I need help with my ELISA

Bryan Kiehl b3748 at cts.com
Wed Feb 12 22:01:51 EST 1997


There are many references, but the following is simple and should work
in most cases.

Bind either antibody or antigen to standard microtiter plate in PBS or
Carbonate buffer. (100 ul) Do not add any other protein or
preservative. Vary concentrations to test dilution of material.

You have a choice between 1-4 hours at 30-37C or overnight at 4C. The
overnight will yield about one-third better binding in most cases.

You can proceed directly to sample here. If you want to store the
plates, remove the material and add 0.5-3% BSA in PBS. Let stand at
room temperature for 30-60 minutes. Remove and let dry (room temp or
30-37C incubator).

Add 100 ul sample in PBS with 0.5% BSA. (The BSA may vary between FCS
(10-20% or various other blockers). For serology (antigen on plate)
BSA is usually fine. For direct antigen capture methods, the correct
blocker is assay specific and can be critical. In addition, treatment
os sample to expose antigen may also be critical (e.g., Chlamydia or
Streptococcus organism extraction). For serology, incubate 30-60
minutes (IgM longer). For direct antigen, you need to test incubation
time. During early development, I usually start at 4 hours or
overnight to assure detection and when working, test shorter times.

Wash with PBS and tween (standard). In a few cases, you may find tween
to interfer.

Either peroxidase or alkaline phosphatase conjuagates are standard and
either can be made to work. The diluent is usually a derivitive of the
specimen dilutent. For serology, commercial IgM or IgG conjugates can
usually be used at 4000 to 15000 dilutions. If you have a specific
conjugate for a direct antigen test, the quality of the antibody and
conjugation will dictate dilutions. If made to the same quality as
commercial conjugates, the same dilutions should hold. Incubation
times are also similar the patient sample times.

Develop color using standard, commercial reagents. In most cases this
will take 30 minutes.

When you are first developing the assay, go for the longest assay time
to assure detection and be sure to test several negatives as well as
one or two positives. The negatives assure that you optimize for
signal/noise. You can never have too many negative controls. After you
have a working assay, you can then opitmize times and concentrations.
Start with patient sample and then move to conjuate and then
developer. The patient sample will contribute the most noise,
conjugate second and the least noise in contributed by color
development.

Hope this helps. 


On 12 Feb 1997 20:55:11 GMT, jlk1117440 at aol.com (Jlk1117440) wrote:

>I'm having difficulty performing an ELISA....I believe I may be using an
>old, perhaps incorrect, protocol.  I would greatly appreciate any
>references to protocols or protocols that you have found successful, or
>any other relevant advice.  Thank you.
>                                   Jim Knoetgen, MD
>                                   St. Luke's - Roosevelt Hospital
>Center/Columbia Univ.
>                                   Department of Surgery

Bryan Kiehl
GenBio, San Diego
b3748 at cts.com



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