JPS6012576B2 - Immunoassay for thymopoietin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides thymopoietin (ratio mopoietin)
) related to immunoassays.

Thymopoietin is a thymic polypeptide hormone.
This promotes the differentiation of prothymocytes into thymocytes and has secondary effects on neuromuscular transmission.

One form of bovine thymopoietin, designated thymopoietin 1 and 0, has been isolated and shown to be immunologically cross-reactive. (Thymopoietin is now more commonly used than its original name, thymin.) G. Goldstein, Nature (G.
．． ld-spin, natme), 247, 11 (1
974). Thymopoietin can be detected by bioassays using its effects on T cell differentiation and neuromuscular transmission, but these assays are complex, time consuming, and difficult to standardize.

Furthermore, unlike immunoassays, and especially unlike radioimmunoassays, bioassays are not easily automated and are therefore not routinely performed in clinical laboratories or other diagnostic facilities where large numbers of samples can be sorted in an economical manner. could not be adopted. A preferred immunoassay uses approximately 0.1 ng of the hormone in a biological fluid sample such as a blood count or serum.
It has a sensitivity of up to /'.

Therefore, this assay can be used as a diagnostic test for disease states exhibiting high (myasthenia gravis) or low concentrations (aging, immunodeficiency or immunologically mediated diseases, cancer, malnutrition, infectious diseases, etc.) of thymopoietin. ■ Can be adopted
One form of thymopoietin cannot be identified by immunoassays. The antigen used to prepare the antibodies for this assay is easily obtained by conjugating thymopoietin with an immunogenic carrier substance in a manner known per se.

Such linkage is preferably obtained by using suitable bifunctional linking groups. A preferred bifunctional linking group for this purpose is a C2-7 dialcanal such as glutaraldehyde. ``Immunogen carrier material'' (immunogen carrier material)
The term real is meant to include substances that have the property of independently eliciting an immunogenic response in the host animal and are capable of binding to L or thymopoietin.

Suitable carrier materials include, for example, proteins; natural or synthetic polymeric compounds such as polypeptides such as polylysine and copolymers of amino acids; polysaccharides, and the like. Particularly preferred carrier materials are proteins and polypeptides, especially proteins. Identification of the protein material used in the preparation of the antigens of the invention is not critical.

Examples of suitable proteins useful in the practice of the invention include corneal mammal serum proteins. For example, human gamma. Glopulin, human serum albumin, bovine serum albumin,
Mytylated bovine serum albumin, rabbit serum albumin, bovine gamma. globulin, and horse gamma globulin. Other suitable proteins will be suggested to those skilled in the art. Although it is generally preferred, it is not necessary to use proteins that are unrelated to the animal host for which the resulting antigen will be used. Coupling of thymopoietin and immunogenic carrier material can be accomplished by methods known in the art.

Preferred methods include equivalent bonding and physical bonding, eg electrostatic. Thus, for example, if the coupling is carried out in such a way that an equivalent bond is obtained, a bifunctional linking group, such as glutaraldehyde, can be attached to the ester. S. Avrameas,
lmm skin o-chemisoy), 6, 43 (1969
) may be used under the conditions described in ). While the use of bifunctional linking groups is the preferred method for obtaining equivalent linkage of thymopoietin and immunogenic carrier material, other methods of linkage may also be utilized.

One such method is “Science”.
ce), Vol. 14, pp. 1344-1346 (published June 12, 196), the carbodimide method may be used. As discussed in this article, carbodiimides can be used to bond materials containing many types of functional groups, including carboxy acids and amines. In this alternative method, the coupling proceeds to form an equivalent bond by coupling thymopoietin with the protein through an amide bond in the manner described in this article in Science. One method of conjugation may also be used, as disclosed in US Pat. For example, electrostatic bonding is carried out using means known to those skilled in the art.The method proceeds by the formation of a Z-anchor compound between the carrier and thymopoietin;
“Methods in Immunology and Immunology” (Methods in Immunology and Immunochemistry) Curtis A. Williams
s) Line, Volume 1, Akade Z Mitsuku Press (Acade
micPress) (1967). Another method is described in US Pat. 3.8 Mouse It is mentioned in scale 7. Of course, it is to be understood that other methods known to those familiar with this technique may also be used to combine thymopoietin and saline.

The antigen of the present invention is used to inject this antigen into a host animal, preferably by known methods.

