A method for the radioimmunoassay of parathyroid hormone in mammalian serum. The method is an improvement on previous double antibody radioimmunoassays in that spurious assay results caused by the nonspecific interaction of serum proteins with the labeled peptide is eliminated by the labeling of a specific portion of either human or bovine parathyroid molecule. The 65-84 portion of human or bovine parathyroid hormone, as radioactively labeled, is incorporated in the assay as the labeled peptide. A chicken antibody with a high affinity for the 65-84 portion of human parathyroid hormone is incorporated in the assay as the first antibody. The invention further pertains to compounds useful in practicing the method, namely X.sup.64 -hPTH.sup.65-84 and Y-X.sup.64 -hPTH.sup.65-84 wherein X is either histidyl, tyramyl, histamyl, or tyrosyl, and wherein Y is either .sup.125 I or .sup.131 I.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to radioimmunoassay measurement of the concentration 
of mammalian parathyroid hormone and certain fragments thereof in 
biological or other fluids. 
2. Description of the Prior Art 
Human parathyroid hormone (i.e. intact hPTH) is an 84 immuno acid sequence 
with a major role in maintaining the constancy (homeostasis) of calcium in 
cells and extracellular fluid, despite marked variations in calcium intake 
and excretion. Parathyroid peptides (both intact hPTH and certain hPTH 
fragments) are secreted from the parathyroid glands in response to a 
lowering of serum calcium; and, in the absence of parathyroid peptide 
secreting tumors, high levels of serum calcium inhibit secretion by the 
glands. Measuring parathyroid peptides in human serum has proven 
clinically to be an extremely helpful and efficient tool in the 
differential diagnosis and management of hypercalcemia; most notably, 
radioimmunoassay (i.e. RIA) can help to diagnose tumors and hyperplasia of 
the parathyroid glands. RIA detection of high blood levels of PTH peptides 
can establish that excess PTH secretion is causing hypercalcemia. RIA's 
can also be useful in localizing hyperfunctioning parathyroid tissue by 
assaying samples obtained via venous catheterization. PTH RIA is also 
useful for the diagnosis and management of hypocalcemia. With rare 
exceptions, hypocalcemic persons with deficient PTH (hypoparathyroidism) 
usually have subnormal blood concentrations of PTH peptides. Measurement 
of the concentration of PTH peptides in blood serum is frequently used to 
assess the status of renal osteodystrophy in renal failure patients on 
chronic dialysis. Serum PTH levels are often markedly elevated in patients 
with renal failure due to chronic negative calcium balance, hypocalcemia, 
and consequent secondary hyperparathyroidism. 
In clinical parathyroid literature, a set of nomenclature has become 
common. N-terminal hPTH is the 1-34 amino acid sequence of hPTH. Antisera 
having a substantially high affinity for this sequence are referred to as 
N-terminal antibodies. Such antibodies will generally also have a high 
affinity for any sequence of amino acids incorporating the 1-34 amino acid 
sequence. Thus, such antibodies will have a high affinity for intact hPTH 
(i.e. hPTH.sup.1-84) as well as N-terminal hPTH. C-terminal hPTH is the 
35-84 amino acid sequence. Antisera having a substantially high affinity 
for the 35-84 sequence are referred to as C-terminal antisera. It is now 
well established that only small amounts of intact hPTH and even less free 
N-terminal hPTH are present in peripheral human serum. The main 
circulating hPTH peptide is C-terminal hPTH. Accordingly, RIA's are 
generally directed at C-terminal hPTH where the purpose is diagnosing 
hyperthyroidism and the other maladies recited above. 
Prior art RIA's which measure the concentration of C-terminal hPTH in human 
serum are common; however, very few are available commercially. The 
commercially available assays generally involve the double antibody 
method. In brief, C-terminal hPTH (and lesser quantities of intact hPTH) 
present in human serum compete with a radioactively labeled peptide 
(generally radioactively labeled beef PTH.sup.1-84) for a particular first 
antibody, thereby inhibiting the binding of the first antibody to the 
radioactively labeled peptide. As a result of the competitive inhibition, 
the ratio of first antibody bound labeled peptide to free labeled peptide 
diminishes as the concentration of C-terminal hPTH (and intact hPTH) 
increases. As a convenient method to circumvent the difficult task of 
separating bound labeled peptide from free labeled peptide, a second 
antibody is added that has a substantially high affinity for the first 
antibody and which forms a precipitate when bound to the first antibody 
bound labeled peptide. The quantity of second antibody added is generally 
only sufficient to bind a portion of first antibody found radioactively 
labeled peptide. The ratio of precipitated radioactively labeled peptide 
to non-precipitated radioactively labeled peptide similarly diminishes as 
the concentration of C-terminal hPTH (and intact hPTH) increases. The 
concentration of C-terminal hPTH (and intact hPTH) in an unknown sample is 
obtained by comparing the inhibition observed with the inhibition produced 
by known amounts of C-terminal hPTH (or intact hPTH), as presented in a 
standard curve. The concentration of C-terminal hPTH is generally 
represented as hPTH.sup.1-84 equivalents. That is, the concentration in 
nanograms/ml (ng/ml) of serum is reported as if C-terminal hPTH were 
intact hPTH. 
Most prior art double antibody PTH RIA's face a common difficulty in 
measuring the concentration of C-terminal hPTH: they often give a spurious 
value because of nonspecific interaction (NSI) of the labeled PTH peptide 
with serum proteins. That is, the labeled peptide is thought to react with 
various proteins present in the serum sample with the result that the 
first antibody, which ordinarily has a high affinity for the labeled PTH 
peptide, has a low or no affinity for the product of the reaction of the 
labeled PTH peptide with serum proteins. 
In some prior art double antibody PTH RIA's, the standardized reagents used 
to prepare the standard curve do not contain human serum (Isotex 
Diagnostics, Friendswood, Texas 77546). In this case, standards are 
generally prepared by dissolving appropriate quantities of PTH peptides, 
whether human, beef, or of other sources, in an appropriate non-serum 
buffer. The NSI for the unknown sample of serum will depress the value of 
the ratio of precipitated to non-precipitated labeled PTH peptide, while 
there will be no similar depression for the standard reagents. This is so 
because the latter do not contain human serum and therefore the serum 
proteins which cause NSI are absent. Thus, the assay will indicate a 
concentration that is higher than the actual concentration. The error 
injected into assay results by NSI can be sufficiently great, on the order 
of several ng's/ml of hPTH.sup.1-84 equivalents, to cause a 
mischaracterization of an unknown sample. That is, an assay of serum taken 
from a person with normal parathyroid glands could indicate 
hyperparathyroid disease. 
