Detection of hepatitis B surface antigen (or antibody to same) with labeled synthetic peptide

• US 4,591,552 A
• Filed: 09/29/1982
• Issued: 05/27/1986
• Est. Priority Date: 09/29/1982
• Status: Expired due to Term

• Current Assignee: New York Blood Center Incorporated
• Original Assignee: New York Blood Center Incorporated
• Inventors: Neurath, A. Robert
• Primary Examiner(s): NOT, DEFINED
• Application Number:
  US06/426,309
• Time in Patent Office: 1,336 Days
• Field of Search: 435/7, 436/828, 436/534, 436/804, 436/820, 260/112.5 R
• US Class Current: 435/5
• CPC Class Codes: G01N 33/5761 Hepatitis B
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/82 Hepatitis associated antigens and antibodies
• CPC Class Codes: Y10S 436/828 Protein A
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins
• CPC Class Codes: Y10S 930/142 Interferon
• CPC Class Codes: Y10S 930/221 Retrovirus related, or human immunodeficiency virus related, or simian immunodeficiency virus related
• CPC Class Codes: Y10S 930/222 Foot and mouth disease related
• CPC Class Codes: Y10S 930/223 Hepatitis related
• CPC Class Codes: Y10S 930/26 Containing cys-cys disulfide bridge between nonadjacent cysteine residues

Abstract

A radiolabeled or enzyme labeled peptide having no more than 60 amino acids in the chain of the peptide; the peptide having covalently linked amino acids disposed in a steric configuration which is recognized by and bound by an antibody. The labeled peptides can be utilized in various processes to detect the presence of a given antibody or antigen in a sample. Hepatitis B surface antigen and antibody to same may be so detected.

Claims

1. A process for the detection of antibodies to Hepatitis B in a sample which comprises:

a. contacting said sample with a solid substrate coated with an unlabeled synthetic peptide comprising the following sequence of amino acids;

Pro Ser Cys Cys Cys Thr Lys Pro Thr or Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser, which peptide is recognized by and bound by the antibodies suspected of being present in said sample, incubating and washing said contacted sample,

b. contacting the incubated, washed mass obtained from step a. above with a radiolabeled or enzyme labeled antibody to human or animal immunoglobulin, incubating and washing the contacted sample, and

c. determining the radioactivity or enzymatic activity of the resultant mass of step b. above.

2. A process for the detection of the presence of an antibody to Hepatitis B in a sample which comprises:

a. contacting said sample with a radiolabeled or enzyme labeled synthetic peptide comprising the following sequence of amino acids;

Pro Ser Cys Cys Cys Thr Lys Pro Thr or Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser, which peptide is recognized by and bound by the antibody suspected of being present in said sample, incubating and washing said contacted sample,

b. adding Staphylococci bearing Protein A to said contacted sample, incubating and centrifuging the resultant mass,

c. washing the resultant mass and

d. determining the radioactivity or enzymatic activity of the resultant pelleted bacteria.

3. A process for detection of the presence of an antibody to Hepatitis B or Hepatitis B surface antigen in a sample which comprises

a. contacting a first portion of a solid substrate coated with antibody with a mixture of said sample and a radiolabeled or enzyme labeled synthetic peptide having no more than 60 amino acids in the chain of the peptide, which amino acids mimic at least one antigenic determinant of a natural antigen, which peptide is recognized by and bound by the antibody in said solid substrate, incubating and washing said contacted first portion, to obtain a first resultant composition, wherein the peptide comprises the following sequence of amino acids:

space="preserve" listing-type="equation">Pro Ser Cys Cys Thr Lys Pro Thr

or

space="preserve" listing-type="equation">Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser;

b. in a control, contacting a second portion of said solid substrate coated with said antibody with the same amount of said radiolabeled or enzyme labeled peptide as in step a. above, incubating and washing said contacted second portion, to obtain a second resultant composition,

c. detecting the radioactivity or enzymatic activity of said first resultant composition from step a. above, and

d. determining the radioactivity or enzymatic activity of said second resultant composition from step b. above, and

e. comparing activities from steps c. and d. above, such that if the radioactivity or enzymatic activity from step c. is less than from step d., then the sample is positive for the antibody in the sample.