By injecting it with Indian supplement fluid, specific antibodies against thymopoietin are generated in the host. Better titers can be obtained by repeating injections over a period of time.
Host animals suitable for this purpose include rope mammals such as rabbits, horses, goats, guinea pigs, rats, cows, and sheep. The resulting antiserum will contain antibodies that selectively bind to either thymopoietin or an antigen made therefrom, as described above. The thymopoietin-specific antibodies produced according to the present invention are useful as reagents in immunoassays for thymopoietin.

In a preferred embodiment, the antiserum diluted in a suitable buffer solution, such as 5% bovine gamma globulin in 0.1 M sulfate-balanced saline 6.5, is combined with the standard or test sample and the radiolabel. None of the known amounts of thymopoietin
Mix with the song dissolved in buffer solution. The amount of thymopoietin present in the test sample is then determined by various methods.

In the first method, the above ingredients are mixed, the mixture is left at room temperature for several hours, and then the antibody-antigen complex is prepared in a cold
% polyethylene glycol. After centrifugation, the supernatant was aspirated and the radioactivity in the precipitate was examined. The thymopoietin content in this sample can then be determined in a manner known per se by comparison with the radioactivity levels found in a standard curve. A suitable standard curve can be constructed by mixing known amounts of thymopoietin with labeled thymopoietin and fixed amounts of an antibody specific for thymopoietin and measuring the degree of binding for each known amount. Two types of separation methods can be used to remove antigen-antibody complexes.

These two methods are in fact preferred over the polyethylene glycol method described above due to the better sensitivity of the assay. One such method uses a double antibody method.

After incubating the above three-component assay reaction mixture, add antibodies obtained from other mammals to the first assay antibody, mix these components, and then leave at room temperature for 5 to 120 minutes. Centrifuge the mixture. After aspirating the supernatant fluid, the radioactivity of the precipitate is measured, and the thymopoietin concentration in the sample is determined from the above standard curve. Another method uses dextran-coated charcoal to aid in the separation of antibody-antigen complexes.

In this method, "dextran-coated charcoal is added to the assay reaction system after incubation. A low temperature of about 4°C is used.
After standing at about 4° C. for 0 minutes, the mixture is spun down and the supernatant liquid is aspirated. Radioactivity is determined on the precipitate and in this method represents "unbound" thymopoietin. In a preferred embodiment of the present assay system, the physiological saline used in the Tako C. Equilibrium system is replaced with MKCI.

This alternative reduces non-specific binding during the assay without the attendant reduction in sensitivity. Labeled thymopoietin suitable for use in the immunoassays of the invention include radioisotope-labeled thymopoietin, particularly tritium (3H), carbon-14 (14C), iodine-125 (1A1).
), or labeled with iodine-131 (1311). For other immunological embodiments according to the invention, using thymopoietin labeled with other unique and detectable labels, such as chromophores, photophores, enzymes, red blood cells, latex particles or electronic resonance groups. Good too. The most preferred radiolabeled thymopoietin is 51 thymopoietin. The 51 label can be introduced into thymopoietin using methods known in the art, such as the chloramine-T method, or more preferably by Biochem. Jay. (Bioc
hemJ. ) i339529, (1973) Bolton-Hunter reagent (1251 P-hydroxyphenylphenyl iodinated propionic acid)
lproplomcacid), N-hydroxysuccinimide. Estelle (N-hydro guy's Mci)
This can be done by using ``nimideester''. This is preferred because direct iodination of the tyrosyl moiety of thymopoietin results in some loss of immune activity. However, since the Bolton G Hunter reagent condenses with free amino groups, it does not affect the immunodetermined tyrosyl moieties.Other labeled thymopoietins as described above are made by means known to those skilled in the art.

For example, enzyme-labeled thymopoietin was released on April 4, 1972.
Date U.S. Patent No. 39 654,090. Also ~ United States Patent Ship dated December 10, 197 France. No. 3,853,987 describes the use of tracer substances, such as crab-light compounds and latex systems, for labeling purposes. These methods of labeling are therefore fully described in the prior art. Therefore, the labeled thymovoietin of the present invention can also be labeled with a radioabesotope ~ with the other three substances mentioned above. The immunoassay of the present invention was tested with various control polypeptides and confirmed to be specific for thymopoietin by observing no migration of the antibody-labeled antigen complex. Shown.

In particular, there was no cross-reactivity with ubiquitin, a substance widely distributed in tissues, and histones extracted from bovine thymus.