Some prior art double antibody PTH RIA's seek to eliminate the effect of 
NSI on assay results. One prior art method (Arnaud, C. D., et al., 50 J. 
Clin. Invest. 21 (1971)) incorporates serum from hypoparathyroid patients 
or pooled serum stripped of PTH peptides in the standard reagents used to 
prepare the standard curve. This attempt to adjust for the effects of NSI 
on assay results is not wholly satisfactory. The serum added to the 
standard reagents is not the unknown serum to be tested. The concentration 
of serum peptides which give rise to NSI may vary significantly from 
person to person. Thus, depending upon whether the NSI for the unknown 
sample is relatively greater or less than the NSI for the serum 
incorporated in the standard reagents, the assay will indicate 
respectively concentrations lower or higher than the actual concentration. 
The error injected into this type of prior art assay can also be 
sufficiently great to mischaracterize an unknown sample. 
Another PTH assay approach seeks to eliminate the effects of NSI on assay 
results by performing the RIA on significantly diluted serum. Diluting the 
serum in this manner does reduce NSI. However, while reducing the effect 
of NSI on assay results, diluting the serum generally reduces the 
sensitivity of the RIA to the degree that the RIA cannot detect the 
concentrations of C-terminal hPTH necessary to distinguish normal from 
abnormal functioning parathyroids. 
At least one prior double antibody PTH RIA has adjusted for the effects of 
NSI while retaining the necessary sensitivity of the assay (PTH II, Immuno 
Nuclear Corp., Stillwater, Minn. 55082). In this RIA, an unknown sample of 
serum is divided into two portions. Intact PTH and C-terminal hPTH are 
removed from one of the two portions, leaving the serum proteins behind. 
Removing the intact hPTH and C-terminal hPTH in this manner is 
accomplished by binding the first antibody to a gel or resin to create a 
specific solid phase absorbent for intact PTH and C-terminal hPTH. 
However, removing the intact hPTH and C-terminal hPTH in this manner 
requires a full cycle of incubation of the portion of the serum to be 
stripped. Following this cycle of incubation, both the untouched portion 
of serum and the stripped portion of serum are assayed according to 
standard RIA methods. The stripped serum serves as a zero reference value 
to be subtracted from the result obtained with the untouched serum to 
yield results adjusted for NSI. While this method of stripping a portion 
of the very serum to be assayed compensates for NSI, it is not wholly 
satisfactory because it involves an extra cycle of incubation which 
increases the total time necessary to reach a final assay result after 
drawing a sample from a patient. 
Double antibody PTH RIA's directed at human serum are primarily useful for 
diagnostic purposes and secondarily useful for research purposes. Similar 
double antibody PTH RIA's directed at the sera of mammals such as rats, 
oxen, or cows are particularly useful for advanced research on the 
functioning of parathyroids, because rats, oxen, and cows can be 
controlled and manipulated in ways inappropriate for human subjects. It is 
commonly known that most regions of bovine PTH and hPTH are structurally 
similar, and that fragments of bovine PTH are present in bovine 
parathyroid glands and circulate in the blood. Furthermore, it is now 
known that some regions of rat PTH and hPTH are structurally similar, and 
that fragments of rat PTH are present in rat parathyroid glands and 
circulate in rat blood. Because of these structural similarities, some 
antibodies have a substantially high affinity for bovine PTH, rat PTH, and 
hPTH. Thus, double antibody PTH RIA's incorporating such antibodies will 
measure the concentration of bovine PTH and rat PTH as well as hPTH. The 
effect of NSI on assay results is common to such RIA's whether directed to 
bovine serum, rat serum, or human serum. It is thought that NSI will 
similarly effect assays of any mammalian serum. Thus, the various attempts 
in prior art double antibody PTH RIA's to adjust for the effects of NSI 
are not wholly satisfactory when such RIA's are directed to mammalian 
serum. 
SUMMARY OF THE INVENTION 
The invention is based on the discovery that NSI can be eliminated in 
double antibody PTH RIA's directed to mammalian serum, especially bovine, 
rat, or human serum, by radioactively labeling only a selected fragment of 
the hPTH sequence or the bovine PTH sequence. A radioactively labeled 
fragment within the range of about 65-84 of bovine PTH or hPTH is 
incorporated in the assay as the labeled peptide. Accordingly, the 
invention includes a method for double antibody radioimmunoassay 
measurement of the concentration of PTH in biological fluids which 
comprises incorporating as the radioactively labeled peptide a 
radioactively labeled fragment within the range of about 65-84 of bovine 
PTH or hPTH, and compounds useful in practicing the method. The compounds 
include those having the formula X.sup.64 -hPTH.sup.65-84 wherein X is a 
member of the group consisting of histidyl, tyramyl, histamyl, and tyrosyl 
and those having the formula Y-X.sup.64 -hPTH.sup.65-84 wherein Y is a 
member of the group consisting of .sup.131 I and .sup.125 I and wherein X 
is a member of the group consisting of histidyl, tyramyl, histamyl, and 
tyrosyl. 
Double antibody PTH RIA's incorporating radioactively labeled fragments 
within the range of about 65-84 of bovine PTH or hPTH follow methods 
otherwise common to double antibody RIA's. A first antibody must be found 
that has a high affinity for the particular radioactively labeled fragment 
and the PTH peptides to be measured. The unknown sample, the first 
antibody, and the radioactively labeled fragment are added together, or 
the addition of the radioactively labeled fragment is delayed for enhanced 
sensitivity of the assay. A second antibody is added which binds a portion 
of first antibody bound radioactively labeled fragment, thereby forming a 
precipitate. Precipitation may be accelerated according to conventional 
means with polyethylene glycol (PEG) and pre-precipitation with normal 
serum. 
