# US 5,705,353 A · Method of reducing interferences in assays

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- Number: US 5,705,353 A
- Document ID: US5705353A
- Status: Granted
- Kind: A
- Application number: 08/483,288
- Filed: 1995-06-07
- Granted: 1998-01-06
- Earliest claimed: 1995-06-07
- Assignees: Beckman Instruments, Inc.
- Inventors: Chan S. Oh; Julie S. Kim; Anthony K. Cheng
- Web page: https://patentgrep.com/patent/US5705353A

## CPC

- G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

An assay for determining the presence of an analyte of interest in a test sample containing an interfering substance that interferes with the assay is disclosed. The assay comprises the steps of forming a reaction mixture by combining in an assay medium (i) an assay system containing components for detecting the analyte of interest, (ii) a test sample containing the analyte and an interfering substance, and (iii) an additional amount of the interfering substance in a quantity sufficient to substantially mask the effect of the interfering substance in the test sample. This reaction mixture is incubated under conditions sufficient to allow complex formation. This resultant complex formation is detected and the presence of the analyte in the test sample is determined.

## Claims

### Claim 1

An assay for detecting the presence of an analyte of rest in a test sample comprising the steps of:
a. forming a reaction mixture by combining in au assay medium:
(i) an assay system comprising a first binding member, a second binding member, a spacer member intermediate between the members, a first binding partner and a second binding partner, the first binding member capable of binding to the first binding partner, the first binding partner capable of binding to the analyte, the second binding member capable of binding to the second binding partner, the second binding partner aiding detection of either bound or unbound analyte in the reaction mixture;
(ii) a test sample containing the analyte and heparin as an interfering substance that is capable of interfering with the assay by interacting with the second binding partner, and
(iii) an additional mount of the heparin in a quantity effective to substantially mask the effect of the interfering substance in the test sample, and wherein the additional amount of heparin is in an effective quantity such that substantially all of the second binding partners in the reaction mixture are non-specifically bound to the interfering substance heparin;
b. incubating the reaction mixture undex conditions sufficient to allow the formation of a complex between the analyte and the first binding partner; and
c. determining the presence of the analyte in the test sample by measuring the extent of any formation of the complex, the extent being proportional to the concentration of the analyte in the sample.

### Claim 2 (depends on 1)

The assay of claim 1, wherein the first and second binding members are members of a bidentate compound.

### Claim 3 (depends on 1)

The assay of claim 1, wherein the first binding member is selected, from the group consisting of theophylline, carbamazepine, gentamicin, acetaminophen, phenytoin, and phenobarbital.

### Claim 4 (depends on 2)

The assay of claim 2, wherein the second binding member is biotin.

### Claim 5 (depends on 2)

The assay of claim 2, wherein the second binding partner is avidin.

### Claim 6 (depends on 5)

The assay of claim 5, wherein the avidin is conjugated to latex.

### Claim 7 (depends on 1)

The assay of claim 1, wherein the first binding partner which specifically binds to the analyte of interest is an antibody or an antibody fragment.

### Claim 8 (depends on 1)

The assay of claim 1, wherein the assay system further comprises a third binding member, the third binding member capable of being attached to the spacer intermediate.

### Claim 9 (depends on 8)

The assay of claim 8, wherein the first, second and third binding members are members of a tridentate compound.

### Claim 10 (depends on 1)

The assay of claim 1, wherein the additional amount of heparin added to the reaction mixture is from about 20 units to about 150 units.

### Claim 11 (depends on 1)

The assay of claim 1, wherein the additional amount of heparin added to the reaction mixture is from about 40 units to about 120 units.

### Claim 12 (depends on 1)

The assay of claim 1, wherein the additional amount of heparin added to the reaction mixture is from about 40 units to about 90 units.

### Claim 13 (depends on 1)

The assay of claim 1, wherein the test sample is selected from the group consisting of serum, plasma, saliva, cerebral spinal fluid, amniotic fluid, urine, feces, mucus, cell extracts, tissue extracts, and pus.

### Claim 14 (depends on 1)

The assay of claim 1, wherein the analyte of interest is selected from the group consisting of drugs, hormones, vitamins, enzymes, proteins, antibodies, polysaccharides, bacteria, protozoa, parasites, fungi, viruses, cell antigens, tissue antigens, blood cell substances, and blood fluid substances.

### Claim 15 (depends on 14)

The assay of claim 14, wherein the analyte of interest is a drug.

