HU205859B - Process for producing hypertonic factor of the parathyroid, diagnostical process for detacting the factor and diagnostical kit for them - Google Patents

Abstract

The present invention relates to a process for the preparation of a substantially untreated parathyroid hypertension factor in a substantially pure state by removing dialysis components from distilled water from distilled plasma by molecular weight less than 1000; separating plasma components with a molecular weight of less than 5000 by ultrafiltration; concentrating the resulting solution by lyophilization; fraction the material on a molecular sieve eluting with dilute ammonium acetate solution; the fraction of parathyroid hypertension factor activity is concentrated by lyophilization. The present invention also relates to a diagnostic method and diagnostic kit for detecting the presence of parathyroid hypertension in blood plasma. EN 205 859 Scope of the description: 24 pages (including 15 sheets)

Description

FIELD OF THE INVENTION The present invention relates to a process for the isolation and production of a novel, unidentified and unidentified parathyroid gland, which regulates cellular calcium uptake and is associated with hypertension in a substantially pure state. The presence of this factor in the serum of humans and mammals suggests the presence of diseases related to the elevation of intracellular calcium or otherwise calcium-mediated disease, and therefore its presence is very diagnostically important. The present invention also provides a diagnostic method for detecting the presence of factor in serum, as well as a diagnostic kit for the method.

High blood pressure is generally defined as an increase in systolic and / or diastolic blood pressure above the nominal 140/90 mmHg (Ski unit: 18,500 / 11,900 Pa). Diseases associated with hypertension include arteriosclerosis, hypertension, renal abnormalities, stroke, myocardial infarction and myocardial infarction. Although several treatments have been found to be effective in reducing arterial blood pressure, the etiology of essential hypertension remains largely unknown. The genetic precursor to hypertension is generally accepted, but the large number of different drugs found to be effective in the treatment of hypertension and the fact that these drugs appear to elicit different pharmacological responses suggest that different baseline levels of essential hypertension there are reasons.

Many studies suggest that one or more factors may play a role in the development or maintenance of hypertension. Wright et al., 1984, Hypertensive Substance Found in the Blood of Spontaneously Hypertensive Rats, 34: 1521-1528; Dahl et al., Humoral Transmission of Hypertension: Evidence from Parabiosis, Cirs. Gap. 24125 (Suppl 1), 21-23 (1969); Greenberg et al., Evidence for Circulating Factors as a Cause of Venous Hypertrophy in Spontaneously Hypertensive Rats, Am. J. Physiol. 241, H421-H430 (1981); Tobian et al., Circulating Humoral Pressor Agent in Dahl S Rats with Salt Hypertension, Clin. Sci. 57: 3450-3470 (1979); Zidek et al., Humorous Factors in the Pathogenesis of Primary Hypertension, Kin. Wochenschr. 63 (Supplement 2), D: 94-96 (1985); Hirata et al., Hypertension Producing Factor in the Serum of Hypertensive Dahl Salt-Sensitive Rats, Hypertension 6, 709-716 (1984). so e.g. in the parabiosis and cross-circulation experiments, an increase in blood pressure can be induced in normal hypertensive animals by transmitting blood from hypertensive animals. Subcutaneous injection of erythrocyte-associated factor from spontaneously hypersensitive rats (SHR) has been shown to induce hypertension in Wistar-Kyto rats (WKY) with normal hypertension and to induce growth in hypertensive hypertensive injection of serum from rats.

