HI,

1. coating as normally done with the specific antibody in 0.1 mol/L carbonate buffer pH 9.6 at 1 ... 10 µg/mL overnight at 4 °C. Do a checker bord titration to find the optimal coating concentration (look optimal signal/noise ratio or positive/negative ratio)

2. removal of all liquid

3. store the plates upside down until use

4. Wash the plate with PBS 0.1% v/v Tween 20 3 - 4 times

5. use as sample incubation buffer an PBS with doubled NaCl-Concentration (0,3 mol/L), add some inert protein like BSA (1 - 3% w/v), add some phenol-red 0,1 mg/L (to see better where you are pipetting). Dilute your samples for the checker board titration (in the second dimension)

6. Incubate (normally 60 - 120 min, 90 min is optimal). Why: you need always some time to fill the plate wells, to empty them is very fast. The antigen-antibody binding is due to the high affinity done within minutes, but if you are acting to short you will see gradients on your plates due to the different times shifts for the pipetting steps. Sometimes it's heplful to use for the dilution a separate plate and to start the assay you have to transfer the samples by multichannel pipette.

7. wash

8 incubate with the second antibody. Optimize the concentration in a second experiment also by checker board titration

8. wash

9. substrate reaction

10. stop

11. measure

The increase of the NaCl could reach up to 0,5 mol/L. But pertussis toxin is a very sticky protein.

Dear: Stephan Kiessig thank you very much for your wise advices, I'll folow it.

I'm still having problems with the ELISA of B. pertussis, to be more specific here I leave some of them.

General problem:

-High background

**Specific problems with high background:

Conjugate (AcM anti-IgG) is joining the coating (fetuin)

Protocol

1. Microtiterplaten Nunc Maxi Sorp (F96 of C96) nieuw (Thermo Scientific,Denmark,cod 439454)

2. Coating: Add 100µl per well of coating solution, incubate for 1 hour at 35oC in a humid chamber.

* Coating solution: fetuin 2mg/ml in 0.1 M Na2CO3 buffer, pH 9.6, incubate 1 hour at 35oC and humid environment.

3. Washing: Wash the plate 4 times with washing buffer.

* Wash buffer: PBS-T 0.05

4. Blocking: Add 150μl per well of blocking solution and incubate for 1 hour at 350C in a humid chamber.

* Blocking solution: 3% skim milk.

5. Application of samples and standard: Incubate for 1 hour at 350C in humid chamber.

* Analyte SN-PRN. Add 100μl per well of a dilution of SN-PT in PBS-T

* Standard Curve. Add 200 ul of standard 1.46 ul / ml PT, first point of the curve. Add to the other wells in that column 100l of PBS-T, serial dilutions 1:2 to the seventh well in the same PBS-T. Antigen using PT.

* Preparation of standard: 0.75 ul standard PT in 200 ul PBS-T

6. Wash the plate 4 times with washing buffer.

7. Primary antibody. Add 100ul per well of polyclonal anti NIBSC Bordetella pertussis PT serum (sheep) (97/572) at a 1:10,000 dilution in PBS-T. Incubate for one hour at 350C in a humid chamber.

* PT 1.2 ul anti 12ml serum in PBS-T

8. Wash the plate 4 times with washing buffer.

9. Conjugate: Add 100ul per well of mAb conjugated sheep anti-IgG peroxidase diluted 1:10 000 in PBS-T-milk 3%. Incubate for 1 hour at 350C in a humid chamber.

* Conjugate: Mix 1.1 ul of conjugate + 11ml milk-PBS-T 3%

10. Wash the plate 4 times with washing buffer.

11. Revealing: Add 100ul per well of developing solution

* Development solution: 10.8 ml of H2O Ac +1.2 ml of 1.1 M pH 5.5 + 0.2 ml of TMB 10mg/ml + 2.4 ul of 30% H2O2.

12. Color development: Incubate for 20 min at room temperature.

13. Detection of the reaction: Add 50 ul of H2O2 2M

* Stop Solution: 80ml of distilled H2O + 10ml of concentrated H2SO4 (98%, density 1.83, about 18M).

14. Read absorbacia at 450nm

I'm still having problems with the ELISA of B. pertussis, to be more specific here I leave some of them.

General problem:

-High background

**Specific problems with high background:

Conjugate (AcM anti-IgG) is joining the coating (fetuin)

Protocol

1. Microtiterplaten Nunc Maxi Sorp (F96 of C96) nieuw (Thermo Scientific,Denmark,cod 439454)

2. Coating: Add 100µl per well of coating solution, incubate for 1 hour at 35oC in a humid chamber.

* Coating solution: fetuin 2mg/ml in 0.1 M Na2CO3 buffer, pH 9.6, incubate 1 hour at 35oC and humid environment.

3. Washing: Wash the plate 4 times with washing buffer.

* Wash buffer: PBS-T 0.05

4. Blocking: Add 150μl per well of blocking solution and incubate for 1 hour at 350C in a humid chamber.

* Blocking solution: 3% skim milk.

5. Application of samples and standard: Incubate for 1 hour at 350C in humid chamber.

* Analyte SN-PRN. Add 100μl per well of a dilution of SN-PT in PBS-T

* Standard Curve. Add 200 ul of standard 1.46 ul / ml PT, first point of the curve. Add to the other wells in that column 100l of PBS-T, serial dilutions 1:2 to the seventh well in the same PBS-T. Antigen using PT.

* Preparation of standard: 0.75 ul standard PT in 200 ul PBS-T

6. Wash the plate 4 times with washing buffer.

7. Primary antibody. Add 100ul per well of polyclonal anti NIBSC Bordetella pertussis PT serum (sheep) (97/572) at a 1:10,000 dilution in PBS-T. Incubate for one hour at 350C in a humid chamber.

* PT 1.2 ul anti 12ml serum in PBS-T

8. Wash the plate 4 times with washing buffer.

9. Conjugate: Add 100ul per well of mAb conjugated sheep anti-IgG peroxidase diluted 1:10 000 in PBS-T-milk 3%. Incubate for 1 hour at 350C in a humid chamber.

* Conjugate: Mix 1.1 ul of conjugate + 11ml milk-PBS-T 3%

10. Wash the plate 4 times with washing buffer.

11. Revealing: Add 100ul per well of developing solution

* Development solution: 10.8 ml of H2O Ac +1.2 ml of 1.1 M pH 5.5 + 0.2 ml of TMB 10mg/ml + 2.4 ul of 30% H2O2.

12. Color development: Incubate for 20 min at room temperature.

13. Detection of the reaction: Add 50 ul of H2O2 2M

* Stop Solution: 80ml of distilled H2O + 10ml of concentrated H2SO4 (98%, density 1.83, about 18M).

14. Read absorbacia at 450nm