### Claim 16 (depends on 15)

The assay of claim 15, wherein the drug is selected from the group consisting of theophylline, carbamazepine, gentamicin, acetaminophen, phenytoin, phenobarbital, digoxin, valproic acid, thyroxine, disopyramide, lidocaine, procainamide, propranolol, guanidine, amikacin, chloramphenicol, kanamycin, tobramycin, tricyclic antidepressants, ethosuximide, primidone, acetylsalicylic acid, and methotrexate.

### Claim 17

An assay for detecting the presence of an analyte of interest in a test sample comprising the steps of:
forming a reaction mixture by combining in an assay medium:
(I) an assay system comprising a first binding member, a second binding member, a spacer member intermediate between the members, a first binding partner and a second binding partner, the first binding member capable of binding to the first binding partner, the first binding partner capable of binding to the analyte, the second binding member capable of binding to the second binding partner, the second binding partner aiding detection of either bound or unbound analyte in the reaction mixture;
(ii) a test sample containing the analyte and heparin as an interfering substance that is capable of interfering with the assay by interacting with the second binding partner, and
(iii) an additional mount of the heparin in a quantity effective to substantially mask the effect of the interfering substance in the test sample, wherein the additional amount of heparin is between about 40 units to about 90 units, and wherein the additional amount of heparin is in an effective quantity such that substantially all of the second binding partners in the reaction mixture are non-specifically bound to the interfering substance heparin:
b. incubating the reaction mixture under conditions sufficient to allow the formation of a complex between the analyte and the first binding partner, and
c. determining the presence of the analyte in the test sample by measuring the extent of any formation of the complex, the extent being proportional to the concentration of the analyte in the sample.

### Claim 18

A test kit for detecting the presence of an analyte of interest in a test sample, the test sample containing an interfering substance that is capable of interfering with the assay, the test kit comprising at least one container containing the following components which can be partially or completely pre-combined or uncombined;
a. an assay medium;
b. a first binding member capable of binding to a first binding partner for the analyte;
c. the first binding partner capable of binding to the analyte;
d. a second binding member capable of binding to a second binding partner;
e. the second binding partner aiding detection of either bound or unbound analyte in the reaction mixture, wherein the interfering substance in the test sample is heparin and is capable of interfering with the assay by interacting with the second binding partner, and
f. an additional amount of heparin in a quantity effective to substantially mask the effect of the interfering substance in the test sample and wherein the additional amount of heparin is in an effective quantity such that substantially all of the second binding partners in the reaction mixture are non-specifically bound to the interfering substance heparin.

### Claim 19 (depends on 18)

The test kit of claim 18, wherein the additional amount of heparin added is from about 20 units to about 150 units.

### Claim 20 (depends on 18)

The test kit of claim 18, wherein the additional amount of heparin added to the reaction mixture is from about 40 units to about 120 units.

### Claim 21 (depends on 18)

The test kit of claim 18, wherein the additional amount of heparin added to the reaction mixture is from about 40 units to about 90 units.

### Claim 22 (depends on 18)

The test kit of claim 18, wherein the test sample is selected from the group consisting of serum, plasma, saliva, cerebral spinal fluid, amniotic fluid, urine, feces, mucus, cell extracts, tissue extracts, and pus.

### Claim 23 (depends on 18)

The test kit of claim 18, wherein the analyte of interest is selected from the group consisting of drugs, hormones, vitamins, enzymes, proteins, antibodies, polysaccharides, bacteria, protozoa, parasites, fungi, viruses, cell antigens, tissue antigens, blood cell substances, and blood fluid substances.

### Claim 24 (depends on 23)

The test kit of claim 23, wherein the analyte of interest is a drug.

### Claim 25 (depends on 24)

The test kit of claim 24, wherein the drug is selected from the group consisting of theophylline, carbamazepine, gentamicin, acetaminophen, phenytoin, phenobarbital, digoxin, valproic acid, thyroxine, disopyramide, lidocaine, procainamide, propranolol, guanidine, amikacin, chloramphenicol, kanamycin, tobramycin, tricyclic antidepressants, ethosuximide, primidone, acetylsalicylic acid, and methotrexate.

### Claim 26 (depends on 1)

The assay of claim 1, wherein the additional amount of the interfering substance heparin is first added to the assay system.

### Claim 27 (depends on 17)

The assay of claim 17, wherein the additional amount of the interfering substance heparin first added to the assay system.

### Claim 28 (depends on 18)

The assay of claim 18, wherein the additional amount of the interfering substance heparin is first added to the assay system.