Dear All,
Is there any difference between Recombinant antigen preparation for ELISA AND Immuno Chromatographic Test.
As we have found that recombinant antigens worked very well in ELISA whereas no activity has been observed in ICT strip test.
In principle there are no essential differences, but you have to optimize each format. Perhaps the ELISA is closer to optimal conditions that the ICT you are trying to develop. You have to find optimal conditions to achieve maximal sensitivity and specificity in your ICT. You need proper controls to make sure that everything (from antigen immobilization on the membrane to antibody detection) is working properly.
There are several differences regarding the nature of the platform (ELISA versus lateral-flow). In ELISA you will have to do a dilution of the antigen, therefore you are coating the plate with 50-100 ng, however in lateral flow you will need a more concentrate sample of the recombinant antigen and good stability if it is going to be used for gold binding. Also, in ELISA you can have your antigen with chaotropic agents such as urea, never in lateral-flow, sometimes detergent is also counterproductive in the last platform, high conductivity, etc (depending of the hands that are producing the lateral-flow test). Regarding the quality of the antigen, is similar, you should obtain the best version of the antigen, with linear and conformational epitopes for detecting IgG and/or IgM in both of them.
What you say is not strange, I have seen lots of antigens which are able to work in ELISA but do not work in immunochromatography. Have a look to the formulation of the storage buffer and an important question is where are you using your recombinant antigen in the immunochromatography? for coating the plastic surface or in the binding to the gold? and the ELISA? is an indirect ELISA, capture ELISA? double antigen sandwich ELISA? all these questions are relevant.
Thank You Dear Ana Camacho for your nice suggestions. we have used indirect ELISA for the detection of specific antibodies from patient sera. while we have tried both direct spotting of antigen to NC membrane and coupling of antigen with gold nano particle (40nm).
As you mentioned above, the presence of chaotropic agents such as urea may inhibit the antigen antibody reaction in lateral flow.
In my case, our recombinant antigens form IB inside the E.coli cell. so we are using urea in the purification of the antigens. Al through, we have removed urea ( ~90%) from the purified proteins via membrane dialysis method.
As you cant remove 100% urea removal form the proteins. So, is there any possibility that very minute quantity of urea might be interfering with antigen antibody reaction in the lateral flow assay???
Thanking You
In my opinion it is very difficult to work with a protein obtained from IB, if there would be any possibility to obtain it in a soluble form, I think that your test would be run more efficiently. If you have eliminated all the urea then there shouldn´t be any problem. The fact is that while the ELISA uses approx. 1 ug/ml for coating the microtiter plates, for NC coating you can use 200-400 ug/ml of protein and if it comes from IB, are very prone to aggregation (the more concentrated, the more aggregation possibilities).
What is the storage buffer of your refolded protein? if it contains detergent or too much conductivity, the binding to the gold particules can be affected...
Thank You Dear Ana Camacho for your quick reply. we have used 1 X PBS/ phosphate buffer (pH7.0) as antigen storage buffer.
Also, we have tried to remove urea from the proteins form 8 M to 0.2 M concentration. we can not able to remove urea completely. As if we go lower concentration from 0.2 M, protein get precipitated even in the low temp (4 degree centrigrate). kindly suggest any other option for lateral flow development..
Once again Thank You Mam..
Dear Anil,
Did you try to produce your protein in a soluble form? I mean by decreasing the culture temperature and/or IPTG concentration?, you will have less protein but of better quality. If you have IB is because your expression is good enough, maybe you can sacrify some expression by solubility. With a protein isolated from IB you are going to have lots of problems, normally they are highly unsoluble and prone to aggregation, making very difficult reproducible results.
Urea makes very difficult conjugation and membrane coating. In my opinion is going to be very difficul to continue with this protein, because if you want to get rid of all the urea, maybe you will need some detergent and some conductivity for the protein stabilization (due to its marked insolubility) and both additives are harmful for LF.
Dear Anil Gupta ,
look the link maybe useful.
http://www.who.int/malaria/areas/diagnosis/rapid-diagnostic-tests/Generic_pf_training_manual_web.pdf
Regards, Shafagat
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