estimation of concentration of Ab in hybridoma culture supernatant? - (Mar/21/2007 )

hi all
want to know how can i estimate the concentration of Ab in the hybridoma culture supernatant.. want to check whether antibody concentration before and after purification is same or not. it would be helpful for my experiment..

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do an ELISA.
coat with the antigen, so you can titrate your antibody in your culture supernatant (do increasing dilutions of the supernatant until you have no more signal : this dilution will give you the titer in antibody)

feel free to ask for more details.

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[ok fine
then i need to use a standard to and comparing with std i have to calculate the unknown concentration???

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no, you don't need a standard.
you will test several dilutions. Let's say start with 50 times diluted, and then dilute twice and again twice and so on :

so you have 1:50 ; 1:100 ; 1:200; 1:400; 1:800 ; 1:1600 : 1;3200

you do that for your sample before and after purification.

let's say that you see a signal with 1:50 1:100 and 1:200, but no more with 1:400.
the titer of your sample is 1:200 (which is not very concentrated)
you don't know exactly the concentration, but you know how much your sample is able to recognize your antigen. you see what I mean?
If you have the same titer before and after purification, you know you have the same concentration in your specific antibody.

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hi nish,
very sinmple dont break you head.......

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