Circulating factors have also been reported in hypertensive rats and hypertensive humans, which increase intracellular calcium [see, e.g. Bános et al., Two Factors Associated with Incidental Uptake of Calcium in Platelets from Essential Hypertensive Patients, Clin. Exp. Hypertens. 9: 1515-1530 (1987); Zidek et al., Effect of Plasma from Hypertensive Subjects on Ca Transport in Permeabilized Human Neutrophils, Clin. Sci., 74, 5356 (1988); Linder et al., Effects of a Circulating Factor in Patients with Essential Hypertension on Intracellular Free Calcium in Normal Platelets, N. Eng. J. Med. 306, 509-513 (1987); Bruschi et al., Cytoplasmic Free Ca is Increased in Platelets of Spontaneously Hypertensive Rats and Essential Hypertensive Patients, Clin. Sci. 68: 179-184 (1985); Wrigt et al., Stimulation of Aortic Tissue Calcium Uptake by an Extract of Spontaneously Hypertensive Rat Erythrocytes Possessing Hypertensive Properties, Can. J. Physiol. Pharmacol. 64: 1515-1520 (1986)]. Because vascular tone is influenced by intracellular calcium levels, it seems reasonable to assume, although not experimentally demonstrated so far, that the factors that increase blood pressure and those that increase intracellular calcium are related. There is already evidence to suggest that calcium regulating hormones are of interest in certain forms of hypertension. Resnick L.M., Am. J. Med. 82 (Supplement 1B) 16 (1987)]. Aparathyroid hormone is a calcium regulating hormone. 30% or more of patients with essential hypertension fall into the subgroup characterized by elevated levels of immunoreactive parathyroid hormone (ir-PTH) (See Laragh et al., Kidney Int. 34 (Suppl. 35), S162 (1988)).

Increases in PTH levels have been reported in SHR rats (see McCarron et al., Hypertension 3 (Suppl. 1) 1162 (1981)), and it has been observed that hyperthyroid patients often exhibit hypertension, the severity of which can be reduced in most cases to parathyroid excision (parathyroidectomy) [see Hellstrom et al., Brit. J. Mole. 30, 13 (1958)]. Similar results of parathyroid excision have been reported in SHR rats [See Schleiffer et al., Jap. Circ. J. 45, 1271 (1981)]. Various researchers have suggested that PTH is involved in the development of essential hypertension, although exogenous administration of PTH causes a reduction in blood pressure in mammals and other vertebrates [see Pang et al., Gene. Comp. Endocrinol. 41, 135 (1980)]. This vasodilator activity of PTH is also related to a specific region of the molecule that differs from that which mediates hypercalcemic effects (see Pang et al., 1983, Endocrinology 7/2, 284). About APTH already

It has been shown to inhibit calcium entry into vascular smooth muscle [see Pang et al; Life Sci. 42, 1395 (1988)] via L-type calcium channels (Wang et al., Accepted). This paradoxical situation is further exacerbated by the fact that hypertensive patients with elevated PTH levels show reduced serum ionized calcium levels (see Resnick et al., New Engl. J. Med., 309, 288 (1983); Hvarfner et al., Acta Med. Scand. 219, 461 (1986)]. It would be expected that the level of ionized calcium in the serum would increase if PTH was increased.

The interest of the parathyroid gland in essential hypertension is obvious, but current literature on the vascular activity of PTH does not concur with the rational role of PTH in essential hypertension. At the time this invention was developed, PTH was the only active hormone reported to be produced by the parathyroid gland.

BRIEF SUMMARY OF THE INVENTION

We present the presence of a circulating factor of parathyroid origin in SHR rats and humans with essential hypertension, which has not yet been reported. The factor is shown to regulate cellular calcium uptake and can be inhibited by increasing calcium in food. The factor is isolated and a method for screening the factor using antibodies raised against the factor is described. The molecular weight of the factor is about 30004000. Biological studies indicate that the factor found in rats and humans is essentially similar.

The circulating factor appears to be present in a significant proportion (but not all) of the human population suffering from essential hypertension, and is mainly associated with low renin hypertension. Circulating factor identification in high blood pressure patients can be used to design and follow therapy that is resistant to this factor.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described briefly.

Figure 1 is the plasma elution profile of SHR rats as in Example 3.

Figure 2a is a reverse phase HPLC separation profile of the plasma sample from SHR rats of Figure 1.

