Is there a standard ratio of concentration of monoclonal antibodies against a particular antigen for an antigen capture assay? To put it plainly how much monoclonal antibody is needed to bind to "x" concentration of antigen.
Hi,
I don't think so. As there are different binding affinities for each monoclonal antibody, ratio also differs. However, probably you would want to use antibody abundant enough to assure "maximum" capture. If you want to optimize your assay, you can use high antigen concentration and titrate monoclonal antibody to find out lowest but efficient concentration.
Go with multiple ratios to pull the right concentration. You can do it by conventional ELISA and then decide the right amount of both.
There are to many variables to predict the correct ratio. You will need to do a block titration to determine optimal molar ratios for antibody and antigen. What is your assay format? Solid phase? antigen precipitation? affinity chromatography? Provide more details and I may be able to provide more assistance and possibly a protocol.
Hi Frank
I am working on a competitive ELISA technique that involves incubation of antigen (blood serum sample) and antibody (monoclonal) in a test tube. The contents of which are then transfered into the ELISA plates coated with pure antigen.
You are using a competitive antibody bridging ELISA which can be a little twitchy to validate. I typically use a slightly different format where the monoclonal antibody is used to coat the micrometer plate the pure antigen is HRP conjugated and the standards and unknowns ore first mixed in test tube and added to the monoclonal antibody coated plate. In this format the greater the colour development the lower the concentration. Your "0" standard in assay buffer is mixed with HRP labeled antigen in assay buffer which is titrated to produce an absorbency of ~1.8. your standards are determined by titration by mixing a volume (50 - 100 ul) of known antigen with HRP conjugated antigen and incubating with monoclonal coated micrometer plate under assay conditions. The monoclonal attached to the solid phase delivers high specificity and this format can typically be adjusted to produces a sensitivity of 1 - 100 ng antigen /ml.
The method, offered by Frank, undoubtedly, is more sensitive than yours. But it is hardly executed when you have too little antigen, so you cannot prepare enough quantity of conjugate. In addition, some Mabs do not attach to plate surface. In your case for obtaining optimal dilution of Mabs you should firstly titrate them on immobilized antigen using indirect ELISA. After that you choose that dilution (as working) which shows an extinction approximately in two times lower of maximal (e.g. max=2.0, chosen=1.0-1.5). Thus you be sure that you do not use excess of Mabs.
As others have said above you will need to empirically determine the amount for each individual antibody. However the essential principles are determined by the laws of mass action and the affinity of your antibody. You can estimate the affinity (or avidity if you allow for multivalent binding by two or more arms of your antibody, depending on class) by titrating the antibody in a suitable assay such as ELISA or FACS etc and then estimating the concentration that gives 50% maximal binding. This concentration of antibody approximates to the dissociation constant or Kd. Most antibodies fall somewhere in the range of micromolar to nanomolar. see also the link below
http://www.path.cam.ac.uk/~mrc7/lecturenotes/abaffinity.pdf
Make three to four concentration of your mab and check it. hopefully you fill find it.
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