4. A process for detecting the presence of Hepatitis B surface antigen in a sample which comprises:

a. contacting a first portion of a composition containing an antibody to said antigen with a mixture of said sample and a radiolabeled or enzyme labeled synthetic peptide having no more than 60 amino acids in the chain of the peptide, which amino acids mimic at least one antigenic determinant of a natural antigen, which peptide is recognized by and bound by said antibody, incubating and washing said first portion, wherein the peptide comprises the following sequence of amino acids;

space="preserve" listing-type="equation">Pro Ser Cys Cys Cys Thr Lys Pro Thr

or

space="preserve" listing-type="equation">Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser,

b. contacting a second portion of said composition containing antibody with the same amount of said radiolabeled or enzyme labeled peptide in an antigen free control, incubating and washing said second portion,

c. adding the same amount of Staphylococci bearing protein A to each of the compositions of steps a. and b. above, incubating both of said compositions, centrifuging each of said compositions and separating the supernatant liquid from the solids therein,

d. determining the radioactivity or enzymatic activities of each of the resultant compositions from step c, above, and

e. comparing the respective radioactivity or enzymatic activity from step d. above such that if the activity for the resultant composition containing the first portion is less than the activity for the resultant composition containing the second portion, then the sample contains antigen.

5. A process for the detection of antibodies to Hepatitis B surface antigen in a sample which comprises:

a. contacting said sample with a solid substrate coated with an unlabeled synthetic peptide having no more than 60 amino acids in the chain of the peptide which amino acids mimic at least one antigenic determinant of a natural antigen, which peptide is recognized by and bound by the antibody suspected of being present in the sample, incubating and washing said contacted sample, wherein the peptide comprises the following sequence of amino acids;

space="preserve" listing-type="equation">Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys,

b. contacting the incubated, washed mass obtained from step a. above with;

a radiolabeled or enzyme labeled peptide having less than 60 amino acids in the chain of the peptide, which amino acids mimic said antigenic determinant or determinants, which peptide is recognized by and bound by the antibody suspected of having been present in said sample, said peptide comprising the following sequence of amino acids;

space="preserve" listing-type="equation">Cys Cys thr Lys Pro Thr Asp Gly Asn Cys Thr Cys,

or with a radiolabeled or enzyme labeled antibody to human or animal immunoglobulin, incubating and washing the resultant mass, and

c. determining the radioactivity or enzymatic activity of the resultant mass of step b. above.

6. A process for the detection of antibodies to Hepatitis B surface antigen in a sample which comprises:

a. contacting said sample with a radiolabeled or enzyme labeled synthetic peptide having no more than 60 amino acids in the chain of the peptide, which amino acids mimic at least one antigenic determinant of a natural antigen, which peptide is recognized by and bound by the antibody suspected of being present in said sample, incubating and washing said contacted sample, wherein the peptide comprises the following sequence of amino acids;

space="preserve" listing-type="equation">Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys,

b. adding Staphylococci bearing Protein A to said contacted sample, incubating and centrifuging the resultant mass,

c. washing the resultant mass and

d. determining the radioactivity or enzymatic activity of the resultant pelleted Staphylococci bacteria.

7. A process for detecting the presence of a Hepatitis B surface antigen in a sample which comprises:

a. contacting a first portion of a solid substrate coated with antibody which is an antibody to the antigen suspected in same sample with a mixture of said sample and a radiolabeled or enzyme labeled synthetic peptide selected from the group consisting of ##STR10## which peptide is recognized by and bound by the antibody in said solid substrate, incubating and washing said contacted first portion, to obtain a first resultant composition,

b. in a control, contacting a second portion of said solid substrate coated with said antibody with the same amount of said radiolabeled or enzyme labeled peptide as in step a. above, incubating and washing said contacted second portion, to obtain a second resultant composition,

c. detecting the radioactivity or enzymatic activity of said first resultant composition from step a. above,

d. determining the radioactivity or enzymatic activity of said second resultant composition from step b. above, and

e. comparing activities from steps c. and d. above, such that if the radioactivity or enzymatic activity from step c. is less than from step d., then the sample is positive for the antigen in the sample.

8. A process for the detection of Hepatitis B surface antigen in a sample according to claim 7 wherein said synthetic peptide is enzyme labeled.