Figure 2b is an HPLC profile of plasma from WKY rats.

Figure 3 is a schematic representation of the mechanism of calcium entry into cells and regulation of the mechanism.

Figure 4 shows the change in mean arterial blood pressure in SD rats infused with plasma from SHR, SD, and WKY rats.

Figure 5 shows the change in mean arterial blood pressure in SD rats receiving bolus plasma injections with SHR, SD, and WKY rats.

Figure 6 shows uptake of ⁴⁵ Ca in Krebspuffer incubated rat tails containing plasma from SD, WKY and SHR rats.

Figure 7 shows the change in mean arterial blood pressure in SD rats after administration of an abnormal fraction of plasma isolated from SHR rats by molecular exclusion chromatography.

Figure 8a is a reverse-phase HPLC profile of a culture medium from a thyroid parathyroid cell culture (using thyroid from SHR rats as the thyroid gland).

Figure 8b is a comparative profile for cultures of thyroid parathyroid cells from WKY rats.

Figure 9a is a reading of SDS-PAGE gel using a 14 kD standard.

Figure 9b is a gel reading of the thyroid extracts of an SD rat thyroid gland after 8 hours of incubation.

Figure 9c: Gel reading of the thyroid adrenal gland extract of an SHR rat after 8 hours of incubation.

Figure 10a is a reading of an SDS-PAGE gel run from culture medium from Sprague-Dawley thyroid cells (12 hour culture).

Figure 10b is a comparative reading using the thyroid gland from SHR rats after 4 hours of culture.

Figure 10c is a reading of culture medium from SHR thyroid culture after 12 hours of culture.

Figure 10d is a gel reading using a 14 kD standard.

Figures 11 and 12 are graphical representations of the data shown in Table 1.

Figure 13 is an isoelectric focusing gel of PHF plasma and cell culture medium.

The present invention is described in detail below.

The presence of a circulating factor in the blood of SHR rats was confirmed in our study by Am. J. Hypertens. 2, 26-31 (1989). In this study, an increase in blood pressure was observed in WKY and SD rats when blood from SHR rats was injected into normal blood pressure rats either by infusion or bolus injection. In addition, it was shown that uptake of ⁴⁵ Ca by a rat carotid artery section increased in a dose-dependent manner as plasma SHR concentrations increased in a buffer-based medium. The results of these experiments clearly show that growth in both blood pressure and calcium uptake in cells is dose-dependent on the amount of SHR plasma present and available in the system. Curiously, the onset of both events was delayed and gradual, despite the presence of known endogenous pressure regulators, e.g. Norepinephrine, angiotensin II and vasopressin have been reported to increase blood pressure almost immediately and very rapidly after administration. Another result observed in this study was

When the infusion of SHR plasma was stopped and replaced by plasma from normal hypertensive rats, the observed blood pressure very quickly dropped to baseline. The observed decrease excluded a simple volume effect. In a related experiment, dialyzed plasma from normal and hypertensive human subjects was injected into normal hypertensive SD rats and shown to induce hypertension. Plasma from these individuals also increased calcium uptake in rat tail arteries in vitro.

The origin of the circulating factor is unknown, but narrative reports that ΡΊΉ increases in hypertensive rats suggested that the parathyroid gland should be the target of the study. Thyroid control of SHR rats was found to lower blood pressure and plasma from SHR rats that had previously undergone parathyroid control did not elevate blood pressure in normal hypertensive rats. In contrast, transplantation of parathyroid gland from SHR in normal hypertensive SpragueDawley (SD) rats induced an increase in blood pressure and resulted in the appearance of factor in plasma as infused with other normal hypertensive rats by Lewis and P : Amer. J. Hypertens. 2, 898 (1989)].

Based on this study, it was concluded that the circulating factor originates from the parathyroid gland and therefore the name of this compound is called "Parathyroid Hypertensive Factor (PHF)".

