Immunoassay on a preblocked solid surface

• US 5,208,143 A
• Filed: 09/30/1991
• Issued: 05/04/1993
• Est. Priority Date: 11/17/1988
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Hopkins, Anne C., McLaurin, Daniel A., Henderson, Glenn L., Hoke, Randal A.
• Primary Examiner(s): Moskowitz, Margaret
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US07/767,351
• Time in Patent Office: 582 Days
• Field of Search: 435/5, 435/7.1, 435/18, 435/7.2, 435/2.97
• US Class Current: 435/5
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/56983 Viruses

Abstract

A method for immunoassay of a viral antigen is performed on a membrane precoated with an inert protein. Nonimmunological capture of antigen takes place by absorption onto the coated membrane. Captured antigen binds to a tracer which includes a label conjugated to a specific antibody, the inert protein concomitantly inhibiting nonspecific binding of tracer. The label may be an enzyme which converts a substrate to a detectable product or converts a blocked inhibitor to an inhibitor whereby a second enzyme is inhibited from converting a substrate to a product. The invention includes a kit of materials for performing an assay in accordance with the method of the invention.

Claims

1. A method for assay of a viral antigen in an aqueous liquid comprising:

a) combining a first aqueous liquid suspected of containing a viral antigen with a membrane precoated with an inert protein but not with a specific capture antibody, said protein being immunologically unreactive toward any other component of the assay, whereby said antigen nonimmunologically attaches to said precoated membrane;

b) separating said membrane having nonimmunologically attached viral antigen from said first liquid by causing said first liquid to pass through said membrane;

c) incubating said membrane with a second aqueous liquid containing an antibody specific for said antigen having a first hydrolase conjugated thereto whereby said viral antigen binds to said antibody to give a bound fraction including said first hydrolase on said membrane;

d) contacting said coated membrane with a third liquid containing a blocked fluoroketone inhibitor, a second hydrolase substantially unreactive toward said blocked fluoroketone inhibitor and a substrate for said second hydrolase whereby said first hydrolase on said support converts said blocked fluoroketone inhibitor to a fluoroketone which inhibits conversion of said substrate to a colored product by said second hydrolase; and

e) detecting said viral antigen by a signal associated with the color of said product.

2. The method of claim 1 wherein said first hydrolase is selected from the group consisting of a peptidase, esterase, phosphatase and glycosidase.

3. The method of claim 1 wherein said second hydrolase is an esterase.

4. The method of claim 3 wherein said substrate is selected from the group consisting of 3-indolyl acetate and butyrate.