Title:  Multiassay method for determining the concentrations of antigens and interferants

United States Patent:  6,174,688

Assignee:  The United States of America as represented by the Secretary of the Navy (Washington DC)

Filed:  December 22, 1999

A method of determining the concentration of a sample antigen in the presence of an interferant by (1) running two immunoassays on the sample: one assay where the interferant influences the binding of both the sample antigen and a labeled antigen and a second assay where the interferant influences the binding of the sample antigen but not the labeled antigen; (2) obtaining a plot of the possible sample antigen concentrations versus the possible interferant concentrations corresponding to the readout for the sample for each of the two immunoassays; and (3) determining the sample antigen concentration and the interferant concentration which correspond to the point that appears on both of the immunoassay plots.

SUMMARY OF THE INVENTION

Accordingly, an object of this invention is to provide a method of accurately quantifying a known antigen and a known interferant in a sample containing a mixture of the antigen and interferant.

Another object of this invention is to provide a method of determining the concentration of a known antigen in the presence of an unknown interferant.

A further object of this invention is to provide a method of determining information about the binding activity of an unknown antigen and an unknown interferant in a sample containing a mixture of the antigen and the interferant.

These and other objects of this invention are achieved by providing

a method for determining the concentrations of a known antigen and a known interferant in a sample comprising:

A. generating a 3-dimensional calibration curve for a competition immunoassay which shows the assay readout for points on a matrix of the antigen concentration versus the interferant concentration;

B. generating a 3-dimensional calibration curve for a non-competition immunoassay selected from the group consisting of inhibition immunoassays and sandwich immunoassays which shows the assay readout for points on a matrix of the antigen concentration versus the interferant concentration:

C. performing the competition immunoassay on the sample using the same conditions and parameters used in generating the 3-dimensional calibration curve in step A to obtain a competition immunoassay readout for the sample;

D. finding the matrix points on the 3-dimensional calibration curve for the competition imunoassay (step A) which have the same readout as the sample (step C) and using the points to form a 2-dimensional curve of the antigen concentration versus the interferant concentration for the competition readout value for the sample;

E. performing the non-competition immunoassay on the sample using the same conditions and parameters used in generating the 3-dimensional calibration curve in step B to obtain a non-competition immunoassay readout for the sample;

F. finding the matrix points on the 3-dimensional calibration curve for the non-competition immunoassay (step B) which have the same readout as the sample (step E) and using the points to form a 2-dimensional curve of the antigen concentration versus the interferant concentration for the non-competition immunoassay readout value for the sample; and

G. finding the point of intersection between the 2-dimensional competition immunoassay curve (step D) and the 2-dimensional non-competition immunoassay curve (step F) and reading the antigen concentration and the interferant concentration corresponding to this point.

If the antigen is known but the interferant is unknown, a substitute known interferant is used with the known antigen to generate the 3-dimensional calibration curves for the competition and the non-competition immunoassays. Otherwise, the method is performed as described above. The resulting matrix point in step G gives the concentration of the known antigen and the concentration of the substitute known interferant which produces the same influence or interference as the unknown concentration of the unknown interferant in the sample.

If the antigen is unknown and the interferant is known. a substitute known antigen is used with the known interferant to generate the 3-dimensional calibration curves for the competition and the non-competition immunoassays. Otherwise, the method is performed as described above. The resulting matrix point in step G gives the interferant concentration and concentration of the substitute known antigen which has the same immunoassay activity as the unknown concentration of the unknown antigen in the sample.

Even if both the antigen and the interferant are unknown, the above method can be adapted to provide some information. A substitute known antigen and a substitute known interferant are used to generate the 3-dimensional calibration curves for the competition and the non-competition immunoassays. Otherwise, the method is performed as described above. The resulting matrix point in step G gives the concentrations of the substitute known antigen and the substitute known interferant which give the equivalent results as the unknown concentrations of the unknown antigen and the unknown interferant in the sample.

Claim 1 of 16 Claims

What is claimed is:

1. An immunoassay method, comprising the steps of:

performing a competition immunoassay wherein a selected antibody which reads specifically with a known target oxygen, on first calibration samples having varying concentrations of both an antigen and an interferant which interferes with the ability of the antigen to bind the antibody of the competition immunoassay and obtaining first calibration readout values;

performing a non-competition immunoassay using the same antibody as in the competition immunoassay, on second calibration samples having varying concentrations of said antigen and said interferant and obtaining second calibration readout values;

performing the competition immunoassay on an assay sample of a specimen to be analyzed and obtaining a first assay readout value;

performing the non-competition immunoassay on an assay sample of said specimen and obtaining a second assay readout value;

determining, from the first calibration readout values, a first function of the values of antigen concentration versus interferant concentration at said first assay readout value;

determining, from the second calibration readout values, a second function of the values of antigen concentration versus interferant concentration at said second assay readout value;

determining the values of antigen and interferant concentrations in the specimen as the intersection of the first function and second function.

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