Synthetic tridecabep polytide based on thymopoietin Zanko 29-41 has the biological activity of thymopoietin, but it did not cross-react. Apparently, there is no residual or tertiary structure in this area that is necessary to reconstitute the antigenic site seen by the anti-thymopoietin antibodies in the antiserum.The invention is further illustrated by the following examples. Example 1 Preparation of antigen and antibody Goldstein Nature (in C℃lds, N
thymopoietin was isolated as described by J.D. atme) 247; 11 (1974).

It is called ``Avrameas; lmmunochemis'' for immunity.
Coupling was carried out with an equal amount of horse gamma-globulin using glutaraldehyde as a coupling agent according to the method of J. T. Sha) 6, 43 (1969). The resulting antigen was then used for the preparation of thymopoietin antibodies. Each of three female San Jouan guinea pigs was immunized with 400 ml of thymopoietin suspended in T.Freund's complete supplement at several locations.

Immunizations were repeated four times at two-week intervals, and the animals were bled to death one week after the last injection. Example 2 Radiolabeling of thymopoietin a Chloramine-T method Thymopoietin was further equilibrated to pH 4.5 in 0.2 ammonium acetate with a linear gradient of 0.9 M, PH.
4.

Ruboxymethyl-cephadex (CM-S)
ephadex) (0.6 x 30 skin column).

Thymopoietin appears immediately after the void volume, and these fractions become lyophilic and L-Sephadex.
) Desalted with G-25. Polyacrylamide. The gel was made pure by electrolysis pH 4.3 and pH 8.9. very<
Highly pure thymopoietin 0.01 9
,and.

Greenwood Nature ~ (Nichihada terand baitee
nwoodF Natme)? 194,495 (19
The radioactivity was labeled using the quaramine method of 62) on Fox Ci carrier free 1 part, and unreacted on Sephadex G-25 (0.6 x 30 columns) in 0.09 phosphate pH 7.5. Armpit 1 thymopoietin was isolated from radionuclide.

The column was prepurified with 0.25% gelatin in phosphate buffer as well.

The radioactivity of one sample taken from each fraction was measured using an automatic gamma spectrometer. The fraction corresponding to the amount of voids was divided into volumes with 0.1 scale steps and stored at 120:00. ,3
Use within a week. b Bolton-Hunter method 2 millicuries of 1251 iodized pea hydroxyphenyl propionic acid dissolved in benzene.
N-hydroxysuccinimide, ester (p-hy
&o phenylpropion acidN-hy
using 150 mmoles of Bolton-Hunter reagent).
Bolton and Hunter, Biochem.G.I., (
Bollin and Hunter, Biochem.

J. ) 133, 529 (1973) 4 by the method described
The thymopoietin was iodized at ℃. Dry the Bolton-Hunter reagent in a steam hood by passing a stream of nitrogen over the mouth of the vial and add 10 μg of L thymopoietin (0.1
0.5 m 9 /similar; p day 8.5) in M sodium sulfate was added and kept at 4°C for 45 minutes. 0 in 0.1M sodium shelfate.

Half a milliliter of 2M glycine pH 8.5 was further added to react with the unconjugated reagents.

After 18 minutes, thymopoietin was separated from the other labeled products of the conjugate by the method described in (a) above.

Only 5% of the radioiodinated thymopoietin by the Chloramine-T method was bound in the presence of excess antibody, and this binding decreased at dilutions of 10-3 and above.

In contrast, 45% of radioiodinated thymopoietin was bound in the presence of excess antibody using the Bolton-Hunter method. This binding also decreased with antibody dilutions of 10-3 and above. Example 3a Radioimmunoassay - Polyethylene Glycol Separation Cultures were performed in triplicate in 12 x 75 sided plastic tubes.

Antiserum diluted with 5% bovine gamma globulin in 0.01M phosphate buffered 0.18M NaCI (B is buffer).
A sample of 0.2' in 5' and 1 ring 1 of 0.3
- Thymopoietin (50,00 pm, in BGG buffer) was added.

When assaying serum samples, 0.1M at pH 8.0
Prepared by molecular chromatography on G-50 Sephadex in ammonium bicarbonate. The fractions after the excluded volume (Vo) and the first included volume (Vi) are pooled, lyophilized and the powder is collected in buffer for thymopoietin.

This preparation serves to exclude molecules with molecular weights larger or smaller than thymopoietin from the assay. The assay tube was sterilized using a vortex mixer and left at room temperature for 2 hours.

