Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OF THE INVENTION
Thymopoie-tin is a polypeptide hormone of the thymus
that indu¢es differen-tiation of prothymocytes to thymocytes
and also has secondary effects on neuromuscular transmission.
Two forms of bovine thymopoietin, designated thymopoietin I
and II have been isolated and shown to be immunologically
cross reactive. (Thymopoietin is now used in preference to
the original name Thymin)~ See G. Goldstein, Nature 247, 11 ~197~).
Although thymopoietin can be detected by bioassay
using either its effects on T cell differen-tiation or
neuromuscular transmission, these assays are complex, time-
consuming, and difficult to standardize. Moreover, unlike
immunoassay, and particularly radioimmunoassay, bioassays are
not readily automated and thus could not be routinely employed
by clinical laboratories or other diagnostic facilities to
screen large numbers of samples in an economic fashion.
DESCRIPTION OF lHE INVENTION
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The present invention relates to an immunoassay
for thymopoie-tin. A pre-ferred immunoassay can give sensitivities
ZO ; down to about O.1 ng/ml. of -the hormone in biological fluid
samples such as plasma or serum. Thus the instant assay can be
employed as a diagnostic test for disease states which exhibit either
elevated (myasthenia gravis) or reduced (aging, immune deficiency
or immunologically mediated diseases, cancer, malnutrition,
infect1ons and the like) levels of thymopoietin. The two forms
of thymopoietin are indistinguishable in immunoassay procedures. ;
The antigen utilized~to prepare the antibody ~or the
instant~assay is readily obtained by bonding thymopoietin to ~-
an 1mmunogenlc carrier mater1al 1n a manner known per se. Such -
bonding is pre~erably achieved by employing a suitable bifunctional ~ ;
l1nking graup. A preferred bifunctional linking group for this
purpose 1S a C2 7 dialkanal such as glutaraldehyde.
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As used herein the term "immunogenic carrier ma-terial"
is meant to include those materials which have the property
of independently eliciting an immunogenic response in a host
animal and which can be coupled to thymopoietin. Suitable
carrier materials include for example, proteins; na-tural or
synthetic polymeric compounds such as polypeptides, e.g.,
polylysine or copolymers of amino acids; polysaccharides; and the
like. Particularly preferred carrier materials are proteins
and polypeptides, ~specially pro-teins. `~
The identity of the protein material utilized in
the preparation of an antigen of the instant invention is not
critical. Examples o~ suitable proteins useul in the practice
o this invention include mammalian serum proteins such as,
for example, human gamma globulin, human serum albumin, bovine
serum albumin, methylated bovine serum albumin, rabbit serum
albumin, bovine gamma globulin and equine gamma globulin. Other
suitable proteins will be suggested to one skilled in the art.
It is yenerally preferred but not necessary that proteins
be utilized which are foreign to the animal hosts in which
the resulting antigen will be employed.
The coupling of thymopoietin to the immunogenic
carrier material can be carried out in a m~nner well known in
the art. Preferred procedures comprise use of covalent bonding
or physical bonding, e.g. electrostatically. Thus, for example,
when the coupling is carried out to achieve covalent bonding, use
of a bifunctional linking group such as glutaraldehyde under the
conditions described by S. Avrameas, Immunochemistry 6, 43 (1969)
may be employed.
While use o a bifunctional linking group is the
preferred manner of achieving covalent bonding of thymopoietin
to the immunogenic carrier materialj other methods for
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bonding may also be utilized. In one such method, use may be
made of the carbodiimide technique as described in "Science",
Vol. 14, pages 1344-1346, June 12, 1964. As reported in this
article, carbodiimides can be used to couple materials containing
many types of functional yroups including carboxylic acids and
amines. In this alternate method, coupling proceeds to form
covalent bonds by coupling of the thymopoietln to the protein
through an amide linkage in the manner described in this
article from "Scien~e". A further method of covalent bondiny~.
utilizing cyanates which may be used is described in U.S.
Patent No. 3,788,948, issued January 29, 1974.
