Method for extending the range of an immunoassay

• US 6,284,472 B1
• Filed: 04/20/1999
• Issued: 09/04/2001
• Est. Priority Date: 10/05/1998
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Dade Behring, Inc. (Siemens AG)
• Inventors: Pankratz, Thomas John, Wei, Tie Quan, Chu, Victor Pichai
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): Do, Pensee T.
• Application Number:
  US09/294,489
• Time in Patent Office: 868 Days
• Field of Search: 422/119, 422/50, 422/62, 422/67, 422/68.1, 422/61, 422/82.08, 436/34, 436/43, 436/50, 436/164, 436/817, 436/901, 436/517, 436/546, 436/518, 436/800, 436/523, 436/538, 436/824, 436/805, 436/808, 435/7.1, 435/7.93, 435/7.94, 435/7.95, 435/962, 435/973, 435/975, 424/7.1, 530/389, 530/390, 530/807
• US Class Current: 435/7.1
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/901 Drugs of abuse, e.g. narcotics, amphetamine
• CPC Class Codes: Y10T 436/11 Automated chemical analysis
• CPC Class Codes: Y10T 436/115831 Condition or time responsive

Abstract

Calibrating an immunoassay by generating two reaction rate measuring curves, from samples having higher and lower relative levels of antigen, extrapolating a combination of the curves to cover sample concentrations known to contain an excess of antigen relative to an amount of capture reagent and combining the low end linear potion of the higher reaction rate measuring curve with the higher end portion of the extrapolated reaction rate measuring curve, thereby eliminating measuring inaccuracies otherwise arising from the hook effect. For antigen concentrations higher than the assay range, a high antigen signal utilizing the two rates avoids reporting false results.

Claims

1. A method for generating a calibration curve for an immunoassay comprising:

separating a sample having an antigen concentration therein into a first larger volume portion and into a second smaller volume portion;

generating first and second reaction rate curves each curve having a low end antigen concentration portion and a high end antigen concentration portion, the first reaction rate curve generated from the first larger volume portion and the second reaction rate curve generated from the second smaller volume portion;

mathematically curve fitting the first and second curves in a low range of antigen concentrations for which both curves are linear;

extrapolating the first reaction rate curve for those antigen concentrations containing an excess of antigen relative to an amount of capture reagent used in the immunoassay to produce an extrapolated reaction rate curve having a low end portion;

combining the low end potion of the first reaction rate measuring curve with the higher end portion of the extrapolated reaction rate curve to produce a single curve; and

,

using the single curve as the calibration curve for an immunoassay thereby eliminating measuring inaccuracies otherwise arising from the hook effect.

2. The method of claim 1 further comprising:

establishing a trip point at the junction of the high end linear potion of the first reaction rate measuring curve and the low end potion of the extrapolated reaction rate measuring curve; and

,

using the low end linear portion of the first reaction rate measuring curve to provide analytical results if the reaction rate measured on a patient sample is equal to or less than the trip point and using the extrapolated reaction rate measuring curve to provide analytical results if the reaction rate measured on a patient sample is greater than the trip point.

3. The method of claim 1 wherein the antigen is C reactive protein.

4. The method of claim 2 wherein the antigen is C reactive protein.

5. The method of claim 3 wherein mathematically curve fitting the two curves in a low range of sample concentrations covers the antigen concentration range below about 100 mg/dL.

6. The method of claim 2 further including means for generating an excess signal in the instance that the antigen concentration within the sample exceeds the trip point.

7. The method of claim 4 wherein the trip point is in the range between 30 mg/dL and 35 mg/dL.

8. The method of claim 1 wherein mathematically curve fitting the two curves comprises use of a regression analysis.

9. The method of claim 1 wherein the immunoassay comprises a turbidimetric measurement.

10. The method of claim 1 wherein the immunoassay is an immunoassay for C reaction protein.

11. The method of claim 1 wherein high antigen concentration is signaled by using the ratio of two or more than two reaction rates.

12. The method of claim 1 wherein high antigen concentration is signaled by using the combination of the differences between the two reaction rates and the rate of one of the two reactions.

13. The method of claim 1 wherein high antigen concentration, not limited to antigen excess and with the cutoff limit determined as needed, is signaled.

14. The method of claim 4 wherein a graduated switch is employed for calculating the combined rate for samples with C reaction protein values near or around the trip point established during calibration.