Then add half a milliliter of cold 30% polyethylene glycol to each tube in a vortex mixer and mix for 3
The mixture was run in a refrigerated centrifuge for 0 minutes until it reached 2.00 specific pm. The supernatant liquid was aspirated, and the radioactivity in the precipitate was measured using an automatic gamma spectrometer. Precipitated 251 thymopoietin (%)
is calculated using the formula a-c(b-c)×100. child)
where a = cpm precipitated with antibody, b; total amount of cpm added,
c=cpm non-specifically precipitated with normal rabbit serum or antibodies and excess unlabeled thymopoietin (these are usually about 10% of total cpm). For the standard curve of binding inhibition, bound thymopoietin was calculated as the percentage of maximum thymopoietin bound to the antibody at 10-1. Thirteen thousand antibody dilutions were used for the standard curve of binding inhibition.

b. Radioimmunoassay Single Duplex Antibody Separation Method The above method (a) was repeated except when using the MKCIB equilibrium solution.

Then, after a 2-hour incubation period, a total of 0.1 secretion of goat anti-rabbit antibody and 0.02 volume of soil normal rabbit were added, the ingredients were mixed in a vortex mixer, and the resulting mixture was stirred for 5 minutes at room temperature. The supernatant was aspirated, and the precipitate was assayed for radioactivity as described in a. c. Radioimmunoassay - Dextran-coated charcoal separation method. (ii) NoritA charcoal 5 times per 100 skins of phosphate buffer containing CI and bovine gamma globulin; (ii) 0.5 times dextran 110 per 100 skins of phosphate buffer containing IMKCI and bovine gamma globulin; Make by mixing.

A total of 0.5 μm of dextran-coated charcoal is incubated as in (b) above for 2 hours and then added to assay tubes at 4°C, and the mixture is left at 4°C for 30 minutes.

These tubes were spun down and the supernatant fluid was aspirated. The radioactivity was measured on the pellet, which in this assay was “
Binding inhibition standard curve for unlabeled thymopoietin in assays employing polyethylene glycol separation of antigen-antibody complexes indicates unbound thymopoietin. He expressed his feelings for the matter.

At a concentration of 10 to 1,00 g/, insulin,
alpha-bung
arotoxin), ubiquitin (u
Biquitin), histones (histofertility), and thymopoietin synthesis tridecapeptide component (residue 29
No significant migration occurred compared to the control polypeptide containing -41).

Binding inhibition curves for unlabeled thymopoietin in assays using dual antibodies and dextran-coated charcoal separation of antigen-antibody complexes showed sensitivity up to 0.1 increased thymopoietin/similar thymopoietin concentrations.

Claims (1)

[Scope of Claims] 1. A labeled thymopoietin for use in an immunoassay of thymopoietin in a sample together with an antibody having specificity for an antigen consisting essentially of thymopoietin and thymopoietin bound to an immunogenic carrier material, comprising: Thymopoietin-specific antibodies are produced by inoculating a host animal with the above antigen,
It is prepared by collecting the serum from the host animal, and the labeled thymopoietin contains tritium, carbon^1^4, iodine^1^3^1, and iodine^.
1^2^5, a labeled thymopoietin selected from the group consisting of a chromophore, a fluorophore, an enzyme, an erythrocyte, a latex grain, and an electron spin resonance group. 2. Labeled thymopoietin according to claim 1, wherein the label is ^1^2^5I. 3. A method for assaying thymopoietin in a sample, in which the sample is mixed with a known amount of labeled thymopoietin and an antibody that selectively binds to the thymopoietin, and the resulting antibody-antigen complex is added to the supernatant. The degree of binding of the labeled thymopoietin in the complex is measured, and the degree of binding is determined by mixing a known amount of thymopoietin with a certain amount of the labeled thymopoietin and the antibody. A method for determining the amount of thymopoietin present in the sample by comparison with a standard curve obtained by measuring the degree of binding. 4. The method of claim 3, wherein a radioimmunoassay is used and radiolabeled thymopoietin is used. 5. The method of claim 4, wherein the antibody-antigen complex is separated from the solution with the aid of 30% polyethylene glycol. 6. The method of claim 4, wherein the antibody-antigen complex is separated from the solution with the aid of a double antibody method using an antibody against the serum of the host animal from which the thymopoietin antibody has been derived. 7. The method of claim 4, wherein the antibody-antigen complex is separated from the solution by the dextran-coated charcoal method, in which case the radioactivity of the precipitate represents unbound thymopoietin. 8 The radiolabeled thymopoietin is ^1^2^5
5. The method of claim 4, wherein the method is thymopoietin I. 9. The method according to claim 4, wherein the sample to be assayed is a plasma or serum sample.