Methods for detecting weakly immunogenic analytes
View Patent ↗GPE antibodies recognize GPE with high specificity. When used in a radioimmunoassay, they reliably measure the concentration of GPE. They may be used to extend the half-life of GPE both in vivo and in vitro, and in methods of purifying the GPE receptor. Two methods of rpHPLC accurately revolve GPE on the basis of its hydrophobicity.
1. A method for detecting Gly-Pro-Glu (GPE) in a sample; comprising:
(a) providing an antibody raised against said GPE derivatized with a reagent selected from the group consisting of keyhole limpet hemocyanin (KLH), diethyl carbodiimide, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 2-[m-azido-o-nitrobenzamido]-ethyl-1,3′-dithiopropionate and N-succinimidyl-3-[4-hydroxyphenyl]propionate;
(b) providing a sample containing an unknown amount of said GPE;
(c) derivatizing said GPE in the sample of step (b) using the reagent selected in step (a);
(d) permitting said derivatized GPE of step (c) to bind with said antibody of step (a); thereby producing a derivatized GPE-antibody complex; and
(e) detecting said derivatized GPE-antibody complex.
2. The method of claim 1 , wherein the antibody affinity for the GPE is 200 ng/tube.
3. The method of claim 1 , wherein the antibody is a polyclonal antibody directed against a Bolton-Hunter (N-succinimidyl-3 -[4-hydroxyphenyl]propionate) derivatized GPE.
4. The method of claim 1 , wherein the antibody is a monoclonal antibody directed against a Bolton-Hunter (N-succinimidyl-3-[4-hydroxyphenyl]propionate) derivatized GPE.
5. The method of claim 1 , wherein the antibody has half maximal binding to an iodinated tracer analog of the GPE with a final titer of at least about 1:600.
6. The method of claim 1 , wherein the antibody has half maximal binding to the derivatized GPE with a final titer of at least about 1:18,000.
7. The method of claim 1 , wherein detection of said derivatized GPE antibody complex has an ED 50 of about 0.01 ng/tube.
8. The method of claim 1 , wherein the ED 50 to displace the derivatized GPE antibody complex with derivatized GPE is about 10 4 times greater than the ED 50 to displace the underivatized GPE antibody complex with underivatized GPE.
9. The method of claim 1 , wherein the antibody does not substantially cross react with derivatized glycine, proline, glutamic acid, gly-pro (GP), pro-glu (PB), IGF-1 or urea.
10. The method of claim 1 , wherein the GPE is derivatized with Bolton-Hunter reagent (N-succinimidyl-3-[4-hydroxyphenyl]propionate).
11. The method of claim 1 , wherein the GPE is derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.
12. The method of claim 1 , further comprising:
(f) eluting the derivatized GPE antibody complex in a chromatographic system;
(g) exciting the derivatized GPE antibody complex at an appropriate UV absorbing wavelength; and
(h) detecting the derivatized GPE antibody complex at an appropriate UV emitting wavelength.
13. The method of claim 11 , further comprising:
(f) eluting the derivatized GPE antibody complex in a chromatographic system;
(g) exciting the derivatized analyte antibody complex at 250 nm wavelength; and
(h) detecting the derivatized analyte antibody complex at 395 nm wavelength.
14. A method for determining the amount of unknown GPE comprising:
(a) providing an antibody raised against said GPE derivatized with a reagent selected from the group consisting of KLH, glutaraldehyde, diethyl carbodiimide,6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 2-[m-azido-o-nitrobenzamido]-ethyl-1,3′-dithiopropionate and N-succinimidyl-3-[4-hydroxyphenyl]propionate;
(b) providing a sample containing an unknown amount of said GPE;
(c) derivatizing said GPE in the sample of step (b);
(d) permitting said derivatized GPE of step (c) to bind with said antibody of step (a); thereby producing a derivatized GPE-antibody complex;
(e) permitting said excess antibody of step (d) to incubate with a radioactive tracer derivatized analyte; thereby producing a radioactive tracer derivatized GPE-antibody complex;
(f) isolating the mixture of the derivatized GPE-antibody and radioactive tracer derivatized GPE-antibody complexes;
(g) eluting the said complexes in a chromatographic system; and
(h) analyzing the eluent and determining the amount of unknown GPE on the basis of the excess of antibody not bound to the derivatized GPE determined on the basis of emitted radioactivity of the tracer derivatized GPE antibody complex.
15. The method of claim 13 , wherein a precipitating antibody is used in step (f) to isolate the mixture of the derivatized GPE-antibody and radioactive tracer derivatized GPE antibody complexes.