hi,
I am facing difficulties in the développement of an ELISA sandwich.
I have several experiences in developing ELISA but that’s the first time I can’t find any solution.
My test is currently as following :
- capture mAb1
- my sample (bacterial antigen)
- detection mAb2 - coupled to biotin
- streptavidin-HRP
Since we can’t use milk as blocking reagent because of its reaction with streptavidin, I use BSA 2,5% .
My problem is that my mAb2 cross-reacts with BSA, and I get blank OD very high, higher than DO>1 !! (Blank = well without antigen).
here is the proof of the unspecific reaction : I did some assays in indirect format as following
- coating my antigen
- mAb2
- anti-mouse -HRP
—> with BSA 2,5% blocking, I get Blank OD > 1
—> with milk 5%, I get Blank OD
Some suggestions:
1) Most simple. Have you tried to optimize concentrations of Mabs and HRP conjugate? In the case of high background lower concentrations of MAb1 (lower than 1 ug/mL) can work well.
2) You can try to remove biotin from milk by dialysis. Add milk solution in buffer for analysis (say, 5%) and dialyse against the same buffer several times to obtain high dilution (e.g. 100 mL of milk solution against 5 L of buffer 3-4 times). Don't dialyse against water. Protein solutions absorb water and tubing can be damaged because of too high pressure. We use casein from bovine milk for the same purpose. We dialyse its solution several times against buffer and use in ELISA with streptavidin. Actually, you can try casein or sodium caseinate (which is more convenient that casein) instead of milk. We performed streptavidin ELISA with commercial caseinate too and it worked well without any dialysis.
3) You can try another label, say FITC-anti-FITC-HRP. Also (if possible) you can change mouse MAb2 to, say, rabbit or goat polyclonals against this antigen. In this case anti-rabbit-HRP or anti-goat-HRP can be used.
MAb are usually pretty easy to develop in ELISA. It could be that your BSA is low quality. You might try a more pure purified product.
It is unlikely that your MAb really cross-reacts with BSA. You might want to double-check and confirm you still have a clonal hybridoma. If that's really the problem, then you need a different blocker. And that's easy to do.
You can't use milk. But you can buy purified casein, as Pavel Khramtsov indicated. Or go over to ovalbumin. 1% serum often works well. You may have some old FBS that you rejected for culture. Or if you have plenty of money, buy SuperBlock (Pierce-Thermo). It almost always works.
Be sure to wash thoroughly. 3x 200ul is standard, but you can do more. Sometimes it helps to do 4x or 5x.
As it was aforementioned, you can try casein. In my lab it is always a good solution when we have problems with milk in ELISA
Fan v. have you tried dialysis?
Regarding quality of BSA. We have recently used different BSA in biotin-streptavidin ELISA and obtained different results (some BSA preparations decreased OD). I think that the key issue is not the reputation of manufacturer, but method of BSA preparation. It can be isolated by cold ethanol fractionation, heat-shock, or both. Sometimes additional steps are added, such sa ultrafiltration. I think that biotin content depends on isolation method. For example, different types of BSA (in terms of fatty acid content) have various blocking properties (https://www.nature.com/articles/s43246-020-0047-9). You can try different BSA. We used this one in our biotin-streptavidin assay: https://www.bioclot.com/s/pi96/pi16/pd129.html
Probably, de-fatted BSA preparations can also be beneficial, because one can expect that biotin is also removed in the course of defatting. They are available from numerous manufacturers.
Fan v. since you concluded that skim milk is the only blocker that works the best with your current setup, I would try changing the detection with no biotin-streptavidin and I would rather conjugate the detection-Ab to HRP, or i might just try a secondary HRP conjugated Ab to avoid any cross reaction with milk or BSA. Like:
-Coat with Capture mAb1
-Add sample (Ag)
-Add detection mAb2 (plain unlabeled)
-Add anti-mouse secondary antibody conjugated to HRP
-Use TMB or any other HRP substrate and try developing an EIA in this format.
I'm not sure if you've already tried this, but I can suggest using the above sandwich EIA with your unlabeled mAbs, if you want to use milk for blocking.
Laharika Katamoni
- we tried to conjugate our detetction Mab2 to HRP , but unfortunately the antibody precipitated. That's why we had to conjugate to biotin
- we cannot use unlabeled detection mAb2 and then anti-mouse secondary antibody conjugated to HRP (as you described), because our two Mabs (both capture and detection) are mouse antibodies.
I've always found that an anti-biotin Ab gives lower background as a probe, compared to avidin/streptavidin label.
Fan v. Did you tried optimizing the conjugation conditions or using different HRP conjugation kits that might yield more stable conjugates.
Or just try optimizing the experimental conditions by either pre-blocking the mAb2 with your blocking buffer before applying it to the ELISA plate to bring down mAbs nonspecific binding or try different concentrations and compositions of BSA or other blockers in your sample diluent and washing buffers to minimize background. Playing around the concentration might help bring down the background noises in most of the sandwich ELISAs.
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