PATENT SPECIFICATION <«> 1 545 332 (21) Application No. 30177/77 (22) Filed 19 July 1977 CO (31) Convention Application No. 709565 CO (32) Filed 28 July 1976 M3 (31) Convention Application No. 810269 (32) Filed 27 June 1977 in S (33) United States of America (US) (44) Complete Specification published 10 May 1979 (51) INT CU C07G 7/00 (G01N 33/16) (52) Index at acceptance C3H 203 241 242 GIB BR (72) Inventor GIDEON GOLDSTEIN

(54) IMMUNOASSAY FOR THYMOPOIETIN

(71) We, SLOAN - KETTERING plasma or serum. Thus the instant assay can INSTITUTE FOR CANCER RESEARCH, be employed as a diagnostic test for disease a Corporation of the State of New York states which exhibit either elevated (myas- of 1275 York Avenue, New York, New York thenia gravis) or reduced (aging, immune defi- 50 10021, U.S.A. do hereby declare the inven- ciency or immunologically medicated diseases, tion for which we pray that a patent may be cancer, malnutrition, infections and the like) granted to us and the method by which it is levels of thymopoietin. The two forms of to be performed to be particularly described thymopoietin are indistinguishible in immuno- in and by the following statement: — assay procedures. 55 10 Thymopoietin is a polypeptide hormone of The antigen utilized to prepare the anti- the thymus that induces differentiation of body for the instant assay is readily obtained prothymoscytes to thymocytes and also has by bonding thymopoietin to an immunogenic secondary effects on neuromuscular transmis- carrier material in a manner known per se. sion. Two forms of bovine thymopoietin, Such bonding is preferably achieved by em- 60 15 designed thymopoietin I and II have been ploying a suitable Afunctional linking group. isolated and shown to be immunologically A preferred Afunctional linking group for cross reactive. (Thymopoietin is now used in this purpose is a C2-r dialkanal such as preference to the original name Thymin). glutaraldehyde. See G. Goldstein, Nature 247, 11 (1974). As used herein the term "immunogenic 65 20 Although thymopoietin can be detected carrier material" is meant to include those by bioassay using either its effects on T cell materials which have the property of in- differentiation or neuromuscular transmis- dependently eliciting an immunogenic res- sion, these assays are complex, time-consum- ponse in a host animal and which can be ing, and difficult to standardize. Moreover, coupled to thymopoetin. Suitable carrier 70 25 unlike immunoassay, and particularly radio- materials include for example, proteins; immunoassay, bioassays are not readily auto- natural or synthetic polymeric compounds mated and thus could not be routinely em- such as polypeptides, e.g., polylysine or co- ployed by clinical laboratories or other diano- polymers of amino acids; polysaccharides. stic facilities to screen large numbers of Particularly preferred carrier materials are 75 30 samples in an economic fashion. proteins and polypeptides, especially pro- According to a first aspect of the present teins. invention, there is provided an antigen con- The identity of the protein material utilized sisting essentially of thymopoietin bonded to in the preparation of an antigen of the instant an immunogenic carrier material. invention is not critical. Examples of suit- 80 35 According to a second aspect of the present able proteins useful in the practice of this invention there is provided an antibody invention include mammalian serum proteins specific to thymopoietin and to an antigen such as, for example, human gamma globulin, consisting essentially of thymopoietin bonded human serum albumin, bovine serum albu- to an immunogenic carrier material, said min, methylated bovine serum albumin, 85 40 thymopoietin specific antibody being pre- rabbit serum albuminmin, bovine gamma globu- pared by innoculating a host animal with the lin and equine gammmma globulin. Other suit- aforesaid antigen and collecting the serum able proteins will be suggested to one skilled from said host animal. in the art. It is generall;enerally preferrepreferred hut not A preferred immunoassay can give sensi- necessary that proteins be utilized which are 90 45 tivities down to about 0.1 ng/ml. of the foreign to the animal hosts in which the hormone in biological fluid samples such as resulting antigen will be employed. 