Radioactively labeled hPTH.sup.65-84 is an example of a radioactively 
labeled fragment within the range of about 65-84 of hPTH. The fragment 
hPTH.sup.65-84 may be radioactively labeled in many different ways, 
including adding a group that is receptive to known radioisotopes. 
Accordingly, hPTH.sup.65-84 can be synthesized by solid state peptide 
chemistry, with, for example, a histidyl, tyramyl, histamyl, or tyrosyl 
(Tyr) group added at the 64th position of the hPTH sequence. The latter 
groups are receptive to radioiodine and other isotopes, which may be 
attached to the receptor groups according to conventional means. Thus, 
.sup.125 I-Tyr.sup.64 -hPTH.sup.65-84 is an example of a radioactively 
labeled hPTH.sup.65-84 and consequently is an example of a radioactively 
labeled fragment within the range of about 65-84 of hPTH. 
The elimination of NSI for an RIA of the present invention is illustrated 
by comparing its assay of mammalian serum with that of a prior art RIA 
(PTH II, Immuno Nuclear Corp., Stillwater, Minn. 55082). The RIA of the 
present invention utilizes .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84 as the 
radioactively labeled peptide and an antibody, raised in chickens against 
intact bovine PTH, having a high affinity (1.times.10.sup.10 l/mol.) for 
hPTH.sup.65-84. This first antibody consequently also has a high affinity 
for .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84, intact hPTH, and C-terminal 
hPTH. The prior art RIA utilizes radioiodinated intact bovine PTH and an 
antibody, raised in chickens against intact bovine PTH, having a high 
affinity for hPTH.sup.44-64. This first antibody consequently also has a 
high affinity for radioiodinated intact bovine PTH, intact hPTH, and 
C-terminal hPTH. 
To compare the RIA of the present invention with the prior art RIA, uremic 
human serum (with elevated C-terminal hPTH) was separated by a superfine 
G-75 Sephadex chromatograph. The contents of each eluate tube were divided 
into two portions to be assayed respectively by the two RIA's. In the area 
of tubes 20-25 (i.e. the void area containing no C-terminal hPTH), the 
prior art RIA showed approximately 4 ng/ml hPTH.sup.1-84 equivalents (a 
spurious result attributable to NSI), while the RIA of the present 
invention showed no hPTH.sup.1-84 equivalents (hence, no NSI). 
The RIA of the present invention, incorporating .sup.125 I-Tyr.sup.64 
-hPTH.sup.65-84, will assay rat serum and bovine serum as well as human 
serum. This is so because its first antibody has a substantially high 
affinity for intact rat PTH and certain rat PTH fragments, and because the 
first antibody also has a substantially high affinity for intact bovine 
PTH and certain bovine PTH fragments. Serum proteins in rat, bovine, and 
human serum are thought to interact nonspecifically on the same region of 
the hPTH molecule when that molecule is radioactively labeled. Therefore, 
the RIA of the present invention by incorporating radioactively labeled 
hPTH.sup.65-84, whether directed at rat, bovine, or human serum, excludes 
that region of the hPTH molecule which interacts with serum proteins. 
Thus, it does not suffer the adverse effects of NSI when directed to such 
sera. 
NSI is similarly eliminated by incorporating as the radioactively labeled 
peptide radioactively labeled fragments within the range of about 65-84 of 
bovine PTH or hPTH. Serum proteins are thought to interact nonspecifically 
on a region of labeled hPTH molecules and labeled bovine PTH molecules at 
least somewhat removed from the region of 65-84. This is evidenced by the 
above comparison of the RIA of the present invention with the prior art 
RIA. NSI in the prior art RIA occurred somewhere in the region of 44-64. 
The antibody therein had an affinity for hPTH.sup.44-64 and NSI suppressed 
binding of this antibody to labeled intact beef PTH. Accordingly, 
radioactively labeled peptides based on fragments smaller than 
hPTH.sup.65-84 or bovine PTH.sup.65-84 in such RIA's would also eliminate 
NSI. Moreover, fragments somewhat larger than hPTH.sup.65-84 or bovine 
PTH.sup.65-84 could serve as the basis for the radioactively labeled 
peptide. For example, radioactively labeled hPTH.sup.63-84 or 
radioactively labeled bovine PTH.sup.63-84 would also eliminate NSI in 
such RIA's. Based on general immunological principles, the first antibody 
of the RIA of the present invention will have a high affinity for 
fragments larger than hPTH.sup.65-84 or bovine PTH.sup.65-84 and may well 
have a high affinity for smaller fragments. Other antibodies could be 
raised with the requisite affinities should this particular first antibody 
not have a high affinity for certain smaller fragments.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
The following is a description of a typical preparation and operation of a 
double antibody PTH RIA which incorporates radioactively labeled 
hPTH.sup.65-84. This RIA will assay rat, bovine, or human serum. It will 
also assay biological or other fluids. 
PREATION OF STANDARD REAGENTS 
0.1 M borate buffer (4.0 Liters) 
24.75 gm boric acid 
8.10 gm NaOH 
9.0 ml concentrated HCl 
Dissolve and dilute to 4.0 liters with distilled water. pH 8.4. 
0.25 M borate buffer (1.6 Liters) 
24.75 gm boric acid 
8.10 gm NaOH 
9.0 ml concentrated HCl 
Dissolve and dilute to 1.6 liters with distilled water. pH 8.4. 
5% BSA-0.25 M borate buffer (4.0 Liters) 
61.88 gm boric acid 
20.25 gm NaOH 
22.5 ml concentrated HCl 
665 ml 30% by volume BSA in distilled water (Bovine Serum Albumin available 
commercially from Sigma Chemical Co., St. Louis, Mo. 63178) 
8.0 ml 1% by volume merthiolate (thimersal) in distilled water. 
Dissolve and dilute to 4.0 liters with distilled water. pH 8.4. 
5% BSA-0.25 M borate buffer in 0.22 M EDTA (100 ml) 
16.5 ml 30% BSA 
8.18 gm disodium EDTA (disodium ethylenediamine tetracetic acid dihydrate) 
0.2 ml 1% merthiolate 
Dissolve and dilute to volume of 100 ml with 0.25 M borate buffer; adjust 
pH to 8.4 with 40% by volume NaOH in distilled water. 
