These are different antibodies that have different affinities for their targets. If all antibodies were the same we would have no need to do a standard curve each time we run an ELISA. However, as you have noted the different kits give different ODs for the same quantity of target. This is the nature of working with antibodies in research they are all different.

I would only worry if your data did not make sense. For example if your three isotypes added up to MORE than total IgG. But your data looks pretty good in that respect.

In short, your data looks good. I think the kits are working fine.

Thanks Jonathan, but as you see my IgG3 alone is more than Total IgG. I would be grateful if you recheck it and if possible give some suggestions.

Sorry i misread your data.

it sounds to me like your standards are not the concentration you think they are. You are right if they are mixed correctly you should get a plateau at the higher concentrations. Can you remake the standards? I would guess that either the protein in the standard is degraded, or a mistake was made in the dilutions.

Thanks for your prompt response. I have tried several times with IgG3. same was the problem with old stock and the one i have used in this experiment was new one. May be the company itself is supplying the poor quality of the standard.

You could try a BCA or other protein concentration measurement on the IgG3 standard to see if that concentration agrees with the advertised concentrations. That is as long as the IgG3 is diluted in a protein free buffer.

Are you making the standards the day of the assay? I was just reading documentation on another IgG3 kit, which said that the diluted standards are not stable.

Thank Jonathan. I dilute the igG3 standard just before my experiment. I soon as I dilute it I use it for coating. I even tried different concentrations. starting fromm 8000 ng/ml, 4000 ng/ml. Interestingly, in the dilution series starting from 8000 ng/ml (2- fold dilution), the first OD was 2.2 while second was around 1 and the third was 0.2 . While in series starting from 4000 also the first OD was same like 2.18 and abrupt fall in second to around 0.4. Both dilutions were done on same conditions and same time. I would expect the second dilution of first series and the first dilution of second( starting from 4000) should have similar OD. but that was not the case. And i have repeatedly done this. As you have mentioned may be Diluted standards of IgG3 are not stable. 

I am attaching a excel file of STD vs OD of different concentrations. PLEASE look at it. 

This series starts with 4000, 3000 and 2000 ng/ml at highest dilutions.

I think some where your dilution is going wrong.It is advice to start every thing fresh from dilution of conjugate unword. Some time repeated freezing and thawing of the diluted antibodies get aggregation and degraded the protein concentration. Further during the standardization one should avoid the saturated concentration dilution. Different Isotypes of IgG should be run in different plates.I think you problem will be solved. If it persist then some thing wrong with original concentration of the supply.

Hi Avishekh,

you cannot add the values of the immunoglobulin subclasses and compare them with the total IgG in antigen specific tests. This would not work due to several facts:

1. ELISA for the detection of specific antibodies depends on the affinity (and nearly not an the concentration) of the antibodies

2. Every subclass will have different affinities to the antigen.

3. In most cases, you would never find an absolute standard. Due to the two points above monoclonals could never be compared (as standards) with a polyclonal.

but

you should be able to add the values of all 4 subclasses to get the same result as for the measurement of total IgG. The only issue is the standardization. Make sure that you get the standards from the same company and they are comparable to the international standards for total IgG as well as for the subclasses. At least you should be able to reach the same magnitude.

In practice, there are many problems here as well. The affinities of the used antibodies to the different immunoglobulin subclasses are different. This results in different detection limits, different shapes of the calibration curves. The conclusion is that you need to hit the "measurement range" (the standard concentrations where you can measure with the lowest coefficient of variation. You need to look also in serial dilutions for the recovery. Do not use a serial dilution with factor 3, the steps are mostly to large. Factor 2 is fine. Two neighboring dilutions should give the same results when recalculating using the respective dilution factors. All others needs to get excluded from the calculation. This has to be done for all ELISAs, IgG1 … IgG4 and total IgG.

You can get some additional information if you are using standards from different sources. Serum, human monoclonals where the concentration is known.

Be careful and extremely precise when you are preparing the high dilutions you need the ELISA. In serum the normal range for IgG is 5 – 20 g/L. The normal measurement range in ELISA is between 1 – 1000 µg/L. The resulting dilution is at 1:100,000. That is why for the measurement in serum mostly nephelometric methods are in use.

Something additional: Do not use the linear fit, try to find an ELISA software which allows you to take a (3) 4-parameter approximation.

Thanks Stephan for your explanation. I will be considering all these valuable information. I am in fact grateful for giving so much time and such a convincing description on Elisa. 

I also had a similar problem, my standards, are at a much lover concentration that what I expected and give off low OD,.which makes it a problem,. Because my samples end up having higher concentrations than the standards.