2 1,545,332 2 The coupling of thymopoietin to the bodiment, antiserum diluted in a suitable immunogenic carrier material can be carried buffer such as 5% bovine gamma globulin in out in a manner well known in the art. Pre- 0.1 M phosphate - buffered normal saline, ferred procedures comprise use of covalent pH 7.4 (BGG - buffer) is mixed with a 5 bonding or physical bonding, e.g. electro- standard or test sample and also a known 70 statically. Thus, for example, when the coupl- amount of radiolabelled thymopoietin, both ing is carried out to achieve covalent bond- being dissolved in BGG buffer. ing, use of a bifunctional linking group such Various methods can then be utilized to as glutaraldehyde under the conditions des- determine the amount of thymopoietin 10 cribed by S. Avrameas, Immunochemistry 6, present in the test sample. In a first tech- 75 43 (1969) may be employed. nique, after mixing of the above components While use of a bifunctional linking group and allowing the mixture to stand for several is the preferred manner of achieving co- hours at room temperature, the antibody - valent bonding of thymopoietin to the antigen complex was precipitated using cold 15 immunogenic carrier material, other methods 30% polyethylene glycol. After centrifuga- 80 for bonding may also be utilized. In one tion, the supernatant is aspirated and the such method, use may be made of the carbo- radioactivity in die precipitate counted. The diimide technique as described in "Science", thymopoietin content of the sample can then Vol. 14, pages 1344—1346, June 12, 1964. be determined by comparing the radioactivity 20 As reported in this article, carbodiimides can level observed to a standard curve in a man- 85 be used to couple materials containing many ner known per se. A suitable standard curve types of functional groups including carb- can be obtained by mixing known amounts oxylic acids and amines. In this alternate of thymopoietin with fixed amounts of method, coupling proceeds to form covalent labelled thymopoietin and the thymopoietin 25 bonds by coupling of the thymopoietin to the specific antibody and determining the degree 90 protein through an amide linkage in the man- of binding for each known amount. ner described in this article from "Science". Alternate separation systems for removing A further method of covalent bonding utilizing the antigen - antibody complex may be cyanates which may be used is described in employed. Such alternate systems are in fact 30 U.S. Patent No. 3788,948, issued January preferred over the polyethylene glycol sys- 95 29,1974. tem described above as they yield greater Bonding of the thymopoietin to the carrier sensitivity for the assay. may also be achieved by physical means, for One such system involves the use of a example, by electrostatic bonding, using double antibody technique. After the incuba- 35 means known to those skilled in the art. This tion of the three component assay reaction 100 method proceeds by formation of a complex mixture as described above, an antibody between the carrier and thymopoietin and elicited in another mammalian species against may be carried out as described in "Methods the primary assay antibody is added, the in Immunology and Immunochemistry", components mixed, then after standing 5 40 edited by Curtis A. Williams, Vol I, Academic up to 120 minutes at room temperature, the 105 Press, (1967). Other methods are described mixture is centrifuged. After aspiration of the in United States Patent No. 3,853,987, supernatant, the precipitate is counted for issued December 10,1974. radioactivity and the thymopoietin level in It is, of course, to be understood that other the sample determined from a standard curve 45 methods known to those skilled in the art as above. 110 may be employed to bond the thymopoietin The other alternative system employs dex- to the carrier. tran coated charcoal to assist in separation The antigens of the present invention may of the antibody - antigen complex. In this be utilized to induce formation of antibodies technique, dextran coated charcoal is added 50 specific to thymopoietin in host animals by to the assay reaction system after incubation. 115 injecting the antigen in such a host, pre- A reduced temperature of about 4°C. is ferably using an adjuvant such as Freund's utilized. After standing at about 4°C. for adjuvant, using known methods. Improved about 30 minutes the mixture is centrifuged litres can be obtained by repeated injections and the supernatant aspirated. The precipi- 55 over a period of time. Suitable host animals tate is counted for radioactivity, which in 120 for this purpose include mammals such as this technique represents "unbound" thymo- rabbits, horses, goats, guinea pigs, rats, cows, poietin. and sheep. The resulting antisera will con- In preferred embodiments of the present tain antibodies which will selectively com- assay systems the normal saline utilized in the 60 plex with thymopoietin or an antigen pre- BGG buffer system is replaced with 4M KC1. 