Assay process with non-boiling denaturation

• US 4,418,151 A
• Filed: 03/30/1981
• Issued: 11/29/1983
• Est. Priority Date: 03/30/1981
• Status: Expired due to Term

• Current Assignee: Micromedic Systems Inc.
• Original Assignee: Rohm and Haas Company (Dow, Inc.)
• Inventors: Forand, Ronald R., Menz, Edward T. Jr.
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/248,826
• Time in Patent Office: 974 Days
• Field of Search: 424/1, 424/12, 23/230 B, 436/505, 436/804, 436/825
• US Class Current: 436/505
• CPC Class Codes: G01N 33/82 involving vitamins or their receptors
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample

Abstract

Competitive protein binding radioassay for sera (or cell) vitamin B₁₂ and/or serum folate (target components, or analytes) or other target components in liquid ambient utilizing a highly alkaline (pH 12-14) environment and reducing agent for denaturing separating the target component(s) from serum without boiling, consistent with other requirements of such assays, then reducing pH to an 8-10 range for effecting the competitive protein binding after which protein bound and unbound groups of radioactively tagged replicates of the target component(s) can be separated and detected to determine content of the target component(s) in the original serum (or cell), i.e. original endogenous analyte(s).

Claims

1. A radioassay process for analysis of the target components folate or Vitamin B₁₂ and folate utilizing competitive protein binding which comprises the steps of:

(a) contacting a precise amount of serum sample having folate or Vitaming B₁₂ and folate endogenous target components with liquid medium containing a mercaptan reducing agent and radioactively tagged replication(s) of folate or Vitamin B₁₂ and folate target component(s) and incubating substantially at room temperature to initiate denaturation (irreversible separation) of the target component(s) from endogenous binder protein(s) in the serum,

(b) providing additional liquid medium containing a means of establishing a pH in the medium at substantially 12.0-14.0 and incubating at substantially ambient temperature to complete the denaturation of the target component(s) from endogenous binder protein(s) in the serum,

(c) reducing the pH to 8-10,

(d) adding simultaneously with the separation or thereafter, binding protein(s) of the target component(s),

(e) incubating to establish binding of target component(s) and tagged replications to the binding protein(s), and

(f) separating unbound tagged replicates from protein bound replicates as separate groups and measuring radioactive emission from one or both groups to provide a count correlatable with the competitive binding result and with target component(s) content of the original serum.

2. The radioassay process in accordance with claim 1 wherein the mercaptan reducing agent comprises dithiotetritol.

3. The radioassay process in accordance with claim 1 or 2 wherein the target component comprises folate.

4. The radioassay process in accordance with claim 3 wherein the tagged replicate comprises a folate derivative tagged with iodine-125 and the binding protein comprises purified beta-lactoglobulin.

5. The radioassay process in accordance with claim 1 or 2 wherein the target components comprise vitamin B₁₂ and folate.

6. The radioassay process in accordance with claim 5 wherein the tagged replicate comprises Vitamin B₁₂ tagged with cobalt-57 and folate tagged with iodine-125 the proteins are purified intrinsic factor and purified beta-lactoglobulin, respectively and a buffer admixed therein to establish a pH of 8-10.

7. The radioassay process in accordance with claim 2 wherein the reduction at 12-14 pH is in the presence of potassium cyanide.

8. A radioassay process wherein the target components comprise folate and vitamin B₁₂ for assay, the tagged replicates comprise folate derivative tagged with iodine-125 and Vitamin B₁₂ tagged with cobalt-57 and the binding protein comprises beta-lactoglobulin and purified intrinsic factor respectively and buffer admixed therewith to establish a pH of 8-10 wherein radioemissions from both tagged replicates are separated based on differing energy levels.

9. A radioassay process for analysis of Vitamin B₁₂ utilizing competitive protein binding which comprises:

(a) contacting a precise amount of serum sample containing Vitamin B₁₂ with liquid medium containing a mercaptan reducing agent radioactively tagged Vitamin B₁₂ and incubating at substantially room temperature to initiate denaturation of the Vitamin B₁₂ from endogenous binder proteins in the serum,

(b) providing additional liquid medium containing a means of establishing a pH in the medium of substantially 12.0-14.0 and incubating substantially at ambient temperature to complete the denaturation,

(c) reducing the pH to 8-10,

(d) adding, simultaneously with the separation or thereafter, purified intrinsic factor,

(e) incubating to establish binding of Vitamin B₁₂ and tagged Vitamin B₁₂ replications to the purified intrinsic factor, and

11. The radioassay process in accordance with claim 9 wherein the reducing agent is dithiotetritol.

10. (f) separating unbound tagged Vitamin B₁₂ replicates from protein bound Vitamin B₁₂ as separate groups and measuring radioactive emission to provide a count correlatable with the competitive binding result and with Vitamin B₁₂ content of the original serum.