Tyrosyl-class antigenic conjugates, their preparation and antibodies raised thereto

• US 3,975,342 A
• Filed: 03/18/1974
• Issued: 08/17/1976
• Est. Priority Date: 05/15/1972
• Status: Expired due to Term

• Current Assignee: Biological Developments Incorporated
• Original Assignee: Biological Developments Incorporated
• Inventors: Gross, Stanley Joseph
• Primary Examiner(s): Turner, V. D.
• Assistant Examiner(s): Fagelson, A. P.
• Application Number:
  US05/451,812
• Time in Patent Office: 883 Days
• Field of Search: 424/8, 424/11, 424/12, 424/13, 424/177, 424/85, 424/88, 260/112 R, 260/112 B, 260/112.5, 260/121, 260/192, 260/207, 260/78 A
• US Class Current: 530/363
• CPC Class Codes: G01N 33/9406 Neurotransmitters
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/818 Human chorionic gonadotropin
• CPC Class Codes: Y10S 436/823 Immunogenic carrier or carrier per se
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins

Abstract

Tyrosyl, thyronine, and histidyl compounds of the formulas: ##SPC1##

Where R'"'"' is selected from the group consisting of --OH and --NHP_p and R" is selected from the class consisting of --H and --COP_p, where P_p is a polypeptide chain having from 1 to 250 peptide groups, with or without pendant groups, X is selected from the class consisting of H and I; when X is I, X'"'"' is selected from the group consisting of H and I; and n is 0 or 1, where X'"'"' is H when n is 0; are conjugated to a carrier by first forming a primary aromatic amine on the carrier, diazotizing the amine, and coupling the tyrosyl, histidyl, or thyronine through the diazo group to form a novel antigenic conjugate. This novel antigenic conjugate can be injected into an animal for production of specific antibodies which are useful in assaying for the target, particularly by radioassaying.

Claims

1. A synthetic antigen of formula:

##SPC3##

wherein the carrier is selected from the class consisting of proteins and synthetic proteins, and confers antigenicity, R'"'"'" is coupled to the carrier through a reactive group, the residue being substantially non-reactive, R'"'"'" being bonded to the aryl with up to three bonds, being substituted or unsubstituted, and having up to four rings and up to 20 aliphatic carbon atoms; and

the hapten has the formula;

##SPC4##

where R'"'"' is selected from the group consisting --OH and --NHP_p, and R" is selected from the class consisting of --H and --COP_p, where P_p is a polypeptide chain having from 1 to 250 peptide groups, with or without pendant groups;

X is selected from the class consisting of H and I;

X'"'"' is selected from the class consisting of H and I when X is I;

n is 0 or 1 and X'"'"' is H when n is 0;

bonding of the hapten to the diazo group being according to the direction of the ring members and

2. The synthetic antigen of claim 1 wherein the bond between the carrier

3. The synthetic antigen of claim 1 wherein the carrier is a protein and the imine moiety of the amide is bound to the carrier, the carbonyl being

4. The synthetic antigen of claim 1 wherein the carrier has a plurality of ##SPC5##

5. The synthetic antigen of claim 1 wherein the carrier is a protein selected from the class consisting of polylysine, bovine serum albumin, keyhole limpet hemocyanin, human immunogammaglobulin, and thyroglobulin.

6. The synthetic antigen of claim 1 wherein the hapten is:

##STR2##

7. An antibody specific to the antigen of claim 1.

14. The process of preparing the synthetic antigen of claim 1 comprising:

1. mixing, in aqueous acid solution at a temperature of from -10°

C. to 60°

C., an antigenic protein and a primary aminoaryloic acid;

2. adding a carbodiimide to the mixture of step (1) and reacting to produce an antigen-aminoaryloic conjugate;

3. removing the unreacted reactants from the reaction product;

4. separating the antigen-aminoaryloic conjugate from the residual reaction medium;

5. diazotizing the amino group of the antigen-aminoaryloic conjugate by reaction in aqueous acid medium at a temperature of from 0°

to 5°

C.; and

,

6. adding the diazotized reaction product to an aqueous alkaline solution of the hapten, maintaining the reaction medium alkaline during the reaction, and stirring to complete the reaction.

15. The process of claim 14 wherein the removal of unreacted reactants is carried out by dialysis.

16. The process of claim 14 wherein the separation of the conjugate from the reaction medium is carried out by lyophilis.

8. 3,5-Diiodothyronine azobenzoyl bovine serum albumin.

9. An antibody specific to the antigen of claim 8.

10. 3'"'"' ,3,5-Triiodothyronine azobenzoyl bovine serum albumin.

11. An antibody specific to the antigen of claim 10.

12. Poly-1-lysine benzoylazoglcagon.

13. An antibody specific to the antigen of claim 12.

17. A process for preparing an antigen of the formula:

##SPC6##

wherein the carrier is selected from the class consisting of proteins and synthetic proteins, and confers antigenicity, R'"'"'" is coupled to the carrier through a reactive group, the residue being substantially non-reactive R'"'"'" being bonded to the aryl with up to three bonds, being substituted or unsubstituted, and having up to four rings and up to 20 aliphatic carbon atoms; and

the hapten has the formula;

##STR3## where R'"'"' is selected from the group consisting of -OH and -NHP_p'"'"' and R" is selected from the class consisting of --H and --COP_p'"'"' where P_p is a polypeptide chain having from 1 to 250 peptide groups, with or without pendant groups;

X is selected from the class consisting of H and I;

X'"'"' is selected from the class consisting of H and I when X is I; and

n is 0 or 1 and X'"'"' is H when n is 0;

comprising;

a. conjugation of a primary aromatic amine with a carrier;

b. separation of the reactants from step (a);

c. lyophilization of the residue to remove the reaction medium;

d. diazotization of the residue; and

,

e. coupling of the hapten to the resulting product.

18. The process of claim 17 further comprising the removal of small molecular products of the amine conjugation step, prior to diazotization.

19. A method for forming a synthetic antigen from a carrier selected from the class consisting of proteins and synthetic proteins, said carrier having an available reactive group for coupling thereto, a hapten having a reactive group selected from the class consisting of amino and carboxyl, and a carbon atom susceptible to diazonium coupling, and a primary aromatic amine compound having a reactive residue capable of coupling to the reactive group on the carrier, wherein the carrier reactive group and the primary amine compound reactive residue consist of a carboxyl group and an amino group, whereby the carrier can be coupled to the primary amine compound by an amide group, the method comprising:

a. conjugation of the primary aromatic amine compound with the carrier to form an amide bond;

b. treatment of the conjugated primary aromatic amine compound to provide for bonding of the hapten by formation of diazonium ion; and

c. coupling of the hapten to the conjugated diazonium ion.

20. The method of claim 19 wherein the carrier is a protein selected from the class consisting of polylysine, bovine serum albumin, keyhole limpet hemocyanin, human immunogammaglobulin, and thyroglobulin.

21. The method of claim 19 wherein the primary aromatic amine compound is para-aminobenzoic acid.