I am planning to use ELISA from Human SErum samples. My aim is quantitative analysis. I have few questions:
1. DO we need to standardise the protocol for our samples or can we directly use the kit for our samples? As the kit is generally for 96 samples, out of which 16-20 will be for standard only.
2.Which ELISA is better for serum samples or in general. I know Sandwich is much more sensitive but bit confused between Sandwich and Competitive (which can be direct , indirect or sandwich)?
Thanks in Advance
Hallo Sandeep Rai Kaushik,
It all depends, what the outcome is supposed to be. For a good quantitative analysis, you should measure standards and samples in triplicates, and include a positive and negative control in your ELISA. You should be aware of, that the seller of the kit means you can measure 96-times (=one microtiterplate, MTP) but with standards and samples measured in triplicates or even in different dilutions, you might need more.
For example: you have 8 calibrators (For ELISA is this the least you should have) and 20 samples executed in triplicates you need 28*3= 84 well. The other you can use for positive and negative controls.
For an ELISA protocol an an MTP, you should also be aware of the so called "plate effect". Randomize the location of the calibrators and samples on the plate to avoid this effect. Means: Don't put a calibration only in Column 1-3 and the samples systematically in the other. It is described for example here:
Article Mitigation of microtiter plate positioning effects using a b...
Your second question: " Which ELISA is better for serum samples or in general "
One cannot say, what is better in general. Both Immunoassay formats are used for completely different things.
The Sandwich assay is used for measuring antigens, that are usually larger then 5000 Da. The antigen is captured between two antibodies, though it needs to present two epitopes. You can (and maybe should) use this assay for determination of proteins, bigger peptides (e.g Interleukins) and so on. The more Analyte ( = Antigene) is bound between the antibody, the higher the signal will be. The signal to-analyte concentration is therefore direct proportional.
The competitive assay is used for determination of small molecules, that can only be recognized by one antibody. Because they are too small to be bound by two antibodies. These are for example bile acids, sterols, steroid-hormones, metabolites, pharmaceuticals etc. There are two different competitive assays:
The direct competitive assay: here the primary antibody is immobilized on the surface of the MTP. The analyte in solution competes with a labelled analyte (Usually a Enzyme-Analyte conjugate named "Tracer") for the binding sites of the immobilized antibody. The lower the concentration of the analyte, more tracer is bound and subsequently the higher the signal will be. So the signal- to-analyte concentration relationship is indirect proportional.
At an indirect competitive immunoassay the analyte is immobilized on the MTP (Usually as a Analyte-protein-conjugate, BSA, OVA etc). The analyte in solution competes with the surface immobilized analyte for the binding sites of the antibody. The higher the concentration of the analyte, the less antibody is bound on the surface. The surface bound antibodies can then be stained with a secondary, enzyme labelled antibody. The signal- to-analyte concentration relationship is indirect proportional as well.
Don't get confused: sometimes in clinical chemistry ELISAs are called "direct" when the primary antibody is labelled with an enzyme and indirekt, when you have to use a secondary labelled antibody, no matter, what the format is.
To conclude: Whether you take a Sandwich assay or an competitive assay, depends what you want to measure. Both have there "crucial points" For example the Sandwich assay is prone for the hook effect: When the concentration of the analyte is too high, you observe a false-negative result, because the immuno-complex is not formed correctly. Also Heterophilic antibodies, Rheumatoid factors and Human-anti-mouse-antibodies in serum of the patients can interfere with your assay.
The competitive is also sensitive to matrix effects: everything that hinders the tracer of binding the antibody will cause a false-positive.
However, if you buy a kit, you will not have any choice anymore. the kit is "ready to use" and you cannot change the step or arrangement of of the format.
Here is some literature for quality assurance of competitive Immunoassays:
Article Quality assurance in immunoassay performance – carbamazepine...
Article Quality assurance in immunoassay performance - Comparison of...
Article Quality assurance in immunoassay performance-temperature effects
Good luck with your ELISA!
Cheers
Peter Carl
when I do Elisa I do have the standard curve and do in duplicate. don't usually standardise my samples but sometimes we take in consideration that the concentration of the sample may fall outside the curve so then I dilute the sample. usually use a wide measuring range . for the second question :
https://www.bio-rad-antibodies.com/elisa-types-direct-indirect-sandwich-competition-elisa-formats.html
In any case you need to run a standard on the same plate. And you should have a standardized protocol (SOP) if you want to quantify. The more important the quantification is the more time you should spend on standardization and assay validation. This really depends on the aim of your study and what kind of information you want to gain with the assay.