1% BSA-0.1 M borate buffer (4.0 Liters) 
24.75 gm boric acid 
8.10 gm NaOH 
9.0 ml concentrated HCl 
133 ml 30% BSA 
1.6 ml 1% merthiolate 
Dissolve and dilute to 4.0 liters with distilled water. 
pH 8.4. 
0.2 M acetic acid-ammonium acetate (500 ml) 
18 ml 1.0 M ammonium acetate 
82 ml 1.0 M acetic acid 
400 ml distilled water 
pH 4.0. 
0.5 M Phosphate buffers (950 ml) 
(a) 68.1 g/liter KH.sub.2 PO.sub.4 
(b) 89.0 g/liter Na.sub.2 HPO.sub.4.2H.sub.2 O (or 71.0 g/liter anhydrous 
Na.sub.2 HPO.sub.4) 
Mix 140 ml of (a) and 810 ml of (b) to obtain phosphate buffer of pH 7.4. 
Store at 2.degree.-8.degree. C. At those temperatures, the salts will 
precipitate; before use, warm solution until salts redissolve. Test 
solution for mold contamination by retaining sample of newly created 
solution at room temperature in an enclosed glass container. If mold is 
present, discard refrigerated solution. 
0.05 M Phosphate buffers (1.0 Liter) 
Dilute 100 ml 0.5 M phosphate buffer to 1.0 liter with distilled water. pH 
7.4. 
15% PEG-0.001% Triton (1.0 Liter) 
150 grams polyethyleneglycol 
0.01 gram Triton 
Dissolve and dilute to 1.0 liter with distilled water. 
Additional reagents necessary to the preferred embodiment may be based on 
the foregoing reagents. 
PREATION OF CHICKEN ANTI-hPTH.sup.65-84 REAGENT 
Chicken Anti-hPTH.sup.65-84 Reagent is the result of raising the chicken 
anti-hPTH.sup.65-84 antibody against intact bovine parathyroid hormone. 
This antibody was chosen for its high affinity for intact hPTH, C-terminal 
hPTH fragments, and radioactively labeled hPTH.sup.65-84. As alternative 
embodiments, other antibodies with a substantially high affinity for these 
three substances, could be utilized in a C-terminal PTH RIA kit. Such 
antibodies may be raised in animals other than chickens. Also such 
antibodies may be raised in chickens or other animals against substances 
other than intact bovine PTH. Such antibodies may be raised, for example, 
against intact hPTH and certain fragments of the foregoing. 
The chicken anti-hPTH.sup.65-84 antibody also has a high affinity for 
intact bovine PTH and bovine PTH.sup.65-84. C-terminal fragments of bovine 
PTH at least as large as those fragments of bovine PTH within the range of 
about 65-84 are designated herein as C-terminal bovine PTH fragments. This 
antibody has a high affinity for such fragments. Furthermore, this 
antibody has a high affinity for intact rat PTH. This suggests that 
certain of the larger C-terminal fragments of rat PTH are sufficiently 
similar in structure to hPTH.sup.65-84 that the antibody will have a high 
affinity for such fragments. C-terminal fragments of rat PTH at least as 
large as those fragments of rat PTH within the range of about 65-84 are 
designated herein as C-terminal rat PTH fragments. It is thought that 
either the chicken anti-hPTH.sup.65-84 antibody or a substitute antibody 
with a high affinity for radio-activity labeled fragments within the range 
of about 65-84 of bovine PTH or hPTH will have a high affinity for the 
parathyroid hormone of any mammal. Fragments of the particular mammalian 
parathyroid hormone which can be measured by an RIA incorporating such 
antibodies are designated herein as C-terminal fragments of the particular 
mammalian parathyroid hormone. 
To prepare Chicken Anti-hPTH.sup.65-84 Reagent, purchase crude bovine PTH 
TCA powder (Inolex Corp., Glenwood, Ill. 60425) and purify it by 
conventional means. Dissolve 40 mg of the powder in 10 ml 0.2 M acetic 
acid-ammonium acetate buffer. Add to the dissolved powder both a 
suspension of blue dextran and a quantity of Na.sup.125 I representing 
approximately 100,000 cpm. Layer this mixture onto a G-75 Sephadex 
60.times.1.5 cm fine column using downward flow and eluate with acetic 
acid-ammonium acetate buffer. The first peak represents the void volume as 
marked by the blue dextran. The second peak represents the included volume 
as marked by .sup.125 I. Collect eluates on a 1.25 minute basis between 
the void and included volume and assay each tube to confirm the presence 
of purified labeled bPTH. Pool tubes containing immunoreactive bovine PTH 
and assay pooled material to determine the quantity thereof. 
Inject 0.1 mg of the purified labeled bovine PTH emulsified with an equal 
volume of Freund's complete adjuvant, weekly for four weeks, in four sites 
near wings and legs on the underside of the boides of Rhode Island or 
Leghorn Red chickens. In the fifth week, bleed the chickens from a vein 
under the wing and test for good titer. 
Centrifuge the chicken's blood to isolate the plasma. Titer the plasma 
against .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84. Prepared dilutions of 
1/500, 1/1,000, 1/5,000, 1/10,000, 1/20,000, and 1/30,000 by volume, by 
adding appropriate quantities of 1% BSA-0.1 M borate buffer to the 
isolated chicken plasma. Add to 200 .mu.l of each dilution of chicken 
plasma 200 .mu.l of .sup.125 I-Tyr.sup.65-84 Reagent (cf. preparation of 
.sup.125 I-Tyr.sup.65-84 Reagent infra). Vortex gently and incubate for 24 
hours at 2.degree.-8.degree. C. Follow the protocol (cf. protocol infra) 
beginning with the addition of RAC-PPT Reagent (cf. preparation of RAC-PPT 
Reagent infra). Plot percent binding of the chicken antibody to .sup.125 
I-Tyr.sup.64 -hPTH.sup.65-84 against dilution of the chicken plasma 
according to the counts registered for precipitated and non-precipitated 
.sup.125 I-Tyr.sup.64 -hPTH.sup.65-84. Good titer is represented by the 
dilution which has at least 40-50% binding. 
Bleed the animal weekly if good titer is present. Maintain animal 
thereafter by injecting 0.05 mg of the purified labeled bovine PTH 
emulsified with an equal volume of Freund's complete adjuvant. 
Substantially maximal sensitivity of the assay kit, on the order of 0.2 
ng/ml hPTH.sup.1-84 equivalents, is achieved by maintaining a pool of 
chicken anti-hPTH.sup.65-84 plasma solution for which there is 
approximately 40-50% binding. The pool is maintained by adding to it 
chicken anti-hPTH.sup.65-84 plasma at a concentration in 1% BSA-0.1 M 
borate buffer which according to its titer will result in 40-50% binding. 
In the case of the presently existing pool, Chicken Anti-hPTH.sup.65-84 
Reagent is prepared by diluting 1.0 ml of the pooled plasma solution to 
100 ml with 1% BSA-0.1 M borate buffer. One ml of the 1/100 dilution is 
diluted to 48 ml with 1% BSA-0.1 M borate buffer. Five ml of the 1/4,800 
dilution is aliquoted per 30 ml Wheaton vial, lyophilized, and stored at 
-20.degree. C. Add 25 ml of distilled water to reconstitute for use as 
Chicken Anti-hPTH.sup.65-84 Reagent. 
PREATION OF RABBIT ANTI-CHICKEN PRECIPITATING COMPLEX REAGENT 
Rabbit Anti-Chicken Precipitation Complex Reagent (RAC-PPT Reagent) is the 
result of raising a rabbit antibody against the gamma globulin of 
chickens. This particular rabbit antibody was chosen for its high affinity 
for chicken anti-hPTH.sup.65-84. The rabbit antibody is added to 
polyethylene glycol (PEG) and normal chicken serum (NCS) to form a 
pre-precipitated accelerated precipitating complex. 
As alternative embodiments, other antibodies with a substantially similar 
affinity for chicken anti-hPTH.sup.65-84 may be utilized in a C-terminal 
PTH RIA kit. Such antibodies may be raised in animals other than rabbits. 
Also such antibodies may be raised in rabbits and other animals against 
substances other than this particular chicken gamma globulin, such as 
whole chicken serum or plasma. 
To prepare the RAC-PPT Reagent, separate gamma globulin from the chicken 
plasma for which there was good titer against .sup.125 I-Tyr.sup.64 
-hPTH.sup.65-84 by the conventional method of salting out with 331/3% 
saturated (NH.sub.4).sub.2 SO.sub.4. Inject 1.0 mg of the gamma globulin 
emulsified with an equal volume of Freund's complete adjuvant, weekly for 
four weeks, in four sites near the legs on the underside of New Zealand 
White rabbits. In the fifth week, bleed each rabbit from a vein under a 
leg and test the titer. 
Centrifuge the rabbit's blood to isolate the plasma. To find the 
concentration or titer of rabbit antibody, prepare dilutions of 1/2, 1/3, 
1/4, 1/5, 1/7.5, 1/10, 1/15, 1/20, 1/25, 1/30, and 1/50 by volume, by 
adding appropriate quantities of 1% BSA-0.10 M borate buffer to the 
isolated rabbit plasma. To reaction vessels corresponding to each 
dilution, add 200 .mu.l of Chicken Anti-hPTH.sup.65-84 Reagent to 200 
.mu.l of .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84 Reagent. Vortex gently and 
incubate 20 hours at 2.degree.-8.degree. C. To each of the reaction 
vessels, add 200 .mu.l of 1/200 normal chicken serum (NCS does not contain 
the chicken anti-hPTH.sup.65-84 antibody), 200 .mu.l of 15% PEG-0.001% 
Triton, and 200 .mu.l of one of the dilutions of the rabbit plasma. Vortex 
gently and incubate for exactly two hours at 2.degree.-8.degree. C. Follow 
the protocol infra from thereon. 
Plot the ratio of the cpm of the precipitate to total cpm of precipitate 
and supernatant against the dilution of rabbit plasma. Maximal binding of 
the rabbit antibody to chicken anti-hPTH.sup.65-84 occurs at the 
concentration of rabbit plasma for which the ratio of cpm's is greatest. 
This concentration is defined as the working concentration or titer of the 
rabbit antibody. 
Bleed the animal weekly if good titer is present. Maintain animal 
thereafter by injecting 0.5 mg of the chicken gamma globulin emulsified 
with an equal volume of Freund's complete adjuvant. 
To prepare RAC-PPT Reagent, mix equal volumes of NCS at 1/40 dilution in 5% 
BSA-0.25 M borate buffer and rabbit plasma at five times the working 
concentration diluted with 5% BSA-0.25 M borate buffer. Mix well and 
aliquot 12 ml per 100 ml vial. Add 22.5 ml of 15% PEG-0.001% Triton to 
each vial. Lyophilize and store at -20.degree. C. Add 100 ml of distilled 
water to reconstitute for use as RAC-PPT Reagent. 
PREATION OF .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84 REAGENT 
The .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84 Reagent is the result of three 
principal steps: (1) synthesizing Tyr.sup.64 -hPTH.sup.65-84 ; (2) 
labeling the synthesized compound with .sup.125 I; and (3) dissolving the 
labeled compound in an appropriate buffer at an appropriate concentration. 
As alternative embodiments, the hPTH.sup.65-84 fragment may be labeled 
radioactively in many conventional ways, including but not limited to, 
attaching histidyl, tyramyl, histamyl, or other similar substances to the 
fragment and labeling with radioiodine. As an alternative to .sup.125 I, 
.sup.131 I may be used. As a further alternative embodiment, a 
radioactively labeled fragment within the range of about 65-84 of bovine 
PTH or hPTH could be substituted for the radioactively labeled 
hPTH.sup.65-84. 
1. Synthesizing Tyr.sup.64 -hPTH.sup.65-84 
The compound Tyr.sup.64 -hPTH.sup.65-84 consists of the following: NH.sub.2 
-Tyr-Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-Asp-Val-Leu-Thr-Lys-Ala-L 
ys-Ser-Gln-COOH. 
Tyr.sup.64 -hPTH.sup.64-84 is synthesized by solid state peptide synthesis, 
a method discovered by R. B. Merrifield and detailed in Stewart & Young, 
Solid State Peptide Synthesis (W. F. Freeman & Co. 1969). 
The Synthesis proceeds from the carboxyl end of hPTH sequence, adding one 
amino acid per cycle of reaction. In general, each amino acid to be added 
is protected by BOC (tertiary butyloxycarbonyl) at its carboxyl end. The 
peptide chain is linked by its carboxyl end to DVB resin (a copolymer of 
styrene and divinyl benzene). The starting material, BOC-Gln-DVB resin, is 
available commercially (Peninsula Laboratories, Inc., Belmont, Calif. 
94002). Place 1.0 mM (i.e. 2.0 gm) BOC-Gln-DVB resin in a reaction vessel. 
The BOC group is cleaved from the Gln-DVB resin by a surplus (hereinafter 
a quantity sufficient to cover the resin in the reaction vessel) of 40% 
solution by volume of trifluoro acetic acid in CH.sub.2 Cl.sub.2. Stir for 
20 minutes and drain. The reaction vessel and contents should be at room 
temperature for this and all succeeding steps in the synthesis. Then wash 
with a surplus of pure CH.sub.2 Cl.sub.2, stir and drain. The Gln-DVB 
resin is neutralized by adding 20 ml of 10% solution by volume of 
triethyamine in CH.sub.2 Cl.sub.2. Stir for 10 minutes and drain. Then 
wash with a surplus of pure CH.sub.2 Cl.sub.2, stir and drain. Repeat 
washing twice with fresh CH.sub.2 Cl.sub.2. 
In the next cycle, add 5 molar equivalents (defined according to the 
Gln-DVB resin) of the next amino acid in the hPTH.sup.65-84 sequence. In 
this particular cycle, the compound added would be BOC-Ser-Bzl (Benzyl 
group). Certain amino acids, such as Ser, Lys, Thr, Asp, Glu, and Tyr, 
require side chain protection to ensure that the amino acid remains intact 
throughout the synthesis. This is accomplished by employing BOC-Ser-Bzl 
rather than BOC-Ser in the initial step of the Ser cycle. Similarly, 
BOC-Lys-Cl-Z (Chlorocarbobenzosy), BOC-Thr-Bzl, BOC-Glu-Bzl, and 
BOC-Tyr-Br-Z (Bromocarbobenzosy) are substituted in the reaction for the 
unprotected BOC-amino acid. No protection is required for Ala, Leu, Val, 
and Gly. The above mentioned unprotected and protected BOC-amino acids are 
available commercially (Peninsula Laboratories, Inc., Belmont, Calif. 
94002). For alternative embodiments, BOC-histamyl, BOC-histidyl, and 
BOC-tyramyl, along with appropriate side chain protection, are available 
commercially (Peninsula). 
To the Gln-DVB resin and the BOC-Ser-Bz, add 5 molar equivalents of 
dichlorohexycarbodiamide dissolved in a surplus of CH.sub.2 Cl.sub.2. Stir 
for 2 hours. Apply ninhydrin test. If the test yields negative results, 
the reaction is complete and the contents of the reaction vessel should be 
washed three times with a surplus of CH.sub.2 Cl.sub.2. If the test yields 
a positive result, the reaction should be continued until complete. After 
washing, the BOC group is cleaved and the peptide-resin is neutralized by 
the steps set forth above. The cycle is repeated with an appropriate 
protected or unprotected BOC-amino acid until Tyr.sup.64 -hPTH.sup.65-84 
-DVB resin, or the appropriate alternative embodiment, is the result. 
The completed peptide, Tyr.sup.64 -hPTH.sup.65-84, is cleaved from the DVB 
resin and the side protecting groups are stripped off the protected amino 
acid by washing the contents of the reaction vessel with 20 ml of 
anhydrous HF. The peptide is purified by countercurrent exchange using 
butanol-acetic acid-water in the ratio of 4:1:5. The purity of the peptide 
can be confirmed by thin layer countercurrent chromatography and HPLC. 
2. Labeling Tyr.sup.64 -hPTH.sup.65-84 
The preferred labeling material is .sup.125 I, although other materials 
could be used. The labeling procedure stems from Yalow and Berson, 212 
Nature 357 (1966) and Hunter & Greenwood, 194 Nature 495 (1962). 
Na.sup.125 I in a standard NaOH solution is manufactured weekly by 
Amersham Searle (Arlington Heights, Ill. 60005). Na.sup.125 I that is 
older than two weeks should not be used. Chloramine T is manufactured by 
Matheson, Coleman and Bell (Cincinnati, Ohio 45212). The stability of 
Chloramine T is unknown. Chloramine T older than six months should not be 
used. Sodium metabisulfite is commonly available. 
Sephadex G-100, fine grade 40-20 microparticle diameter, should be utilized 
in the labeling procedure. A 60.times.1 cm column at room temperature 
should be packed in a level position. The Sephadex slurry is poured down a 
glass rod into the column. After the column is poured, add 4 ml of 5% 
BSA-0.1 M borate buffer and approximately 0.5 ml of 0.5% blue dextran. Run 
two volumes of 0.1 M borate buffer through the column to pack it 
thoroughly and to wash excess BSA off the column. The blue dextran checks 
column packing and determines void volume. To achieve this, the eluate is 
collected into a graduated cylinder beginning with the addition of the 
blue dextran until the blue dextran comes off the column. 
Pick a gamma counter with linear response in the range of 50,000 to 200,000 
cpm (Tracor Analytic, Elk Grove Village, Ill. 60007). Place a two 
millicurie sample of Na.sup.125 I in the standard NaOH solution in a 
V-vial. Adjust the gamma counter so that the counts are kept between 
50,000 and 200,000 cpm by adjusting the distance of V-vial from the gamma 
counter, the geometry of the well of the counter, and the gamma counter's 
window. Add 50 .mu.l of 0.5 M phosphate buffer to the V-vial. The strong 
buffer is necessary to neutralize the NaOH. Cover tightly and vortex until 
the meniscus disappears. Add 60 .mu.l of 0.1 .mu.g/.mu.l of Tyr.sup.64 
-hPTH.sup.65-84 in 0.1 N acetic acid by placing the pipet tip containing 
that solution below the meniscus in the V-vial and very gently blowing. 
After ten seconds, add 10 .mu.l of Chloramine T solution by similarly 
gently blowing it below the meniscus. Cover tightly and mix. The 
Chloramine T solution should be prepared within 1 hour of its use by 
diluting 12.5 mg of Chloramine T to a volume of 10.0 ml with 0.05 M 
phosphate buffer. After mixing the contents of the V-vial for 30 seconds, 
add 25.0 .mu.l of sodium metabisulfite solution below the meniscus and 
vortex. The sodium metabisulfite solution is made by diluting 12.5 mg of 
sodium metabisulfite to a volume of 10.0 ml with 0.05 M phosphate buffer. 
Transfer the V-vial's contents to the top of the dry Sephadex bed with a 
Pasteur pipet that was rinsed with 5% BSA-0.1 M borate buffer. The latter 
helps prevent labeled Tyr.sup.64 -hPTH.sup.65-84 from sticking to the 
pipet. Allow the V-vial's contents to soak into the bed. Rinse vial with 
200 .mu.l of 5% BSA-0.25 M borate buffer by repeatedly washing down sides 
of V-vial with the same BSA. Transfer the 200 .mu.l wash to the column 
with the same Pasteur pipet. Allow to soak into bed. Operate the fraction 
collector manually during sample and rinse application. Rinse sides of 
column with a small amount of 0.1 M borate buffer (usually 0.25-0.5 ml) 
and allow it to soak into the bed. Fill the area of column above the bed 
with 0.1 M borate buffer and attach reservoir. With a fraction collector, 
collect 0.5 ml eluates per 1.25 minutes. Collect approximately 100 
fractions. 
Count V-vial in which labeling reaction took place and count each eluate. 
Plot eluate counts on plain graph paper with counts on the ordinate and 
eluate number on the abscissa. The first peak is generally considered 
damaged PTH fragment and is discarded. The third peak represents free 
salt. The second peak is the good labeled hormone and is saved and pooled 
into a plastic bottle using a Pasteur pipet to transfer the solution. Use 
20 ml minus the estimated volume of the pooled eluate of 5% BSA-0.25 M 
borate buffer for rinsing the eluate tubes and the Pasteur pipet. Mix the 
pooled eluates, and any unused 5% BSA-0.25 M borate buffer. 
After plotting the eluate counts against the eluate number, separate the 
protein counts (first two peaks) from free salt (the third peak). The 
percent transfer may be calculated by the formula: 
##EQU1## 
The specific activity may be calculated by the formula: 
##EQU2## 
A specific activity within the range of 100-200 .mu.c/.mu.g is acceptable. 
A count of the reaction vial should not yield a result that is more than 
5% the total counts of the original vial and contents. If the count 
exceeds 5%, the number in excess of 5% should be considered to be protein 
associated and therefore must be deducted from the amount of protein 
applied to the column. The amount of protein applied to the column may be 
calculated by the formula: 
##EQU3## 
The amount of labeled protein per container may be calculated by the 
formula: 
##EQU4## 
3. .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84 Reagent 
Dilute the pooled eluate representing the second peak to a sufficient 
volume with 5% BSA-0.25 M borate buffer in 0.22 M EDTA so that the 
concentration of .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84 is 1.25 
nanograms/ml based on the preceding formula for calculating the amount of 
labeled protein per container. Aliquot 5 ml of .sup.125 I-Tyr.sup.64 
-hPTH.sup.65-84 at a concentration of 1.25 nanograms/ml into 30 ml serum 
vials. Store this solution at -20.degree. C. until it is lyophilized. 
Store at -20.degree. C. after lyophilization. The lyophilized .sup.125 
I-Tyr.sup.64 -hPTH.sup.65-84 stored at -20.degree. C. has a useful life of 
6 weeks. Reconstitute the lyophilized .sup.125 I-Tyr.sup.64 
-hPTH.sup.65-84 with 25 ml of distilled water as .sup.125 I-Tyr.sup.64 
-hPTH.sup.65-84 Reagent. Perform a check on the labeling procedure by 
directing an assay incorporating the newly prepared .sup.125 I-Tyr.sup.64 
-hPTH.sup.65-84 Reagent against known amounts of Tyr.sup.64 
-hPTH.sup.65-84. 
PREATION OF CONTROLS AND STANDARDS 
Prepare the Normal Control Reagent from plasma units available 
commercially. Centrifuge to remove fibrin or filter through glass wool. 
Mix pool well and aliquot 2.0 ml into a 5 ml Wheaton vial using a 
sufficiently accurate pipetting device. Assay to determine ng/ml of 
hPTH.sup.1-84 equivalents. Store at -20.degree. C. until lyophilized, and 
store at -20.degree. C. thereafter. Reconstitute with 2.0 ml of distilled 
water as Normal Control Reagent. 
Prepare the High Control Reagent from plasma units available commercially. 
Centrifuge as indicated above. Add 1000 ng of bovine PTH to 1000 ml of 
pooled plasma, mix well. Assay to determine ng/ml of hPTH.sup.1-84 
equivalents. Aliquot 2.0 ml into 5 ml Wheaton vials using sufficiently 
accurate pipetting device. Store at -20.degree. C. thereafter. 
Reconstitute with 2.0 ml of distilled water as High Control Reagent. 
Prepare the Zero Standard and the Nonspecific Binding Standard Reagent (NSB 
Reagent) by placing 8.0 ml of freshly prepared 1% BSA-0.25 M borate buffer 
into each of two vials, lyophilize, and store at -20.degree. C. 
Reconstitute each with 20 ml of distilled water as Zero Standard Reagent 
and as NSB Reagent respectively. 
Prepare Standard Reagents of 0.312, 0.625, 1.25, 2.5, 5.0, and 10.0 ng/ml 
of hPTH.sup.1-84 equivalents as follows. Dissolve and dilute 0.5 mg 
Tyr.sup.64 -hPTH.sup.65-84 to 5.0 ml with 0.1 N acetic acid. One hundred 
.mu.l of this solution is further diluted to 100 ml with 1% BSA-0.1 M 
borate buffer. This results in a 100 ng/ml concentration of Tyr.sup.64 
-hPTH.sup.65-84. Dilute 1.0 ml of this solution with 39.0 ml of 1% BSA-0.1 
M borate buffer to achieve a working dilution of 2.5 ng/ml which is equal 
to 10.0 ng/ml of hPTH.sup.1-84 equivalents. Serially dilute the 10.0 ng/ml 
of hPTH.sup.1-84 equivalents standard with 1% BSA-0.1 M borate buffer to 
give 5, 2.50, 1.25, 0.625, and 0.312 ng/ml of hPTH.sup.1-84 equivalents 
standards. Aliquot 500 .mu.l of each standard in 2.5 ml serum vials. 
Dispensing volume should remain in range of 500.+-.5.0 .mu.l. Store at 
-20.degree. C. before and after lyophilizing. Reconstitute each standard 
with 500 .mu.l of distilled water as the respective Standard Reagent. 
All of the lyophilized standards have a useful life of at least one year. 
PROTOCOL 
1. Reconstitute lyophilized reagents as indicated. 
2. Label 12.times.75 mm glass tubes in duplicate according to the needs of 
protocol. Commercially available tubes with lowest nonspecific binding are 
those of CMB Dispo, SP Dispo, and Kimble (Curtis Matheson Scientific, 
Inc., Houston, Texas 77001). 
3. Add reagents to the tubes as follows: 
a. Nonspecific binding (NSB) 
250 .mu.l of NSB Reagent 
b. Zero standard 
50 .mu.l of Zero Standard Reagent. 
200 .mu.l of Chicken Anti-hPTH.sup.65-84 Reagent. 
c. hPTH standards (0.312 ng/ml-10 ng/ml) 
50 .mu.l/tube respectively of each Standard Reagent. 
200 .mu.l of Chicken Anti-hPTH.sup.65-84 Reagent. 
d. Quality control 
50 .mu.l/tube respectively of High Control and Normal Control Reagents. 
200 .mu.l of Chicken Anti-hPTH.sup.65-84 Reagent. 
e. Unknown sera 
50 .mu.l of unknown serum 
200 .mu.l of Chicken Anti-hPTH.sup.65-84 Reagent. 
4. Vortex gently and incubate 4 hours at 2.degree.-8.degree. C. Incubation 
can be extended to 24 hours for increased sensitivity. 
5. Add 200 .mu.l of .sup.125 I-Tyr.sup.64 -hPTH.sup.65-84 Reagent to all 
tubes. 
6. Vortex gently and incubate 20 hours at 2.degree.-8.degree. C. 
7. Add 500 .mu.l of RAC-PPT Reagent. 
8. Vortex gently without foaming and incubate at room temperature for a 
minimum of 2 hours or at 2.degree.-8.degree. C. for a minimum of 16 hours. 
9. Centrifuge the tubes for 20 minutes at a minimum of 760.times.g. 
10. Decant supernatants in separate 12.times.75 mm tubes. 
11. Count the supernatant and its related precipitate, each for a 
sufficient time to achieve statistical significance. 
As an alternative embodiment, the above protocol may be altered so that the 
200 .mu.l of Chicken Anti-hPTH.sup.65-84 Reagent and 200 .mu.l of .sup.125 
I-Tyr.sup.64 -hPTH.sup.65-84 Reagent are added as one step to the NSB 
Reagent, various standard reagents, control reagents, and unknown serum. 
This initial step may be followed by vortexing gently and incubating for 
16 hours at 2.degree.-8.degree. C. However, greater assay sensitivity is 
generally obtained by the order of reactions indicated by the preferred 
embodiment. 
RESULTS 
The concentration of intact hPTH and C-terminal hPTH fragments in the 
unknown human serum are determined by reference to a standard curve. 
Standard curves may be obtained by several methods, for example, by 
plotting percent binding vs. concentration or by plotting cpm of 
precipitate, or the supernatant, or the ratio therebetween vs. 
concentration. FIG. 3 shows a sample standard curve where percent binding 
is plotted vs. concentration in ng/ml of hPTH.sup.1-84 equivalents. The 
percent binding may be calculated by the formula: 
##EQU5## 
As an alternative embodiment, the results of the nonspecific binding (NSB) 
sample may be incorporated in the calculation of the percent binding. NSB, 
not to be confused with NSI, is the result of radioactive supernatant 
residing in the precipitate and protein binding to the walls of the assay 
tubes, as well as other factors. An alternative calculation should be used 
when the NSB sample results in precipitate cpm's indicating greater than 
5% binding. The % binding for a sample may be calculated in the 
alternative by the formula: 
##EQU6## 
The alternative calculation of % binding is used for % binding of NSB 
samples that exceed 5%. For binding of NSB samples of 5% or less, the 
distortion due to NSB is thought to be substantially uniform for all 
samples. The RIA's based on the foregoing protocol typically result in 5% 
binding for NSB samples. 
As another alternative embodiment, the assay may be run with 100 or 200 
.mu.l of standards and unknown serum for greater sensitivity at low 
concentrations. FIG. 4 compares standard curves for 50, 100, and 200 .mu.l 
of standard and unknown serum. Conversely, if the concentration of an 
unknown serum sample exceeds 10 ng/ml, a second assay of the serum pool 
diluted by 0.4% BSA-0.1 M borate buffer will yield the concentration after 
applying an appropriate dilution factor. 
Data for the purposes of illustration are shown below. These numbers were 
used to plot the standard curve in FIG. 3 which results in a concentration 
of 3.5 ng/ml of hPTH.sup.1-84 equivalents for the unknown serum sample. 
______________________________________ 
Average Known 
(2 tubes) 
Average ng/ml Calculated 
cpm of (2 tubes) hPTH.sup.1-84 
ng/ml 
super- cpm of % equi- hPTH.sup.1-84 
Sample natant precipitate 
binding 
valents 
equivalents 
______________________________________ 
NSB 14,265 735 4.9% -- -- 
Standard 
8,100 6,900 46.0% 0 -- 
8,175 6,825 45.5% 0.312 -- 
8,550 6,450 43.0% 0.625 -- 
9,000 6,000 40.0% 1.25 -- 
9,945 5,055 33.7% 2.5 -- 
11,250 3,750 25.0% 5.0 -- 
13,125 1,875 12.5% 10.0 -- 
Quality 
Control - 
Normal 8,445 6,555 43.7% 0.5 0.5 
High 9,555 5,445 36.3% 2.0 2.0 
Un- 
known 10,500 4,500 30.0% -- 3.5 
______________________________________