US 5,116,725 AGranted

Assay for Epstein-Barr virus infection with solid phase bound synthetic polypeptides

AssigneeScripps Clinic and Research Foundation

InventorsJohn H. Vaughan, Dennis A. Carson, Gary Rhodes, Richard Houghten, Richard S. Smith, John E. Geltosky

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Abstract

Antigens, immunogens, inocula, antibodies, and particularly diagnostic methods and systems relating to Epstein-Barr virus nuclear antigen (EBNA) are disclosed. The diagnostic methods and systems utilize a synthetic, random copolymer polypeptide containing about 8 to about 40 amino acid residues that includes the overlapping five and six amino acid residue sequences (i) --Gly--R.sup.1 --Gly--R.sup.2 --Gly-- wherein R.sup.1 and R.sup.2 are amino acid residues selected from Ala, Asn, Arg, Gly, Leu, Pro, Ser, and Thr with the provision that R.sup.1 and R.sup.2 are not both Gly; and (ii) --Gly--Ala--Gly--Gly--Ala--Gly--. The polypeptide contains at least 50 mole percent Gly residues. The diagnostic method and system are particularly useful for assaying for the stage of mononucleois disease, and the presence of nasopharynegeal carcinoma. In an assay for determining the state of Epstein-Barr virus infection, the synthetic polypeptide is fixed to first and second solid phases supports which can be provided by a double-welled spoon having two adjacent solid phases. The solids phases are each coated with the synthetic polypeptide. After contacting each solid phase with a body sample, one is contacted with anti-human IgG an This invention was made with the support of the U.S. Government, and the U.S. Government has certain rights in the invention.

Claims2

Dependent claims

A method for determining the state of Epstein-Barr virus infection in a patient in about six minutes comprising the steps of:

(a) providing a solid phase matrix in the shape of a double-welled spoon, said spoon hvaing a generally flattened upper portion extending for about one-half the length of said spoon and adapted for being held by a user's fingers, the remaining, lower, one-half length portion of said spoon including a cylindrically-shaped portion that is narrower than said upper portion and extends about two-thirds the length of said lower portion, the lowest about one-third of said spoon containing a second flattened portion that is about the width of said upper portion and defines two dish-shaped depressions that are slightly smaller in diameter than the width of the second flattened portion, said two depressions being separated by a dam that extends at approximately right angles to said second flattened portion, said two depressions including a coating of a synthetic polypeptide containing about 15 to about 40 amino acid residues and having an amino acid residue sequence, written from left to right and in the direction from amino-terminus to carboxyterminus, that is selected from the group consisting of:
##STR6## the pharmaceutically acceptable salts thereof;
said synthetic polypeptide-coated depressions constituting adjacent solid phase supports;
(b) admixing an aliquot of a human body sample to be assayed with each of said solid supports in liquid medium to form first and second solid/liquid phase admixtures;
(c) maintaining said two solid/liquid phase admixtures for about two minutes;
(d) separating the solid and liquid phase of each of said solid/liquid phase admixtures to provide two separated solid phase-bound immunoreactants;
(e) admixing anit-human IgG antibodies having a linked indicating means to one of the two separated solid phase-bound immunoreactants of step (d) and admixing anti-human IgM antibodies having a linked indicating means to the other of said two separated solid phase-bound immunoreactants to form two second solid/liquid phase admixtures;
(f) maintaining said two solid/liquid phase admixtures for about two minutes;
(g) separating the solid and liquid phases, and washing the separated solid phases;
(h) admixing hydrogen peroxide and a color-forming dye precursor substantially simultaneously with each of said solid phases to form two color-forming solid/liquid reaction admixtures;
(i) stopping said two color-forming reactants after about two minutes;
and
(j) comparing the color intensity of the resulting two depressions, a more intense color in the depression containing anti-human IgG antibodies indicating that the patient is in convalescence, a more intense color in the depression containing anti-human IgM antibodies indicating that the patient is in the acute phase, and colors of about the same intensity indicating acute phase passing into convalescence.

10 more elements

A method for determining the state of Epstein-Barr virus infection in a patient in about six minutes comprising the steps of:

(a) providing a solid phase matrix in the shape of a double-welled spoon, said spoon having a generally flattened upper portion extending for about one-half the length of said spoon and adapted for being held by a user's fingers, the remaining, lower one-half length portion of said spoon including a cylindrically-shaped portion that is narrower than said upper portion and extends about two-thirds the length of said lower portion, the lowest about one-third of said spoon containing a second flattened portion that is about the width of said upper portion and defines two dish-shaped depressions that are slightly smaller in diameter than the width of the second flattened portion, said two depressions being separated by a dam that extends at approximately right angles to said second flattened portion, said two depressions including a coating of a synthetic polypeptide having an amino acid residue sequence, written from left to right and in the direction from amino-terminus to carboxy-terminus, H-Ala-Gly-Ala-Gly-Gly-Gly-Ala-Gly-Gly-Ala-Gly-Ala-Gly-Gly-Gly-Ala-Gly-Gly- Ala-Gly-OH, said synthetic polypeptide-coated depressions constituting adjacent solid phase supports;
(b) admixing an aliquot of a human body sample to be assayed with each of said solid supports in liquid medium to form first and second solid/liquid phase admixtures;
(c) maintaining said two solid/liquid phase admixtures for about two minutes;
(d) separating the solid and liquid phase of each of said solid/liquid phase admixtures to provide two separated solid phase-bound immoreactants;
(e) admixing horseradish peroxidase-conjugated anti-human IgG antibodies to one of the two separated solid phase-bound immunoreactants of step (d) and admixing horseradish peroxidase-conjugated anti-human IgM antibodies to the other of said two separated solid phase-bound immunoreactants to form two second solid/liquid phase admixtures;
(f) maintaining said two solid/liquid phase admixtures for about two minutes;
(g) separating the solid and liquid phases, and washing the separated solid phases;
(h) admixing hydrogen peroxide and ortho-phenylenediamine substantially simultaneously with each of said solid phases to form two color-forming solid/liquid reaction admixtures;
(i) stopping said two color-forming reactions after about two minutes;
and
(j) comparing the color intensity of the resulting two depressions, a more intense color in the depression containing anti-human IgG antibodies indicating that the patient is in convalescence, a more intense color in the depression containing anti-human IgM antibodies indicating that the patient is in the acute phase, and colors of about the same intensity indicating acute phase passing into convalescence.

8 more elements

Dependencies are inferred from the claim text.

Facts

Application: 07/117,241Filed 4 Nov 1987 · continuation in part
Priority: 8 Aug 1984
Granted: 26 May 1992
Term ends: Not estimatedThe source records don’t support an estimate.

Classification25 codes

A61K 39/245
Viral antigens: herpetoviridae, e.g. herpes simplex virus
A61K 39/12
Medicinal preparations containing antigens or antibodies: viral antigens
C07K 14/005
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: from viruses
C07K 16/085
DNA viruses: orthoherpesviridae , e.g. pseudorabies virus or Epstein-Barr virus