Bonding of ~he thymopoietin to the carrier may also -
be achieved by physical means, for example, by electrostatic
bonding, using means known to those skilled in the art. This
method proceeds by formation of a complex between the carrier
and thymopoietin and may be carried out as described in "Methods
in Immunology and Immunochemistry", edited by Curtis A. Williams, ~ -
Vol. I, Academic Press, (1967). Other methods are described in
United States Patent No. 3,853,987, issued December 10, 1974.
It is,of course, to be understood that other methods
known to those skilled in the art may be employed to bond the
thymopoietin to the carrier.
The antigens of the present invention may be utilized
to induce formation of antibodies specific to thymopoietin in
host animals by injecting the antigen in such a hostl preferably
using an adjuvant such as Freund's adjuvant, using known methods.
Improved titres can be obtained by repeated injections over a
period of time. Suitable host animals for this purpose include
mammals such as rabbits, hoxses, goats, guinea pigs, rats, cows,
sheep, etc. The resulting antisera will contain antibodies which
will selectively complex with thymopoietin or an antigen prepared
therefrom, as described above.
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The specific antibodies for thymopoietin prepared in
accordance with the present invention are usefu] as reagents
in an immunoassay for thymopoietin. In a preferred embodiment,
antiserum diluted in a sui-table buffer such as 5% bovine gamma
globulin in 0.1 M phosphate - buffered normal saline, pH 7.4
(BGG - buffer) is mixed with a standard or test sample and also
a known amount of radiolabeled thymopoietin, both being dissolved
in BGG buffer.
Various methods can then be utilized to determine the
amount of thymopoietin present in the test sample. In a first
technique, after mixing of the above components and allowing the
mixture to stand for several hours at room temperature, the anti-
body - antigen complex was precipitated using cold 30~ poly-
ethylene glycol. After centrifugation, the supernatant is
aspirated and the radioactivity in the precipitate counted. The
thymopoietin content of the sample can then be determined by
comparing the radioactivity level observed to a standard curve
in a manner known per se. A suitable standard curve can be
obtained by mixing known amounts of thymopoietin with fixed
amounts of labeled thymopoietin and the thymopoietin specific
antibody and determining the degree of binding for each known
amount.
Alternate separation systems for removing the antigen-
antibody complex may be employed. Such alternate systems are
in fact preferred over the polyethylene glycol system described
above as they yield greater sensitivity for the assay.
One such system involves the use of a double antibody
technique. After the incubation of the three component assay re-
action mixture as described above, an antibody elicited in another
3~ mammalian species against the primary assay antibody is added, the
components mixed, then after standing 5 up to 120 minutes at
room temperature, the mixture is centri~uged. After aspiration
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of the supernatant, the precipitate is counted for radioactivity
and the thymopoietin level in the sample determined from a
standard curve as above.
The other alternative system employs dextran coated
charcoal to assist in separation of the antibody-a}ltigen complex.
In this technique, dextran coated charcoal is added to the assay
reaction system after incubation. A xeduced temperature of about
4C. is utilized. After standing at about 4C. for about 30
minutes the mixture is centrifuged and the supernatant aspirated.
The precipitate is counted for radioactivity, which in this
technique represents "unbound"~thymopoietin.
In preferred embodiments of the present assay systems
the normal saline utilized in the BGG buffer system is replaced
with 4M KCl. This substitution reduces non-specific binding in
the assay without concomitank loss of sensitivity.
Suitable labeled thymopoietins for use in the immuno-
assay of the present invention include radioisotopically labeled
thymopoietins, particularly those labeled with tritium (3H),
carbon 14(14 C), iodine 125 (125~), or with iodine 131(131I).
For other immunoassay embodiments i~ accordance with this invention
one may employ thymopoietins labeled with any other unique and
detectable label such as for example chromophores, fluorophors,
enzymes, red blood cells, latex partiales or electron spin ~
resonance groups. -
A most preferred radiolabeled thymopoietin is 1
thymopoietin. The introduction of the 125I label into thymopoietin
can he carried out by procedures known in the art such as by
using the chloramine-T method, or more preferably by using the
Bolton-~unter reagent (125I iodinated p-hydroxyphenylpropionic
acid, N-hydroxysuccinlmlde ester) as described in Biochem. J. 133,
529 (1973~. This preference is based on the fact ~hat direct
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iodination of the tyrosyl moieties of thymopoietin results in
some loss of immunoreactivityO However, since the Bolton-
Hunter reagent condenses with free amino groups it does not
affect the immunodeterminant tyrosyl regions.
