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ELISA development for prg4/lubricin

ELISA development for prg4/lubricin - ELISA Assay Forum

ELISA development for prg4/lubricin - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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Old 02-13-2011, 03:40 AM
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Default ELISA development for prg4/lubricin



Hi,
I have been trying to develop a sandwich ELISA to measure human lubricin/prg4 concentrations. However, so far, I have been unable to detect any specific antigen binding, just what appears to be a lot of background signal. I am coating R&D ELISA plates with a C-terminal epitope rabbit polyclonal IgG in PBS at 0.5 and 2 ug/mL. Then blocking with 5% Tween 20 in PBS. I am then adding lubricin in PBS at several standard concentrations ranging from 0 to 100 ng/mL for 2 hours. Then adding a N-terminal epitope goat polyclonal IgG at 0.4 and 1.6 ug/mL, in the R&D reagent diluent formulation for 2 hours. Then adding a HRP conjugated mouse anti-goat IgG in diluent for 30 minutes at the same concentration as the previous promary detection antibody. Then standard substrate and stopping. I am washing thrice with the R&D wash buffer between each step. I end up with no differentiation between any of the lubricin concentrations but an OD which increases with antibody concentration (both capture and detection). If anyone could shed any light on this issue it would be very much appreciated. Thanks in advance.
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Old 02-13-2011, 04:18 AM
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Default Re: ELISA development for prg4/lubricin

First off you aren't blocking. 5% Tween20 in PBS won't do that you need non-fat dry milk, casein, albumin etc to block---Try Superblock or StartingBlock from Pierce.

Foolproof way to get a sandwich ELISA (for me) is:
1. High quality polystyrene plate.
2. Carbonate/Bicarbonate buffer pH 9.0 (Pierce BupH packs are convenient)
3. Coat capture antibody at 10ug/ml diluted in Carb/Bicarb buffer overnight at 4 deg C.
4. Block with StartingBlock incubate 15 min to 30min.
5. Wash with TBS-0.2%Tween20.
6. Add antigen (range for pure rec. protein standard 6-300ng per well); Temps can be 4C, room temp, 37C--protein dependent.
7. Incubate from 2 hours to Overnight, you pick.
8. Wash TBS-T.
9. Add detector antibody at 4ug/ml. Incubate 30-60 minutes at 37C.
10. Wash w/ TBS-T.
11. Add HRP or AP conjugate antibody at concentration recommended by manufacturer for ELISA (usually 1:40K to 1:60K dilution), incubate 37C 30min.
12. Wash TBS-T
13. Add color development solution. wait for color etc. Read at appropriate wavelength, subtract background.

Last edited by danfive; 02-13-2011 at 04:24 AM.
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Old 02-13-2011, 04:22 AM
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Default Re: ELISA development for prg4/lubricin

My method is foolproof. I use a lot of Pierce products, and go through a lot of antibody as well.

But once you have a working ELISA, you can optimize and lower antibody concentration, incubation times and temps etc.

I've made tons of ELISAs, let me know if you need more help.
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Old 02-13-2011, 04:45 AM
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Default Re: ELISA development for prg4/lubricin

Thanks so much for the help. I will try that this week. But just out of curiosity, why would a higher capture antibody concentration lead to a higher signal, independent of the antigen concentration. Could that mean that the secondary is binding to the capture as well?
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Old 02-13-2011, 04:54 AM
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Smile Re: ELISA development for prg4/lubricin

Quote:
Originally Posted by iceman967 View Post
Thanks so much for the help. I will try that this week. But just out of curiosity, why would a higher capture antibody concentration lead to a higher signal, independent of the antigen concentration. Could that mean that the secondary is binding to the capture as well?
Higher capture antibody is used to coat the wells and leave as little empty space available for non-specific binding.

Your assay is sound but you really need excellent blocking (and a working antibody pair and positive control protein) to get good results.

Good blocking will come easier by coating the well with a lot of capture antibody and then blocking with a great blocking solution.
(both the 10ug/ml concentration and startingblock recommendations are to save you time from optimizing one variable/step at a time)

Secondary antibody won't bind to the capture antibody, but it could (and may currently be doing so) bind to empty polystyrene space (polystyrene naturally and strongly binds protein) thus leading to false signal; which you'll see in the "blanks or neg ctrls".
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