Hallo Sandeep Rai Kaushik,
It all depends, what the outcome is supposed to be. For a good quantitative analysis, you should measure standards and samples in triplicates, and include a positive and negative control in your ELISA. You should be aware of, that the seller of the kit means you can measure 96-times (=one microtiterplate, MTP) but with standards and samples measured in triplicates or even in different dilutions, you might need more.
For example: you have 8 calibrators (For ELISA is this the least you should have) and 20 samples executed in triplicates you need 28*3= 84 well. The other you can use for positive and negative controls.
For an ELISA protocol an an MTP, you should also be aware of the so called "plate effect". Randomize the location of the calibrators and samples on the plate to avoid this effect. Means: Don't put a calibration only in Column 1-3 and the samples systematically in the other. It is described for example here:
Article Mitigation of microtiter plate positioning effects using a b...
Your second question: " Which ELISA is better for serum samples or in general "
One cannot say, what is better in general. Both Immunoassay formats are used for completely different things.
The Sandwich assay is used for measuring antigens, that are usually larger then 5000 Da. The antigen is captured between two antibodies, though it needs to present two epitopes. You can (and maybe should) use this assay for determination of proteins, bigger peptides (e.g Interleukins) and so on. The more Analyte ( = Antigene) is bound between the antibody, the higher the signal will be. The signal to-analyte concentration is therefore direct proportional.
The competitive assay is used for determination of small molecules, that can only be recognized by one antibody. Because they are too small to be bound by two antibodies. These are for example bile acids, sterols, steroid-hormones, metabolites, pharmaceuticals etc. There are two different competitive assays:
The direct competitive assay: here the primary antibody is immobilized on the surface of the MTP. The analyte in solution competes with a labelled analyte (Usually a Enzyme-Analyte conjugate named "Tracer") for the binding sites of the immobilized antibody. The lower the concentration of the analyte, more tracer is bound and subsequently the higher the signal will be. So the signal- to-analyte concentration relationship is indirect proportional.
At an indirect competitive immunoassay the analyte is immobilized on the MTP (Usually as a Analyte-protein-conjugate, BSA, OVA etc). The analyte in solution competes with the surface immobilized analyte for the binding sites of the antibody. The higher the concentration of the analyte, the less antibody is bound on the surface. The surface bound antibodies can then be stained with a secondary, enzyme labelled antibody. The signal- to-analyte concentration relationship is indirect proportional as well.
Don't get confused: sometimes in clinical chemistry ELISAs are called "direct" when the primary antibody is labelled with an enzyme and indirekt, when you have to use a secondary labelled antibody, no matter, what the format is.
To conclude: Whether you take a Sandwich assay or an competitive assay, depends what you want to measure. Both have there "crucial points" For example the Sandwich assay is prone for the hook effect: When the concentration of the analyte is too high, you observe a false-negative result, because the immuno-complex is not formed correctly. Also Heterophilic antibodies, Rheumatoid factors and Human-anti-mouse-antibodies in serum of the patients can interfere with your assay.
The competitive is also sensitive to matrix effects: everything that hinders the tracer of binding the antibody will cause a false-positive.
However, if you buy a kit, you will not have any choice anymore. the kit is "ready to use" and you cannot change the step or arrangement of of the format.
Here is some literature for quality assurance of competitive Immunoassays:
Article Quality assurance in immunoassay performance – carbamazepine...
Article Quality assurance in immunoassay performance - Comparison of...
Article Quality assurance in immunoassay performance-temperature effects
Good luck with your ELISA!
Cheers
Peter Carl
Thank you all for suggestions.
My proteins are >60kDa. So, I think Sandwich is better for me Peter Carl .
And yeah, we have to use in duplicates/triplicates. I have around 30 samples; but if use 2 plates then there can be some plate error. so, i want to use duplicates. IS it okay and acceptable??
Yes, Sandwich ELISA is the method of choice.
Regarding using, whether duplicates are ok:
Statistically? No. But I am very picky with this.... others will say its ok.
For the purpose of your measurement? It depends what you need. Do you do this for research, or as "accreditated analysis?". You need to decide, which level of uncertainty is adequate to your question.
If you want to run two plates, you need one calibration curve on each plate. Especially, if you do it by hand and do not have a pipetting robot and fully-automated ELISA (Then you only need a curve from time to time and a control sample on each plate)
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