This is an extension from a previously asked question of mine- I want to elute antibodies from an ELISA complex to use in a functional assay.
The ELISA uses streptavidin coated plates, where biotinylated peptides of interest were bound. To this, diluted human serum is added. What I want is to remove the antibodies in the serum from the peptides, collect them, and use them in a functional assay.
Additionally, I'm interested in separating the collected peptide-specific antibodies, to isolate IgG1 and IgG2 (separately) to add to the neuronal cell culture. I've found a little literature about affinity columns or using protein A, but I'm having trouble thinking of a good way to separate these two human sub-classes of antibodies to use individually on the functional assay.
The helpful answers I got previously were to use urea or glycine at a low pH to elute the antibodies from the ELISA complex, as the biotin-streptavidin bond is very strong and shouldn't be affected. I think this is the best first step- and I'm after any advice on how/whether to neutralize the pH of the collected antibodies, and techniques for isolating the subclasses, so to (hopefully) not corrupt their function for use in the culture assay.
Dear Kaija,
for neutralization, just add the eluted antibodies to a small volume of 1M tris base or 0.5M NaH2PO4. (Calculate and then check with pH paper).
Concerning the separation of isotypes, use two small (100ul each) affinity columns with anti isotype antibodies (covalently bound to eg NHS activated Sepharose) connected in series, elute separately and neutralize (as above).
However, don't expect much antibodies to be retrieved from a single microplate well. If you can identify single candidate peptides as bait, better do large-scale affinity purification of the antibodies recognizing them (immobilize the peptides on streptavidin Sepharose), if you have access to enough material (100 ul of serum and up). A HPLC or better a biochromatography machine ("FPLC") will be very useful.
Dear Kaija,
I agree with the suggestions by Wolfgang, but alternatively you could consider using magnetic nanoparticles. You can bind isotype specific antibodies to amine-functionalized particles. The chemistry for conjugation is available by most suppliers and is rather straight forward.
As pointed out by Wolfgang, eluting only from a well may not give you sufficient antibodies for your assay. Would be good to use more.. Hope it helps.
The amount of antibodies bound on a surface of an solid phase assay (here ELISA) is in a femtomolar range. I wouldn't expect that you can earn a sufficient amount of antibodies for functional assays.
The suggestion to use microspheres (e.i. magnetic latex) as a solid phase for an small scale affinity chromatography. Due to the small particles you have a large surface and you can elute a sufficient amount of antibodies. The binding conditions should be closed to physiological conditions (pH 7.2 in PBS). Wash the particles with PBS. The elution at pH 2 ... 2.5 (0.01 Glycine-HCl buffer) is the best way. You can neutralize the eluate immediately after elution by adding a Tris-HCl buffer (0.15 mol/L, pH 7.5) best. Check only the volumes you need for neutralization to pH 7.2 ahead of the experiment.
During the elution process in recombinant antibody purification, adding the eluted antibodies to a small volume of 1M tris-HCl base(pH 9.0) can be neutralize the acid elution buffer, about 150uL(Tris): 1mL(elution). It can avoid aggregation and denaturation.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA