradients over the plates, Make sure that all buffers are at room temperature to avoid temperature gradients.

If possible, use a chromogenic substrate, so you can follow the reaction visually. Other substrates didn't improve the test performance (detection limit). The detection limit depends (according to the law of mass action) only on the affinity of the first antibody.