125 pared therefrom, as described above. This substitution reduces non-specific bind- The specific antibodies for thymopoietin ing in the assay without concomitant loss of prepared in accordance with the present sensitivity. invention are useful as reagents in an immuno- Suitable labelled thymopoietins for use in 65 assay for thymopoietin. & a preferred em- in the immunoassay of the present invention 130 2 1,545,332 3 include radioisotopically labelled thymo- immunization, it was coupled to an equal 65 poietins, particularly those labelled with tri- weight of equine gamma globulin with glu- tium (3H), carbon 14 (14C), iodine 125 (125I), taradehyde as the coupling reagent accord- or with iodine 131 (I31I). For other immuno- ing to the procedure of Avrameas, Immuno- 5 assay embodiments in accordance with this chemistry 6, 43 (1969). The resulting anti- invention one may employ thymopoietins gen was then employed in preparing thymo- 70 labelled with any other unique and detectable poietin antibody. label such as for example chromophores, Three female San Juan rabbits were each fluorophors, enzymes, red blood cells, latex immunized with 400 p.g of thymopoietin 10 particles or electron spin resonance groups. antigen emulsified in Freunds complete adju- A most preferred radiolabelled thymo- vant and injected intradermally in multiple 75 poietin is 125I thymopoietin. The introduc- sites. Immunization was repeated four times tion of the 125I label into thymopoietin can at bi-weekly intervals and the animals were be carried out by procedures known in the bled one week after the last injections. 15 art such as by using the chloramine-T method, or more preferably by using the Bolton- EXAMPLE 2 Hunter reagent (125I iodinated p - hydroxy- Radiolabelling of Thymopoietin 80 phenylpropionic acid, N - hydroxysuccin- (a) Chloramine-T Procedure imide ester) as described in Biochem, J. 133, Thymopoietin was further purified on 20 529 (1973). This preference is based on the carboxymethyl - Sephadex (CM - Sephadex fact that direct iodination of the tyrosyl (Registered Trade Mark)) (0.6x30 cm moieties of thymopoietin results in some loss column) equilibrated in 0.2 M ammmonium 85 of immunoreactivity. However, since the acetate, pH 4.5 and developed with a linear Bolton-Hunter reagent condenses with free gradient to 0.5 M, pH 4.5. Thymopoietin 25 amino groups it does not aSect the immuno- appeared closely behind the void volume and determinant tyrosyl regions. these fractions were lyophilized and desalted The remaining above-mentioned labelled on Sephadex G-25. Purity was established by 90 thymopoietins are prepared by means known polyacrylamide gel electrophoresis at pH 4.3 to those skilled in the art. For example, and pH 8.9. 30 enzyme labelled thymopoietins may be pre- Ten micrograms of the highly purified pared as described in United States Patent thymopoietin was radiolabelled with 2 mCi No. 3,654,090, issued April 4, 1972. Also, carrier free 125I by the chloramine method of 95 United States Patent No. 3,853,987, issued Hunter and Greenwood, Nature, 194, 495 December 10, 1974, describes methods for (1962) and 125I thymopoietin was separated 35 use of tracer materials such as fluorescent from unreacted radionuclides on Sephadex compounds and latex systems for labelling. G-25 (0.6 x 30 cm column) in 0.05 M phos- Accordingly, such methods of labelling are phate, pH 7.5. The column was prewashed 100 fully described in the prior art. Thus labelled with 0.25% gelatin in phosphate buffer. The thymopoietins of the present invention in- radioactivity of 1 u.l sample from each frac- 40 elude those radioisotopically labelled, as well tion was determined in an automated gamma as thymopoietins labelled with the other spectrometer and the fraction corresponding materials mentioned. to the void volume was divided into 0.1 105 The immunoassay of this invention was ml. aliquots and stored at —20°C for use shown to be specific for thymopoietin by est- within three weeks. 45 ing with various control polypeptides and observing no displacement of the antibody- (b) Bolton-Hunter Procedure labelled antigen complex. In particular no Two millicuries of 125I iodinated p - cross-reaction was obtained with ubiquitin, a hydroxyphenylpropionic acid N - hydroxy- 110 material which is widespread in tissues, and mccinimide ester (Bolton-Hunter reagent 50 with histones, which are extracted from «4500 Ci/mmole) dissolved in benzene were bovine thymus. A synthetic tridecapeptide utilized to iodinate thymopoietin at 4°C. by based on residues 29—41 of thymopoietin, the procedure described by Bolten and which has the biological activities of thymo- Hunter, Biochem, J. 133, 529 (1973). The 115 poietin did not cross react; apparently this Bolton-Hunter reagent was dried in a fume 55 region lacks either the residues and/or the hood by passing a stream of nitrogen over tertiary structure required to reconstitute the the mouth of the vial and 10 insulin, alpha-bungarotoxin, ubiquitin, The assay tubes are agitated on a vortex histone and a synthetic tridecapeptide frag- 30 mixer and left standing for two hours at ment of thymopoietin (residues 29—41) at room temperature. Half milliliter of cold 30% concentrations of 10 to 1,000 ng./ml. polyethylene glycol was then added to each Binding-inhibition curves for unlabelled 95 tube which was agitated in a vortex mixer thymopoietin in the assays employing the and centrifuged at 2,000 rpm in a refriger- double antibody and dextran coated charcoal 35 ated centrifuge for 30 minutes. The super- separation of the antigen-antibody complex natants were aspirated and the radioactivity both showed sensitivity to thymopoietin con- in the precipitates was determined in an centrations down to 0.1 ng thymopoietin/ 100 automated gamma spectrometer. l25I thymo- ml. These latter two procedures are thus poietin precipitated (%) was calculated especially suitable for measuring thymopoietin 40 according to the formula a—c (b—c)xl00 levels in serum samples. where a=cpm precipitated with antibody, b=total cpm added, and c=cpm precipitated WHAT WE CLAIM IS:— nonspecifically with normal rabbit serum or 1. An antigen consisting essentially of 105 with antibody and excess unlabelled thymo>- thymopoietin bonded to an immunogenic 45 poietin (these were usually approximately carrier material. 