Use of intramolecularly, covalently cross-linked proteins as binding partners in immunoassays
View Patent ↗The invention concerns the use of intramolecularly, covalently cross-linked proteins and covalently cross-linked reverse transcriptase from HIV as immunological binding partners in immunoassays. It also concerns immunological test procedures for detecting an analyte in a sample in which intramolecularly, covalently cross-linked proteins are used as binding partners, and it further concerns intramolecularly, covalently cross-linked reverse transcriptase from HIV and a method for producing this reverse transcriptase.
1. A method for detecting an analyte in a sample comprising the steps of:
a. combining the sample with an intramolecularly, covalently cross-linked HIV reverse transcriptase in the absence of detectable intermolecular multimers of the HIV reverse transcriptase, wherein the HIV reverse transcriptase specifically binds with the analyte or with a substance bound to the analyte to form a complex,
b. adding to the combination formed in step (a) a binding partner provided with a label that combines with the complex formed in step (a) to produce a detectable signal, and
c. determining the signal produced in step (b) as a measure of the analyte in the sample.
2. The method of claim 1 wherein the analyte is an anti-HIV reverse transcriptase antibody.
3. A method for detecting an analyte in a sample comprising the steps of:
a. contacting the sample with a composition comprising an intramolecularly, covalently cross-linked HIV reverse transcriptase, wherein said composition comprises less than about 5% of total intermolecular multimers of HIV reverse transcriptase relative to the eluted peak intramolecularly, covalently cross-linked HIV reverse transcriptase as determined in a gel permeation chromatograph, wherein the intramolecularly, covalently cross-linked HIV reverse transcriptase specifically binds with the analyte or with a substance bound to the analyte to form a complex,
b. adding to the combination formed in step (a) a binding partner provided with a label that combines with the complex formed in step (a) to produce a detectable signal, and
c. determining the signal produced in step (b) as a measure of the analyte in the sample.
4. The method of claim 3 wherein the intermolecular multimers of HIV reverse transcriptase are undetectable using SDS-PAGE analysis when 500 ng of said composition is loaded per lane.
5. The method of claim 3 wherein the intermolecular multimers of HIV reverse transcriptase are undetectable in said composition as determined by gel permeation chromatography analysis.
6. The method of claim 4 wherein the analyte is an anti-HIV reverse transcriptase antibody.
7. The method of claim 6 wherein HIV reverse transcriptase is cross-linked, to form said intramolecularly, covalently cross-linked HIV reverse transcriptase, using a cross-linking agent selected from the group consisting of MHS (3-maleimidobenzoyl-N-hydroxysuccinimide ester), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DSS (disuccinimidylsuberate), HSAB (N-hydroxysuccinimidyl-4-azidobenzoate), and sulfo-SANPAH (sulfosuccinimidyl-6(4′-amido-2′-nitrophenylamido)hexanoate).
8. The method of claim 6 wherein the intramolecularly, covalently cross-linked HIV reverse transcriptase is further modified to comprise a signal generating group or a ligand for binding to a solid support.
9. The method of claim 8 wherein the signal generating group is selected from the group consisting of enzymes, fluorescent groups and chemiluminescent groups.
10. The method of claim 8 wherein the ligand is biotin or streptavidin.
11. The method of claim 1 wherein the intramolecularly, covalently cross-linked HIV reverse transcriptase is further modified to comprise a signal generating group or a ligand for binding to a solid support.
12. The method of claim 1 wherein the intramolecularly, covalently cross-linked HIV reverse transcriptase is covalently linked to biotin or streptavidin.