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ELISA peptide coating problem =(


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#1 edilberto ojeda

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Posted 14 July 2010 - 09:11 AM

I dont know what to do to bound the peptide to the plates.
I have a problem coating the ELISA's plates with the antibody that I used to vaccinate my rodents. What happens is when I coat the plate with the peptide, it doesn't bound to the plate because when I am going to read the plates are just clear as they can be, so it shows that there is not any immune response from my samples.
I tried to coat the plates with different concentrations of peptide: 15g/ml, 30 g/ml, 60 g/ml, and 90 g/ml with different concentrations of blocking solution (PBST-Milk): Milk 2%, 1.5%, 1%, y 0.5% but my peptide didnt bind to the plate.
I thought that it was because the rodents didnt have any immune response against the peptide but they did have immune response but the response showed up when I did the ELISA only using the serum to coat the plate instead of coating first with my peptide.
What I would like to know is if that response was specific due to peptide because what I know now is that I have response to the vaccine but I dont know if that response is specific to the peptide, that is why I would like to bind the peptide first to the plates.
The plates I am using are the F96 MAXISORP NUNC-IMMUNO PLATE
The peptide weighs 985.03 g/mol with 9 amino acids
First, I coat the plate with the peptide and PBS pH 7.4 overnight at 4C then I wash the plates with PBSTM 2% three times, then I coat the plate with my serum (serum + PBSTM) at different concentrations. I leave the plate 1 hr at RT then wash them with PBSTM. I coat the plate with IgG peroxidase (IgG peroxidase + PBSTM). I leave it 1 hr at RT. I wash the plates again and I add phosphate citrate solution + H2O2 and I put the plates in incubation at 37C during 20 min. What I have are clear plates =(.

Thank you. I hope you can help me

#2 sgt4boston

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Posted 14 July 2010 - 09:29 AM

It may be that your peptide does not bind/stick to the surface of the wells. I have coated proteins > 50K in size. It may also be that when you block the peptide gets masked by the larger proteins such as milk and serum. I am also unsure of your sequence to making the plates?

It should be
1. coat with protein of interest**
2. wash PBS
3. BLOCK with BSA or Milk/Casein or other blocking agent
4. wash PBS/surfactant
5. Test

Test:
1. Add serum samples +/- specific antibody (not coat?)
2. Wash several times
3. Probe with antibody-conjugate
4. Wash.
5. Substrate
** I would explore coupling the peptide to carrier protein and passively adsorbing that to your plates

#3 K.B.

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Posted 14 July 2010 - 10:34 AM

Conjugate peptide with carrier protein (different from carrier protein you used for immunisation) and use it for coating.

#4 edilberto ojeda

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Posted 15 July 2010 - 12:30 AM

Thanks for the information but I need a little extra help =). I would like to coupling the peptide to a carrier protein like you said but I dont have any idea on how to conjugate it and which carrier protein I have to use to do so. Sorry but we dont have a lot of experience doing ELISAS. .
This is the peptide WI-8 WEPDDNPI
Thanks guys.
I really appreciate it =)

#5 K.B.

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Posted 15 July 2010 - 03:08 AM

I'm not an expert on this specific topic, but from what I've heard from my friend, if you fail to obtain immunization with (free) peptide, you need to conjugate it with carrier protein eg. BSA, OVA, KLH. If you fail to get signal on ELISA then you need conjugate of your peptide with different protein for coating.

For conjugation and ELISA - check out:
http://mitchison.med...tocols/ab1.html
http://mitchison.med...tocols/ab2.html

#6 klinmed

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Posted 15 July 2010 - 04:28 AM

I dont know what to do to bound the peptide to the plates.
I have a problem coating the ELISA's plates with the antibody that I used to vaccinate my rodents. What happens is when I coat the plate with the peptide, it doesn't bound to the plate because when I am going to read the plates are just clear as they can be, so it shows that there is not any immune response from my samples.
I tried to coat the plates with different concentrations of peptide: 15g/ml, 30 g/ml, 60 g/ml, and 90 g/ml with different concentrations of blocking solution (PBST-Milk): Milk 2%, 1.5%, 1%, y 0.5% but my peptide didnt bind to the plate.
I thought that it was because the rodents didnt have any immune response against the peptide but they did have immune response but the response showed up when I did the ELISA only using the serum to coat the plate instead of coating first with my peptide.
What I would like to know is if that response was specific due to peptide because what I know now is that I have response to the vaccine but I dont know if that response is specific to the peptide, that is why I would like to bind the peptide first to the plates.
The plates I am using are the F96 MAXISORP NUNC-IMMUNO PLATE
The peptide weighs 985.03 g/mol with 9 amino acids
First, I coat the plate with the peptide and PBS pH 7.4 overnight at 4C then I wash the plates with PBSTM 2% three times, then I coat the plate with my serum (serum + PBSTM) at different concentrations. I leave the plate 1 hr at RT then wash them with PBSTM. I coat the plate with IgG peroxidase (IgG peroxidase + PBSTM). I leave it 1 hr at RT. I wash the plates again and I add phosphate citrate solution + H2O2 and I put the plates in incubation at 37C during 20 min. What I have are clear plates =(.

