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
What you would like to detect? The specific antibodies in a serum sample?
1. forget the Maxisorp plates, all other plates are working as well and in most cases the background is lower.
2. Coating: 100µl is fine, but incubate overnight at 4 °C in your carbonate buffer. Cover the stack of plates with an empty plate. but: use 2 µg/mL only, not 2 mg!!!!!!!!
3. Washing is fine but not before using. If you would like to store the coated plates, remove as much from the coating solution and store the plates bottom up frozen.
4. Blocking: forget this, blocking is required in commercial assays only. Your plate has a binding capacity of about 100 ng/cm², so you are coating with an excess of protein anyhow. If you need really a blocking, make sure that there are no other proteins in the blocking buffer reacting with your coating protein. Beware only: fetuin is a very sticky protein.
5. Sample (serum/plasma containing the specific antibodies????) incubation buffer: use your washing buffer add NaCl to a concentration of 0.3 mol/L (if necessary you can use up to 0.5 mol/L) This doubled NaCl-concentration will l reduce unspecific binding. Add an inert protein like BSA at a concentration of 1 - 3%. Add phenol red (a pitch) only to get a slightly colored buffer. So will see much better where you are pipetting. Incubate at room temperature. Anything else will lead to edge-effects due to temperature / himidity grafdients over the plate.
Use an empty plate to prepare your samples and standards & controls. Don't do the dilution of those in the coated plates. You will get scratches at your coated surface and you will have different incubation times for the different wells. If I would like to run a 100 µL-assay, I used 150 µL - 250 µL as volume for the sample preparation.
6. Washing is fine.
and now I've a issue: just now the anti-pertussis-antibody?
7. conjugate incubation (anti-species-IgG-Antibody-HRP labelled (anti-human-IgG-HRP if you would like to detect human antibodies, Protein-A/G-HRP if you like to detect antibodies from rodents....) Your polyclonal anti NIBSC Bordetella pertussis PT serum (sheep) can be used as standard, but due to the fact that it is from sheep, you need to have an anti-Sheep-IgG-HRP. This requires also that all your samples are from sheeps?
8. Washing
9. substrate reaction: TMB, H2O2 (use an ready to use solution: http://www.seramun.com/products/substrates/horseradish-peroxidase.html
12. Color development: Incubate for 20 min at room temperature.
13. Stop: sure you can kill the peroxidase by adding that much H2O2, but a 1 mol/L H2SO4 is much easier and cheaper.
14. Read at 450 nm is fine.
Have a short lock for the ELISA schemes in the attached paper ... and send me an information via RG.
Article Enzyme Immunoassay Techniques. An Overview
Thank you very much for your help Stephan Kiessig, what we are doing is trying to quantify fetuin, but we are having problems with background. Regards
Sorry Stephan Kiessig what we want to detect is pertussis toxin, no fetuin, fetuin is used only to covering.
Than you still can stick with a couple of points:
5. Sample incubation buffer: as describes above
7. anti-pertussis-IgG incubation: a normal concentration for such an antibody is about 1 µg/ml if purified or monoclonal. Here you have to check for an optimal dilution by checkerboard titration.
8. Washing
9. substrate reaction: see above
12. Color development: see above
13. Stop: 1 mol/L H2SO4.
14. Read at 450 nm is fine.
some alternatives:
optimize the coating concentration. you are about 1000 time to high! checkerboard titration.
use for coating a monoclonal antibody vs. pertussis toxin line in a classical two site binding assay.
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