Possible complexity factors in the experiments described above include the presence of calcium and other low molecular weight pressure regulating compounds in elevated concentrations in plasma used for transfusion. To eliminate this possibility, all plasma for transfusions is dialyzed overnight using a membrane with a molecular weight exclusion of about 1000. This procedure must be effective in removing calcium and most known endogenous pressure regulators, e.g. to remove ouabain-like factors, which have been reported to have molecular weights of between 400 and 500. All known endogenous pressure regulators work quickly.

For the purpose of isolating and identifying PHF from plasma, SHR rats, WKY rats and SD rats were decapitated and bled, and plasma collected from each type was centrifuged after treatment with heparin. Collected plasma was first dialyzed (molecular exclusion 1000) on an ultrafiltration system with an upper exclusion molecular weight of about 5000, and the collected filtrate was concentrated by lyophilization. Concentrated dialysates are applied to a molecular sieve column, e.g. Charged with Bio-Gel Ρ-6. Fractions were collected by elution with 0.05 M NH ₄ OAc (pH 7.0). Elution was measured photometrically at 280 nm using a Pharmacia UV detector and a fraction was detected in plasma from SHR rats that is not present in samples obtained from normal blood pressure rats (see Figure 1).

Abnormal fractions from SHR rats were pooled, concentrated by lyophilization and injected into normal hypertensive rats. An increase in blood pressure is observed with a delayed onset (30-45 minutes), which is typical of plasma from SHR rats. Plasma preparations obtained from rats in the same elution situation but with normal blood pressure do not show such activity. The active component in the abnormal SHR fraction has an estimated molecular weight of about 3500 daltons and is chromatographic, using both a liquid chromatography column and HPLC.

In order to further characterize the active fraction, plasma samples were lyophilized and fractionated on a Brownlee RP-P (C-8) reverse phase HPLC gradient using 0.1% trifluoroacetic acid: acetonitrile and the eluate again 280. nm. The results are shown in Figures 2a and 2b.

Thyroid parathyroid gland was excised from SHR rats and cultured for 7 days in Hank's medium (Gibco) with daily replacement of medium. APHF production can be stimulated by reducing calcium in the medium. The collected media were subjected to dialysis and filtration in an analogous manner to the plasma sample treatment, then lyophilized and fractionated by HPLC using a reversed phase column. Figure 8a shows the isolation. Figure 8b shows the results obtained using the glands of WKY rats.

Primary cell cultures prepared from SHR and SD rats were grown as described above. At 4 hour intervals, the medium is sampled and lyophilized. After resuspending in a minimal amount of distilled water, the extracts were dropped onto SDSPAGE gel (15-18% acrylamide / bis-acrylamide) and developed (Bio-Rad Mini Protein II) for about 1 hour. After staining with coomassie blue or preferably silver, the gel is counted. The results are shown in Figure 10. A unique peak appears in the SHR rat cell medium, which is not present in the SD rat cell medium, and is not present in the SHR rat cell medium before about 12 hours. The peak is estimated to have a molecular weight of approximately 3,300 daltons.

In a related experiment, thyroid parathyroid cells were cultured as described. After 8 hours, the cells were removed from the culture, homogenized in 50 mM acetic acid, centrifuged at about 5000 xg to remove cell debris and run on a 10-18% acrylamide / bis-acrylamide gel. The results are shown in Figure 9.

The isoelectric point was determined for PHF from SHR Thyroid Parathyroid Culture Medium on two gels, IEF and SDS-PAGE, using a Bio-Rad mini isoelectric focusing system and an ampholytic gradient of pH 3-10. The result4

Figure 17 shows a common patch at about pH 6.