Thank you. I hope you can help me

It can be very problematic to get such small peptides to bind to plastic surfaces. Sometimes impossible.

I have found a very useful method is to get the peptide synthesized with a biotin tag (most companies do this). You can then bind it by incubation on streptavidin coated microplates.
You could also reverse your assay by 1) coating plates with anti-mouse Ab,washing 2) incubating with test sera, wash, 3) incubating with peptide-biotin, wash 4) detect with streptavidin-HRP.

A less elegant way is to get synthesized a peptide-carrier protein conjugate (different carrier protein to that in the immunogen). The carrier protein will then facilitate peptide immobilization on the plates.

Hope this helps.

#7 braincow

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Posted 15 July 2010 - 07:18 AM

You could try different coating buffers. Proteins bind to the plate by non-covalent interactions. It may be that PBS pH 7.4 may result in your peptide being neutrally charged, and thus, weakly interacting with the binding surface.

You could try carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), which works well for me. Or you could try an acidic buffer, like citric acid buffer.

#8 edilberto ojeda

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Posted 12 August 2010 - 01:41 AM

Hi,
After trying the suggestions that you told me, Im still working on getting the right technique for my ELISA test. I dont know what to do. Here are the techniques that Ive been using to couple the BSA to my peptide and the technique that I used for the ELISA technique is at the bottom. If somebody can tell me what I am doing wrong, please let me know because I cant figure it out by myself.
Thanks for all the help that youve given me.

Coupling peptide to BSA (Peptide used WI-8 WEPDDNPI PM:985.03 g/mol)

Technique 1
1 ml of 0.1 M NaHCO3 + 2mg BSA
1 ml of 0.2% glutaraldehyde
0.5 mg peptide/0.5 ml DMSO
1. Mix, adding glutaraldehyde last.
2. Incubate 90 at 37 C
3. Add 250 l of 0.1M NaBH4. After 15 add another 250 l of 0.1M NaBH4

Technique 2
1 mg of peptide + 10 l of PBS pH7.4 (I used the formula for a 40-fold excess of peptide for BSA)
1.69 mg of BSA + 1.69 ml of PBS
1. Mix and cool the solution 2-6 C. Add a stir bar and stir at medium speed.
2. Add 1.69 ml of 2% glutaraldehyde drop by drop
3. Stir the mixture while maintaining the temperature at 2-6 C for one hour.
4. Stop the reaction by adding 6.76 mg of NaBH4

After using technique 1 or 2 to coupling the peptide I followed the next technique to prepare the plates
1. I coated the plate with different concentrations of peptide (10, 15, 20, 25, 30, 35 and 40 g/ml PBS for both techniques)
2. I left the plates over night at 4 C
3. The next day, I washed the plates 3 times with PBST (0.05% Tween 20)
4. Blocking with PSBTM (2% milk) 200 l each well
5. I left the plates 1 hr at RT
6. While the wells were blocking I prepared the serum solutions
a This applies to each sample of serum
b 250 l of PBSTM to the first well and 125 l to the rest of them
c I added 2.5 l of serum to the first well
d I mixed it well and then I made dilutions 1:1
7. After 1 hr of blocking, I washed the plates 3 times with PBST and I added the dilutions to the plates (100 l to each well)
8. I left the plates 1 hr RT
9. I washed the plates 3 times with PBST
10. I prepared the conjugate anti mouse IgG peroxidase with PBSTM (1:1000)
11. I added 100 l of conjugate to each well
12. I left the plates 1 hr TR
a I prepared tampon 0.05 M phosphate-citrate (56:44) to prepare substrate solution
i. Na2HPO4 anhydrous 365 mg
ii. Citric acid anhydrous 215 mg
iii. qs 50 ml of H2O
iv. 1 tablet of ABTS
v. 12.5 l of H2O2 right before adding it to the wells
13. I washed the plates 4 times
14. I added 100 l of substrate solution to each well
15. I left the plates incubating at 37 C 20 min
16. I read in 405 nm




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