General questions - General questions (Feb/26/2010 )
I have some questions that I hope I can have some feed back on..
Just to get thinks strait I am working developing new IVD assays on ELISA and LF platform. As I am part of healthcare (more or less the similar of CDC in the US)..I work with infections diseases.
I am primary working with polyclonal antibodies..
Well back to questions:
One of the things we often se is that when we are producing antibodies against an antigen (polysaccharide) that is excreted in the urine we often get a very fine dilution curve even if we spike the urine with the antigen, but when looking at patients urine we often don’t detect a thing, but other commercial kits do… this has probably something to do with the fragmentation of the antigen ?… but often we se in spiked urine that we have a better detection limit than the commercial kits…( I could upload some examples if you like) anybody have a antibody screening idea ?? In addition can you immunization human urine (patients with antigen) in rabbits and get high avidity antibodies? – should I switch during immunization from antigen to urine at say 4 immunization to select the plasma cells??? or would this just end up giving tolerance ??
the antigen may be fragmented or modified that is passed in urine.
you indicated that you do not see the antigen at all. the concentration in the urine may be dependent upon the time of collection...did the patient urinate earlier? was this a first morning sample?
I would explore the difference between the antigen that you used to immunize and the antigen that appears in urine. I would not use urine as an immunogen marix. Can you concentrate the antigen that appears in the urine and use the purified form for immunization?
I was waiting for your responds as you tend to have some nice ideas…
Well the problem / challenge is very interesting and really a puzzle..
We have a commercial kit that we know works… we know the antigen and if we uses the purified antigen in the commercial kit we get a fine curve with detection limit etc… if we uses our own antibodies on the purified antigen we get a better detection limit etc even in the Urine matrix… when we the move to clinical samples the commercial kit still detects the antigen but our antibodies don’t…
The question is how do we screen our antibodies for the antigens epitopes present in urine…
I think I will set up the following study…
1. Coat ELISA plates with antigen (concentration close to what we think is in the urine 500 ng/mL)
2. block with skim-milk
3. adsorption of antibody in positive clinical urine and negative clinical urine (one hour) then add this to well
4. Secondary anti-rabbit antibody
In principal if the antibodies reacts with the antigen in the clinical sample the results will decrease OD value compare to the negative urine…
Any other suggestions ??
The antigen is a polysaccharide as it is heart stabile…we can boil the urine and the commercial kit still detects it.
In a high urine sample we estimate that there is app. 500 ng/mL antigens… but how would you purify for immunization??? – boil to denaturize proteins, dialyses against PBS and then ethanol perception ?? perhaps… have you tried to use urine in immunization protocols. ??
If you are still working on problem.
Something that you should examine is how screening is performed for hCG and LH antibodies. Both appear in urine, concentrations vary with time of collection and 'concentration' of the urine sample. hCG is know for various types/fragments. How individuals solved this problem will help you solve yours.
For screening you would need to have purified forms of the antigen
Coat wells in parallel with all types
Screen your abs with these forms.
With the commercial kit you should call the mfg to determine the cross reactivity of the abs they use in the system...which they should tell you. (ie the different forms of the antigen that their kit detects). They should provide info on cross reactivity and interferring substances which should help you. They should be able to tell you the form of the calibrator/standard they use.