The problem I am encountering is, whenever I dilute the standard three fold I always find the absorbance of the first two wells almost similar despite the fact that the second well contains less antibody compared to first. This has confounded me as the standard curve is not optimal and the sample which has low absorbance appears to be in higher concentration than the ones which have higher absorbances. I expect if the antibody is diluted three fold OD or absorbance should also be at around 3 fold less range.
Sometimes even upto first 3-4 wells absorbance does not seem to vary much. I have tried this several times and sometimes I question my attention in the work and repeat again but despite that I am not getting a good standard curve.
I would be grateful for any suggestions.
Quantification against a standard is only valid when using the slope of the sigmoidal OD curve. It sounds like your standard at the initial dilutions is giving a signal at the plateau of the curve.
I agree with Andrew, your first standards are too concentrated. Try diluting your standard more and see how it looks. What is the concentration of your first standard?
Thanks Andrew and jonathan for your suggestions, the concentration i have been using is 1 microgram per microliter. I would try out to halve this concentration and try again.
There are several reasons possible:
1. optical: due to the instrument
2. Physics / Mathematics: The absorbance is the recirpocal value of the transmission. This will result in typical curves Y=1/x. Therefore the absorbance should not be higher than 2. Sure, some instruments can meausre up to 4.0, it's only a marketing gag.
3. lack of Substrate (rare event)
4. maximal binding capacity is reached. So start with the first dilution where you can see a difference to the next one. anf. it#s mostly better to us a twofold dilution. You get a mor precise calibration curve.
and the curves are in lin/log fromates always sigmoidal, in log/log in a disinct region only linerar and lin/lin hyperbolic. So you need for the equations the Rodbard model (or comparable models) which are included in most ELISA software packages.
Thank you so much Stephen. I am planning to work on quantification again and I am already engrossed with anxiety about the curve. I have always tried three fold dilution. Today I shall be planning 2 fold.
Hi Avishekh,
the measurement range in ELISA is mostlfy founf within 2 order of magnitude. By using 2 as the base dilution factor you can cover this (using 7 dilutions: 2 ... 128) by using 3 you will end up with 3 ... 2187 (using also 7 dilutions.
So start with a chekerbaord titration to define the measurment range. In some cases you can use also factors lower than 2 (e.i. 1,5) (1,5 ... 364). The better you can narrow the measurment range the more preciseliy your measurment will be.
I have posted my latest part of Elisa quantification result. Just the nature of O.D of the standard seems to affect the result a lot. Even if the O.D seems quite similar among different isotypes the quantity differs immensely just because of the standard curve. Particulary in IgG3 i get similar type of standard optical density and curve. I ordered the new one and again the result is same. The company i use is BD Pharmingen. The IgG3 level seems to exceed total IgG level.
Interestingly, we can see plateau at intial dilutions of some isotypes and that is not the case with total and IgG3. Please check this out and kindly give me some suggestions as before. Thanks.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA