Method for enzyme immunoassay and peptide-enzyme conjugate and antibody therefor

• US 4,517,290 A
• Filed: 09/19/1983
• Issued: 05/14/1985
• Est. Priority Date: 03/31/1980
• Status: Expired due to Fees

• Current Assignee: WAKO Pure Chemical Industries Limited (Fujifilm Holdings Corporation)
• Original Assignee: Takeda Chemical Industries, Ltd. (Takeda Pharmaceutical Company Limited)
• Inventors: Kondo, Koichi, Iwasa, Susumu, Yoshida, Isamu
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/533,619
• Time in Patent Office: 603 Days
• Field of Search: 435/7, 435/188, 435/810, 424/85, 424/88, 436/547, 436/510, 436/814, 260/112.5 R
• US Class Current: 435/7.93
• CPC Class Codes: C07K 14/59 Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: C07K 16/26 against hormones ; against hormone releasing or inhibiting factors
• CPC Class Codes: C07K 5/1008 the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/76 Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/963 Methods of stopping an enzyme reaction or stabilizing the test materials
• CPC Class Codes: Y10S 435/967 Standards, controls, materials, e.g. validation studies, buffer systems
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/814 Pregnancy
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins
• CPC Class Codes: Y10S 930/11 Gonadotropin; related peptides

Abstract

In an enzyme immunoassay, when a specific antibody produced by contacting a peptide essential to the formation of a specific antibody to a peptide antigen, a freeze-dried material of β-D-galactosidase-enzyme conjugate or a peptide-enzyme conjugate prepared by coupling a labeling enzyme with a peptide of the general formula:

H-R.sub.1 -Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH

wherein R₁ is a peptide fragment consisting of 1 to 14 amino acid residues including Gly in the 14-position of the peptide Ala¹ -Pro² -Pro³ -Pro⁴ -Ser⁵ -Leu⁶ -Pro⁷ -Ser⁸ -Pro⁹ -Ser¹⁰ -Arg¹¹ -Leu¹² -Pro¹³ -Gly¹⁴ is used, a high reproducibility of the result of the enzyme immunoassay is obtained.

Claims

1. An enzyme immunoassay method involving the use of an antibody as a reagent, which comprises using an antibody obtained by inoculating human chorionic gonadotropin into an animal to produce an anti-human chorionic gonadotropin antibody, contacting a body fluid containing the anti-human chorionic gonadotropin antibody thus produced with a peptide of the formula:

space="preserve" listing-type="equation">H-R.sub.1 -Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH

wherein R₁ is a peptide fragment consisting of 1 to 14 amino acid residues including Gly in the 14-position of

space="preserve" listing-type="equation">Ala.sup.1 -Pro.sup.2 -Pro.sup.3 -Pro.sup.4 -Ser.sup.5 -Leu.sup.6 -Pro.sup.7 -Ser.sup.8 -Pro.sup.9 -Ser.sup.10 -Arg.sup.11 -Leu.sup.12 -Pro.sup.13 -Gly.sup.14,

wherein said peptide is synthetically prepared by chemically combining at least one member selected from the group consisting of amino acids and peptide fragments of fewer amino acids than said peptide, said peptide being insolubilized on a carrier, and eluting the anti-human chorionic gonadotropin antibody thus specifically absorbed.

2. A method as claimed in claim 1, wherein the peptide is H-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH.

3. An assay kit for the kit for the detection of human chorionic gonadotropin, which comprises:

(1) A portion of a peptide-β

-D-galactosidase conjugate which has an enzymatic activity of about 0.1 to 500 μ

U, the conjugate being prepared by coupling β

-D-galactosidase with a peptide of the formula;

space="preserve" listing-type="equation">H-R.sub.1 -Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH

wherein R₁ is a peptide fragment consisting of 1 to 14 amino acid residues including Gly in the 14-position of Ala¹ -Pro² -Pro³ -Pro⁴ -Ser⁵ -Leu⁶ -Pro⁷ -Ser⁸ -Pro⁹ -Ser¹⁰ -Arg¹¹ -Leu¹² -Pro¹³ -Gly¹⁴ ;

(2) A portion of anti-human chorionic gonadotropin antibody which binds about 5 to 100% of the enzymatic activity of the conjugate, the antibody being produced by contacting a peptide of the formula;

space="preserve" listing-type="equation">H-R.sub.1 -Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH

wherein R₁ is a peptide fragment consisting of 1 to 14 amino acid residues including Gly in the 14-position of Ala¹ -Pro² -Pro³ -Pro⁴ -Ser⁵ -Leu⁶ -Pro⁷ -Ser⁸ -Pro⁹ -Ser¹⁰ -Arg¹¹ -Leu¹² -Pro¹³ -Gly¹⁴, wherein said peptide is synthetically prepared by chemically combining at least one member selected from the group consisting of amino acids and peptide fragments of fewer amino acids than said peptide, said peptide being insolubilized on a carrier, with a body fluid containing an antibody reactive to human chorionic gonadotropin, and then eluting the thus-specifically absorbed antibody;

(3) From 0 to 500 Iu of standard human chorionic gonadotropin;

(4) A buffer solution which is used for diluting the above reagents (1) to (3) and the sample fluid to be assayed;

(5) A given amount of an insoluble anti-animal IgG-carrier conjugate which is sufficient to bind all of first antibody (2);

(6) From about 1 to 100 μ

G of a substrate;

(7) A buffer solution for washing the second antibody-carrier conjugate (5) and dissolving the substrate (6); and

4. (8) A buffer solution for terminating the enzymatic reaction.

6. The improvement as claimed in claim 4, wherein R₁ is a peptide fragment selected from the group consisting of Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly, Ser-Pro-Ser-Arg-Leu-Pro-Gly and Gly.

7. The improvement as claimed in claim 4, wherein the labeling enzyme is a glycosidase or phosphatase.

8. The improvement as claimed in claim 4, wherein the labeling enzyme is β

-galactosidase or alkaline phosphatase.

9. The improvement as claimed in claim 4, wherein the peptide-enzyme conjugate is a freeze-dried peptide-β

-D-galactosidase conjugate.

11. A method as claimed in claim 9, wherein the peptide is H-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH.

5. In a method for enzyme immunoassay of human chorionic gonadotropin involving the use of a peptide-enzyme conjugate as an assay reagent, an improvement which comprises using a peptide-enzyme conjugate prepared by coupling a labeling enzyme with a peptide of the general formula:

space="preserve" listing-type="equation">H-R.sub.1 -Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH

wherein R₁ is a peptide fragment consisting of 1 to 14 amino acid residues including Gly in the 14-position of the peptide Ala¹ -Pro² -Pro³ -Pro⁴ -Ser⁵ -Leu⁶ -Pro⁷ -Ser8-Pro⁹ -Ser¹⁰ -Arg¹¹ -Leu¹² -Pro¹³ -Gly¹⁴, and an antibody obtained by inoculating human chorionic gonadotropin into an animal to produce an anti-human chorionic gonadotropin antibody, contacting a body fluid containing the anti-human chorionic gonadotropin antibody thus produced with a peptide of the formula;

space="preserve" listing-type="equation">H-R.sub.1 -Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH

wherein R₁ is a peptide fragment consisting of 1 to 14 amino acid residues including Gly in the 14-position of Ala¹ -Pro² -Pro³ -Pro⁴ -Ser⁵ -Leu⁶ -Pro⁷ -Ser⁸ -Pro⁹ -Ser¹⁰ -Arg¹¹ -Leu¹² -Pro¹³ -Gly¹⁴, wherein said peptide is synthetically prepared by chemically combining at least one member selected from the group consisting of amino acids and peptide fragments of fewer amino acids than said peptide, said peptide being insolubilized on a carrier, and eluting the anti-human chorionic gonadotropin antibody thus specifically absorbed.

10. A method for producing a specific antibody, which comprises inoculating human chorionic gonadotropin into an animal to produce an anti-human chorionic gonadotropin antibody, contacting a body fluid containing the anti-human chorionic gonadotropin antibody thus produced with a peptide of the formula:

space="preserve" listing-type="equation">H-R.sub.1 -Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-OH

wherein R₁ is a peptide fragment consisting of 1 to 14 amino acid residues including Gly in the 14-position of Ala¹ -Pro² -Pro³ -Pro⁴ -Ser⁵ -Leu⁶ -Pro⁷ -Ser⁸ -Pro⁹ -Ser¹⁰ -Arg¹¹ -Leu¹² -Pro¹³ -Gly¹⁴, wherein said peptide is synthetically prepared by chemically combining at least one member selected fromfhe group consisting of amino acids and peptide fragments of fewer amino acids than said peptide, said peptide being insolubilized on a carrier, and eluting the anti-human chorionic gonadotropin antibody thus specifically absorbed.