The remaining above-mentioned labeled thymopoietins
are prepared by means known to those skilled in the art. For
example, enzyme labeled thymopoietins may be prepared as described
in United States Patent No. 3,654,090, issued April 4, 1972.
Also, United St~ates~Patent No. 3,853,987, issued December 10,`~1974,
describes methods for use of tracer materials such as fluorescent
compounds and latex systems for labeling. Accordingly, such
methods of labeling are fully described in the prior art. Thus
labeled thymopoietins of the present invention include those
radioisotopically labeled, as well as thymopoietins labeled with
lS the other materials mentioned. ,! "
The immunoassay of this irvention was shown to be
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specific ior thymopoietin by testing with various control
polypeptides and observing no displacement of the antibody-
labeled antigen complex. In particular no cross-reaction was
obtained with ubiquitin, a material which is widespread in tissues,
and with histones, which are extrac~d from bovine thymus. A
synthetic tridecapeptide based on residues"29-41 of thymopoietin,
which has the biological activities of -thymopoietin did not cross
react; apparently this region lacks either the residues and/or
the tertiary structure required to reconstitute the antigenic
sites recognized by the antithymopoietin antibodies in the
antiserum.
The present invention is further illustrated by the
following examples.
EXAMPLE 1
Antigen and Antibody Prepara-tion
Thymopoietin was isolated as described by Goldstein,
Nature 247, 11 ~1974)~ For immunization, it was coupled to an
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equal weight of equine gamma globulin with glutaradehyde as the
coupling reagent according to -the procedure of Avrameas,
Immunochemistry 6, 43 (1969). The resulting antigen was then
employed in preparing thymopoietin antibody.
Three female San Juan rabbits were each immunized with
400 ~g of thymopoietin antigen emulsified in Freunds complete
adjuvant and injected intradermally in multiple sites.
Immunization was repeated four ti.mes at bi-weekly intervals
and the animals were bled one week after the last injections.
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EXAMPLE 2
Radiolabeliny of Thymopoietin
(a) Chloram1ne-T Procedure
Thymopoietin was further purified on carboxy-
methyl-Sephadex~ CM-Sephadex) (0.6 x 30 cm column) equilibrated
in 0.2 M ammonium acetate, pH 4.5 and developed with a linear
gradient to 0.5 M, pH 4.5. Thymopoietin appeared closely
behind the void volume and these fractions were lyophilized
and desalted on Sephadex G-25. Purity was established by
polyacrylamide gel electrophoresis at pH 4.3 and pH 8.9.
Ten micrograms of the highly purified thymopoietin
was radiolabeled with 2 mCi carrier free 125I by the chloramine
method o Hunter and Greenwood, Nature, 194, 495 (1962) and
1 5I thymopoietin was separated from unreacted radionuclides
on Sephadex G-25 ~0.6 x 30 cm column) in 0.05 M phosphate,
pH 7.5. The column was prewashed with 0.25% gelatin in phosphate
buffer. The radioactivity of 1 ~lsample from each fraction
was determined in an automated gamma spectrometer and the fraction
corresponding to the void volume was divided into 0.1 ml.
aliquots and stored at -20C. for use within three weeks.
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(b) Bolton-Hunter Procedure
Two millicuries of 125I iodinated p-hydroxy-
phenylpropionic acid N-hydroxysuccinimide ester (Bolton-~unter
reagent ~1500 Ci/mmole) dissolved in benzene were utilized
to iodinate thymopoietin at 4C. by the procedure described
by Bolton and Hunter, Biochem. J. 133, 529 (1973). The Bolton-
Hunter reagent was dried in a fume hood by passing a stream
of nitrogen over t~e mouth of the vial and 10 ~1 of thymopoie~tin
(0.5 mg/ml. in 0.1 M sodium borate, pH 8.5) were added and held
at 4C. for 45 minutes. Half milliliter of-0.2M ~lycine i~
O.1 M sodium borate, pH 8.5 was then added to react with
unconjugated reagent. After 15 minutes I thymopoietin
was separated from the other Iabeled products of the conjugation
by the method described in (a) above.
