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Molecular Biology Techniques Q&A
Immunology and Immunochemistry: ELISA

This months question from the Molecular Biology Forums (online at molecularbi-
ology.forums.biotechniques.com) comes from the Immunology and Immunochem-
istry Methods section. Entries have been edited for concision and clarity. Mentions of
specific products and manufacturers have been retained from the original posts, but do
not represent endorsements by, or the opinions of, BioTechniques.

How do I determine the sensitivity of an ELISA? (Thread 22460)

Q I am running simple ELISAs to detect the presence of antibodies against an enzyme

in serum. The assay works well, with positive controls showing strong signals, clean
negative controls, and clear detection of antibodies in the test samples. However, I have
some questions regarding interpretation of the results.

I am not certain of the proper method for determining whether a sample is positive or negative
for the antibody. I understand that it must be based on the cutoff or endpoint of the varying
dilutions of the sample. As I dilute the sample in the wells across the plate, at some point, I
lose the colored signal. Is that the endpoint used to calculate the titer? If so, is there a standard
way of calculating the titer by comparing the dilution factor of the sample to the negative
controls? Will someone please explain to me exactly how to determine an antibody titer
in an ELISA?

Also, when discerning between positive and negative wells, is a well considered positive
simply because a color is produced in that well? Would that also be true for barely diluted
samples? Or is there a standard ratio of antibody titers that forms a threshold between the
positive control and test samples?

A You are talking about the sensitivity of the ELISA. In general, as you dilute the standard
with a known amount of antigen, at some point, it will reach an absorbance that is twice
higher than that of the negative control. This is considered the minimal amount of antigen the
ELISA can detect and indicates the sensitivity limit of your assay. You may consider a sample Stem Cell Promotion !
as positive if its absorbance is twice as much as the absorbance of the negative control. www.promocell.com/promotion

A You cannot say that a sample is automatically positive just because the absorbance is twice
as high as the negative control. A sample might be positive even if it is only 1.5 higher. There
are also ELISA tests where samples are defined as positive when they are three times higher
than the negative controls. It all depends on the manufacturer and the test system you are
Go for PromoCell !
using; the cutoff has to be calculated with defined samples, standards, and programs. Your partner for stem cell cultivation
Huge variety of human stem & blood cells:
A Yes, but a sample is positive if its absorbance is repeatedly twice that of the control. Mesenchymal Stem Cells
This is the common practice. If you want to be sure, you can run statistics. If you are using
commercial kits, they should provide suggestions for determining the cutoff. Pericytes
CD34+ and CD133+ Progenitor Cells
Q One of the things I am worried about is that my positive control, which is pooled Mononuclear Cells
CD14+ Monocytes new
from reacting patients, has a very high titer and needs to be diluted a great deal to reach
the endpoint. Signals from my positive samples are far lower than the signal from the
positive control. I know that pooled positive sera will give a very strong signal, but because www.promocell.com
the signals from my test samples are so far from the positive control signal, it makes me
question whether I should really consider those samples positive. When compared exclu-
sively against the negative control, the signals are clearly positive. Is it correct to use only
the negative controls to determine whether or not the sample is positive, and use the
positive control only to determine if the ELISA worked properly?

A I usually use the positive control to check if the ELISA is working well, just in case all
of my test samples turn out negative. When a sample is only weakly positive, you should
repeat the test to ensure that it is indeed positive. PromoCell GmbH
North America: 1 - 866 - 251 - 2860 (toll free)
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Q I have been following this conversation
on calculating the antibody titer in an
ELISA, but I still have a few questions.
(They are listed below). I am working on
a previously established protocol, but have
some concerns about the experimental
design that Id like to resolve before
proceeding with my experiment. I am new
to ELISA, so Im not yet familiar with the
best practices for these assays.
1. I found a method for determining
endpoint cutoff values (Frey et al.
1998. A statistically defined endpoint
titer determination method for
immunoassays. J. Immunol. Methods
221:35-41) and wondered if this is
widely accepted or if there is a more
straightforward method. Specifically,
is twice the negative control signal a
generally accepted threshold?
2. Is the negative control referred to in
the previous answers the blank or
background value? Or is this a measure
of the baseline reactivity?
3. To determine the percentage over
baseline, should I compare mean
endpoint OD values of the baseline
versus samples?
A The cutoff for an ELISA is usually calcu-
lated using a sufficient number (~50100)
of negative specimens from the population.
Normally, a cutoff is equal to the mean OD
of the negative samples plus 3 standard
deviations. The cutoff can be made more
robust by adding a higher factor to the
mean. This will make the ELISA more
specific, but with a slight loss of sensitivity.
That is more desirable in most cases.
To determine antibody titer, a positive
specimen is serially diluted 5-fold or
more and then tested on the ELISA. The
endpoint titer is determined by the last
diluted specimen that gives positive results
on the ELISA.
A You should look in The ELISA
Guidebook by John R. Crowther to
find answers to all of these questions. It
is common to use the average of negative
samples + 3 sd to determine the cutoff.
All the ELISA tests used in serology for
antibody detection should come with
details regarding their sensitivity and
specificity. You can determine the cutoff by
whatever means works best for you; just be
certain to reference it when presenting your
results. You will need to mention how you
calculated it, how many negative samples
you ran, how you knew they were negative,
and if you compared ELISA results with
other tests.
556
Q2 I am running a sandwich ELISA for
TNF- in 96-well ELISA plates. I put
my standards in rows A and H with the
samples in between. Recently, one row of
standards develops normally, but the other
row does not develop at all. I load from the
same reagent reservoir using a multichannel
pipet, so everything should be the same for
the samples. I did the capture overnight in
a refrigerator with a glass door, but I dont
think that the capture antibodies are light-
sensitive. What could be causing this?
A Is your plate reader working properly?
Does it work if you move the standard from
row H to row B?
Q For some of the ELISAs, row A does not
develop and in others, the problem was in
row H. I also could not see any color in those
wells. I tried using rows A and B and didnt
have a problem, but then I am concerned
about how much I can trust the data from
samples in row H. This only happens with
the TNF- ELISA.
A Check the multichannel pipet to see if
it takes up the reagent equally in all tips.
If one tip is loose, it might not deliver the
same amount of reagent as the others.
Q I usually check the multichannel by
eye while adding reagent and will check
the volume in all of the tips on my next
attempt.
A Row A and H are the leftmost and
rightmost pipetting channels. Will your
plate reader accept plates that are rotated
180?
A If this only happens in row A or H,
it might be caused by how you hold the
pipettor or how the plate is oriented. The
plate reader is probably innocent since it
cannot read if there is no color.
A This looks like a case of one loose tip or a
pipettor with one clogged channel. When
did you last have it serviced and calibrated?
Make sure all of your tips are tight and keep
an eye on the liquid level as you pipet.
Selected and edited by Kristie Nybo, Ph.D.
BioTechniques 49:555-556 (August 2010)
doi 10.2144/000113475
www.BioTechniques.com