Total RNA from PHF-producing cells can be extracted by conventional techniques (Maniatis T. et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982). The poly (A) RNA can be isolated by chromatography, e.g. an oligo- (dT) cellulose column and can be converted to cDNA using reverse transcriptase and then double-stranded using conventional techniques [e.g. Land et al., Nucl. Acids. Gap; 9, 2251 (1981)]. The DNA may be inserted into a suitable transformation vector and used by suitable cloning hosts, e.g. E. coli K12. The digested plasmids obtained from the host are used to construct a cDNA library and screened with a synthetic labeled probe according to standard protocols. Southern: J. Mole. Biol. 98, 503 (1975)]. Highly hybridizing fragments can be used to construct an expression vector. Cells transformed with the expression vector can be used as a high-level source of PHF.

Using purified PHF from plasma, cultured production cells or transformed cells, polyclonal and monoclonal antibodies to PHF can be generated and used in assays for PHF.

Male Balb / C mice were immunized by implantation of aminophenol thiol ether plates on which PHF partially purified from a thyroid parathyroid culture was fixed according to the procedure of Viamontes et al. Immunol. Meth. 94, 13-17 (1986)]. Mice are vaccinated with antigen in Freund's incomplete adjuvant at two-week intervals and antibody titers are determined by enzyme-linked immunosorbent assay (ELISA) using PHF isolated from plasma as antigen. A detectable amount of polyclonal antibodies is observed within 1 month, after which the titer increases.

Monoclonal antibodies can be prepared from the spleen of mice producing polyclonal antibodies.

The desired hybridoma and monoclonal antibodies can be obtained using the method of Langone and Van Vanakis (1986) Methods in Enzimmology, 27,1-947, with minor modifications well known to those skilled in the art.

Methods using polyclonal and / or monoclonal antibodies are not limited to some solutions; solutions include radioimmunoassay, enzyme immunoassay, enzyme-linked immunosorbent assay, and immuno-precipitation assay. A particularly desirable embodiment of such assay systems can be in the form of a diagnostic kit that can be used in a medical office or clinic without the need for sophisticated analytical equipment. Such methods can be used to detect hormones and other immunoreactive compounds in body fluids. An example of such a kit is a commercial kit for early pregnancy detection. Accordingly, PHF can be detected qualitatively or quantitatively using such methods.

The kit for enzyme immunoassay includes: (a) an anti-hypertensive antibody to the parathyroid gland, preferably bound to a solid phase; (b) an enzyme-labeled secondary antibody; c) a substrate for said enzyme label or said secondary antibody; and d) standard solutions of parathyroid high blood pressure factor. The RIA Assay Kit includes: a) an anti-parathyroid antibody to high blood pressure factor; b) an anti-parathyroid antibody to high blood pressure factor; (c) standard solutions of radiolabeled parathyroid high blood pressure factor; and (e) precipitating reagent. The kit for immunoprecipitation includes: (a) an anti-hypertensive antibody to the parathyroid gland; b) a solid phase to which said antibody is attached; and c) standard solutions of parathyroid high blood pressure factor.

The identification of PHF explains the seemingly contradictory reports in the literature that dietary calcium is often effective in lowering blood pressure, while calcium channel blockers are also effective in the same patient. It is expected that more serum calcium ions will eventually lead to higher intracellular calcium levels and higher blood pressure. While not wishing to be bound by any particular theory at present, it appears that PHF acts on open calcium channels as shown in Figure 2, and high levels of dietary calcium inhibit the release of PHF. Moreover, SHR rats are shown to have a calcium-deficient diet that results in an increase in plasma PHF, which is not present in the plasma of SHR rats fed a high-calcium diet [Lewanczuk and Pang, 4th Annual Meeting of the American Society for Hypertension; Abstract (1989).

This theory is supported by infusion studies of other self-acting compounds. When SD rats are infused with dialyzed SHR plasma and norepinephrine, arginine vasopressin (AVO) or angiotensin II (A-II), the increase in mean arterial pressure (MAP) is enhanced and the maximum increase in the peak of SHR plasma is high. during the blood pressure response.

The presence of PHF appears to be characteristic of low renin and salt-sensitive forms of hypertension, but not of high-renin and salt-sensitive forms.