Only 5% of thymopoietin radioiodinated by the
chloramine-T method were bound in the prçsence of excess antibody,
this binding decreasing at dilutions greater than 10 . By
contrast 45~ of thymopoietin radioiodinated by the solton-Hunter
method was bound in the presence of excess antibody; this
binding also decreased at antibody dilutions greater than 10 3.
EXAMPLE 3
ta) Radioimmunoassay-Polyethylene Glycol Separation
Incubations were carried out in triplicate in 12 x 75
mm plastic tubes. To 0.5 ml. of antiserum diluted in 5%
bovine gamma globulin in 0.01M phosphate-buffered 0~15M NaCl
(BGG buffer) was added 0.2 ml. of sample and 0O3 ml. of
125I-thymopoietin (~50,000 cpm in BGG buffer). When serum
samples are to be assayed they are prepared by molecular sieve
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chromatography on G-50 Sephadex in O.lM ammonium bicarbona-te,
pH 8Ø The fractions behind the excluded volumn (Vo)
and ahead of the included volumne (Vi) are pooled and
lyophilized and -the resulting powder -taken up in buffer
for thymopoietin. This preparation serves to exclude molecules
of molecular weights higher or lower than thymopoietin from
t~e assay.
The a~ssay tubes are agitated on a vortex mixer
and left standing for two hours at room temperature. Half
milliliter of cold 30% polyethylene gl~col was then added ~ ~-
to each tube which was agitated on a vortex mixer and
centrifuged at 2,000 rpm in a refrigerated centrifuge for
30 minutes. The supernatants were aspirated and the
radioactivity in the preci~itates was determined in an automated
lS gamma spectrometer. 125I thymopoietin precipi-tated (%) was
calculated according to the formula a-c (b-c) x 100 where a = cpm
precipitated with antibody, b - total cpm added, and c = cpm
precipitated nonspecifically with normal rabbit serum or with
antibody and excess unlabeled thymopoietin (these were usually
approximately 10~ of total cpm). For the standard curve of
binding inhibition the thymopoietin bound was calculated as
a percentage of maximal thymopoietin binding with antibody
at 10 1. An antibody dilution of 1:3000 was used for the
standard curve of binding inhibition.
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' 25 (b) Radioimmunoassay - Double ~ntibody Separation
The procedure of (a) above was repeated except
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! that 4M KC~ BGG buffer was used and after the two hour
incubation period a total of 0.1 ml. of goat anti-rabbit
antibody and 0.02 ml. of normal rabbit were added, the components
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mixed on a vortex mixer, the resulting mixture was allowed
to stand at room temperature for 5 minutes and then was
centrifuged. The supernatant was aspirated and the precipitate
was counted for radioactivity as in (a) above.
(c) Radioimmunoassay - Dextran Coated Charcoal Separation
Dextran coated charcoal is prepared by mixing
equal volumes of (i) 5 gm of Nori-t A charcoal per 100 ml of
phosphate buffer containing 2MKCl and bovine gan~a globulin
and (ii) 0.5 gm of dextran 110 per 100 ml. of phosphate buffer ~ ,
containing lM KCl and bovine gamma globulin.
A total 0.5 ml. of dex-tran coated charcoal is added
at 4C. to the assay tube after -the two hour incubation as
in (b) above and the mixture allowed to stand at 4C. for
30 minutes. The tubes are centrifuged and the supernatants
aspirated. The pellets are counted for radioactivity which
represents "unbound" thymopoietin in this assay~
The binding-inhibition standard curve for unlabeled
thymopoietin in the assay employing polyethylene glycol
separation of the antigen-antibody complex showed sensitivity
to thymopoietin concentrations greater than 5 ng/ml. No
significant displacement was produced by control polypeptides
which included insulin, alpha-bungarokoxin, ubi~uitin, histone
and a synthetic tridecapeptide fragment of thymopoietin
(residues 29-41) at concentrations of 10 to 1,000 ng./ml.
Binding-inhibition curves for unlabeled thymopoietin
in the assays employing the double antibody and dextran ~oated
charcoal s~paration of the antigen-antibody complex both
showed sensitivity to thymopoietin concentrations down to 0.1 ng
thymopoietin/ml. These latter two procedures are thus
especially suitable for measuring thymopoietin levels in serum
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