10% of total cpm). For the standard curve 2. An antigen according to claim 1 where- of binding inhibition the thymopoietin bound in bonding of said thymopoietin to said car- was calculated as a percentage of maximal rier material is by covalent bonding. 110 thymopoietin binding with antibody at 10-1. 3. An antigen according to claim 1 where- 50 An antibody dilution of 1:3000 was used in said covalent bonding is through a bi- for the standard curve of binding inhibi- functional linking group. tion. 4. The antigen of claim 3 wherein said bifunctional linking group is dialkanal. 115 (b) Radioimmunoassay - Double Antibody 5. The antigen of claim 4 wherein said Separation C2-7 dialkanal is glutaraldehyde. 55 The procedure of (a) above was repeated 6. The antigen of claim 1 wherein said except that 4M KC1 BGG buffer was used immunogenic carrier material is a protein. and after the two hour incubation period a 7. The antigen of claim 6 wherein said 120 total of 0.1 ml of goat anti-rabbit antibody protein is a mammalian gamma globulin. and 0.02 ml. of normal rabbit were added, 8. The antigen of claim 6 wherein said 60 the components mixed on a vortex mixer, protein is bovine serum albumin. the resulting mixture was allowed to stand 9. An antibody specific to thymopoietin at room temperature for 5 minutes and then and to an antigen consisting essentially of 125 was centrifuged. The supernatant was aspir- thymopoietin bonded to an immunogenic car- 2 1,545,332 5 tier material, said thymopoietin specific anti- antibody-antigen complex is separated from body being prepared by innoculating a host solution with the assistance of 30% poly- animal with the aforesaid antigen and collect- ethylene glycol. 40 ing the serum from said host animal. 16. The method of Claim 15 wherein the 5 10. The antibody of claim 9 wherein said antibody-antigen complex is separated from antigen consists of thymopoietin covalently solution with the assistance of a double bonded to a protein. antibody technique using an antibody against 11. The antibody of claim 10 wherein said the serum of the host animal in which the 45 covalent bonding is by a bifunctional linking thymopoietin antibody was elicited. 10 group. 17. The method of Claim 14 wherein the 12. The antibody of claim 10 wherein said antibody-antigen complex is separated from protein is selected from the group consisting solution with the assistance of a dextran coated of equine gamma globulin and bovine serum charcoal technique wherein the radioactivity 5C albumin and said bifunctional linking group of the precipitate represents unbound thymo- 15. is glutaraldehyde. poietin. 13. A method for the assay of thymo- 18. The method of Claim 14 wherein poietin in a sample, which method com- said radiolabelled thymopoietin is I125-thymo- prises mixing said sample with a known poietin. 55 amount of labelled thymopoietin and an anti- 19. The method of Claim 14 wherein the 20 body as defined in any one of Claims 9 to sample being assayed is a plasma or serum 12, which will selectively complex with said sample. thymopoietin, separating the resulting anti- 20. Labelled thymopoietin for use in the body-antigen complex from the supernatant, assay of thymopoietin as defined in any one 6( measuring the degree of binding of the said of Claims 13 to 19, when labelled with a 25 labelled thymopoietin in said complex and labelling medium selected from a group con- determining the amount of thymopoietin pre- sisting of tritium, carbon14, iodine131, sent in said sample by comparing said degree iodine125, chromophores, fluorophores, en- of binding to a standard curve obtained by zymes, red blood cells, latex particles and 65 mixing known amounts of thymopoietin with electron spin resonance groups. 30 fixed amounts of said labelled thymopoietin 21. Labelled thymopoietin according to and said antibody and determining the degree Claim 20 wherein the label is I25I. of binding for each known amount of thymo- poietin. HULSE & CO., 14. The method of Claim 13 wherein a Chartered Patent Agents, 35 radioimmunoassay is employed and • radio- Cavendish Buildings, labelled thymopoietin is used. West Street, 15. The method of Claim 14 wherein the Sheffield, SI 1ZZ. Printed for Her Majesty's Stationery Office, by the Courier Press, Leamington Spa, 1979 Published by The Patent Office, 25 Southampton Buildings. London, WC2A IAY, from which copies may be obtained.