# US 4,397,960 A · Immunoassays using F(AB').sub.2 fragments

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- Number: US 4,397,960 A
- Document ID: US4397960A
- Status: Granted
- Kind: A
- Application number: 06/362,334
- Filed: 1982-03-26
- Granted: 1983-08-09
- Earliest claimed: 1978-01-26
- Assignees: Technicon Instruments Corporation
- Inventors: Claude H. Moussebois; Pierre L. Masson; Jean-Pierre Vaerman; Joseph Limet; Cesar L. Cambiaso
- Web page: https://patentgrep.com/patent/US4397960A

## CPC

- A61K 39/44: Antibodies bound to carriers
- G01N 33/563: involving antibody fragments
- G01N 33/6857: {Antibody fragments}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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

Immunoassays for antigens or haptens are effected using, instead of an immunoglobulin antibody, the F(ab').sub.2 fragments thereof. In this way, interference from endogenous RF and Clq in the fluid under assay is avoided without the necessity of pre-treating the fluid to remove or inactivate the RF or Clq. The assays may be of various types including latex agglutination and competitive binding assays. A reagent for such use comprising F(ab').sub.2 fragments insolubilized on a water-insoluble substrate, especially a particulate substrate, is described.

## Claims

### Claim 1

A method of immunoassay which comprises reacting an antigen in a fluid with insolubilized F(ab').sub.2 fragments of an immunoglobulin which is specific to said antigen, the reaction being effected in the substantial absence of the (whole) immunoglobulin and of F(c) fragments thereof.

### Claim 2

A method of assaying a fluid by agglutination for an antigen therein, which comprises the steps of:
(a) mixing a sample of the fluid with F(ab').sub.2 fragments of an immunoglobulin which is specific to said antigen to form a reaction mixture, said fragments being bound to water-insoluble particles, wherein, to avoid interference from exogenous Clq and RF, the reaction mixture is substantially free from the whole immunoglobulin and the F(c) fragments thereof;
(b) incubating the mixture to allow reaction between the said F(ab').sub.2 fragments and any said antigen present to cause agglutination of said particles; and
(c) directly determining the extent to which agglutination of said particles has occurred in the mixture and thereby the presence and/or amount of said antigen in the fluid sample.

### Claim 3 (depends on 2)

A method according to claim 2, wherein said particles are latex particles.

### Claim 4 (depends on 2)

A method according to claim 2, wherein the extent of agglutination is determined by selectively counting the agglutinated or unagglutinated latex particles.

### Claim 5 (depends on 2)

A method according to claim 2, wherein said particles are magnetically attractable and wherein, after reaction between the antigen and the fragments, the particulate material is separated by using a magnetic field.

### Claim 6 (depends on 2)

A method according to claim 2, wherein the fluid under assay is a biological fluid.

### Claim 7 (depends on 2)

A method according to claim 2, wherein the fluid under assay is human serum.

### Claim 8 (depends on 2)

A method according to claim 2, which is effected by continuous flow techniques.

### Claim 9 (depends on 2)

A method according to claim 2, which is effected by a discrete manual technique.

### Claim 10

A reagent for use in immunoassay which comprises a suspension of finely divided particulate material having bound thereto the F(ab').sub.2 fragments of an immunoglobulin, the suspension being substantially free from the said whole immunoglobulin and F(c) fragments thereof.

### Claim 11 (depends on 10)

A reagent according to claim 10 in which the said particulate material comprises magnetically attractable material.

### Claim 12 (depends on 10)

A reagent according to claim 10 which is in the form of a latex suspension.

### Claim 13

A method of assaying a fluid by agglutination for an antigen therein, which comprises the steps of:
(a) mixing a sample of the fluid with labelled F(ab').sub.2 fragments of an immunoglobulin which is specific to said antigen to form a reaction mixture, the labels being water-insoluble particles, wherein, to avoid interference from exogenous Clq and RF, the reaction is substantially free from the whole immunoglobulin and the F(c) fragments thereof;
(b) incubating the mixture to allow a competition binding reaction between the F(ab').sub.2 fragments and any said antigen present;
(c) analyzing the amount of labelled fragments which have reacted with the antigen or remain unreacted in the mixture to determine the presence and/or amount of antigen in the fluid.