I was wondering if anyone knows of any alternative methods of calculating antibody specific titres. Currently, we are using an ELISA tittering technique where I compare the titration of serum specific antibodies to that of a negative control serum lacking the requisite antibodies. I would then find the cut off OD using the negative control, and from there I would determine the dilution of the serum specific antibodies required to reach that OD. I understand this is a common technique, so if anyone can provide me with any alternative methods, or ways of improving my data analysis of my current method it would be greatly appreciated. Thank you.
Hello Justin,
I think in your case you don't have commercial or reference/standard antibodies with known concentrations for quantification of specific antibody concentrations in samples by ELISA. I would recommend the below method of titre determination for sera samples which lack appropriate reference/standard antibodies.
Test different dilutions (3 or 5 fold starting from 100 fold dilution) of both pre-immune sera ('0' day) and post-immune sera samples by ELISA. Plot the OD values against log dilutions (separately for both sera samples) in 4-parameter logistic curve. Pick the dilution which shows 1 OD unit (0.1 OD or 0.5 OD or 3 std. dev. of blank can also be used). The reciprocal of that particular dilution is the titre of the antibody present in the sera. Represent the antibody induction as fold rise by diving the post-immune sera titre with pre-immune sera titre (post vs pre-immune sera). This method will clear tell you the induced antibody titre as compared against the control sera.
Good Luck.
My apologies i forgot to mention that we coat the plate in the antigen in question, in order to calculate antigen specific titres.
Also do you happen to have an article that uses this method? Any help is greatly appreciated
Hello Justin,
Please specify which analyte you are going to quantify. Is it Antigen or Antibody. Whatever the method i told you above is for Antibody determination in sera samples lacking appropriate reference antibody standards..
We are quantifying our serum antibodies specific to an antigen. Therefore we coat the plates with said antigen. We have no reference antibody standards.
Hi Justin,
If you see any of the commercial secondary antibodies from the market they will define a working dilution for the product by direct ELISA using purified analytes for coating. This they will say using a particular end point, for e.g. serum dilution achieving 1 OD.
I have attached one file from Sigma for the product of anti-mouse secondary Ab. You can find the relevant information in the product profile section of this file.
Hope this helps you.
Good Luck.
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