The origin of the PHF cell in the parathyroid gland is confirmed by parathyroid excision (parathyroidectomy) of SHR rats, which results in a reduction in mean arterial blood pressure (MAP). In addition, a unique cell type can be observed in the parathyroid gland of the SHR but not in the parathyroid gland of SD or WKY rats. The new cells can be observed under both light and electron microscopes. The cells have a dense and irregular shape

They have a more intense staining of the cytoplasm using fucinaldehyde or iron hemothoxylin.

The availability of the APHF detection procedure allows the physician to perform a diagnostic test to identify the cause of each essential hypertension and to select and monitor appropriate therapy.

In addition to identifying essential hypertension, the presence of PHF and the detection of the presence of PHF can also be used to study and treat other conditions that may be associated with hypertension but are not necessarily our primary symptom. so e.g. non-insulin dependent diabetics often suffer from hypertension. Conversely, people with hypertension show reduced glucose tolerance. In both cases, increased intracellular free calcium was observed.

PHF can be detected in the plasma of Ob / Ob mice, which are obese, suffer from hypertension and non-insulin dependent diabetes mellitus. APHF can be isolated from these mice from the same serum subfraction as PHF from SHR rats. Detecting PHF may be useful in the diagnosis of non-insulin dependent diabetes mellitus (NIDOM) and may open up a new area of research to clarify the role of PHF in NIDOM.

Some forms of cancer are characterized by an increase in intracellular free calcium (see Okazaki et al., Canc. Gap. 46 (12 Pt 1), 6059-6063 (1986); Lipton and Morris, Canc. Chemother. Phaimacol. 18 (1): 17-20 (1986); Chien and Warren, Canc. Gap. 46 (11), 5706-5714; Shirakawa et al., Canc. Gap. 46 (2): 658-661 (1986); and Meyer, J. Hypertens. 5 (Supplement 4), S3-S4 (1987)]. Activation of the parathyroid gland is also associated with certain forms of cancer [see Palmer et al., Am. J. Epidemiol. 127 (5): 1031-1040 (1988), and Feig and Gottesman (Cancer 60 (3): 429-432 (1987)). Because PHF is a parathyroid gland, and preliminary data suggest that PHF can increase intracellular calcium, PHF may be interested in these and other forms of cancer. Accordingly, testing for PHF can be valuable in understanding the pathology of such cancers and in developing detection methods and therapeutic treatment systems.

The following examples illustrate the present invention in detail, but are not limiting thereof. Various modifications may be apparent to those skilled in the art; these modifications are within the scope of the invention.

Example 1

Detection of the presence of PHF in SHR rats

Male rats of SHR, Wistar-Kyto (WKY), and Sprague-Dawley (SD) were decapitated and bled, and blood collected from each strain was treated with heparin (100 International Units / ml) and centrifuged at 3Kxg for 10 minutes at 4 ° C. temperature. The resulting plasma is dialyzed against distilled water using a membrane with a molecular weight exclusion of 1000 Daltons.

SD rats were anaesthetized with Na-pentobarbital (50 mg / kg i.p.) and a catheter was inserted into the jugular vein for injection of plasma and drugs, and a catheter was inserted into the carotid artery for blood pressure measurement.

Plasma is administered either as an infusion of 3 ml / kg / hr or as a series of bolus injections; in the latter case, a bolus of 2.5 ml / kg.

Figure 4 shows the change in mean arterial blood pressure over time after infusion of plasma from SHR, WKY, and SD rats into SD rats. After 105 minutes, SHR plasma infusion was terminated and replaced with SD plasma; this results in a return to baseline blood pressure within 30 minutes.

The effect of bolus administration of SHR, WKY and SD plasma on SD rats is shown in Figure 5. The function of the change in blood pressure is similar to Figure 4, but not to the relative effect.

Example 2

Regulation of calcium uptake

Calcium uptake ( ⁴⁵ Ca) is measured in tail arteries in vitro using the low affinity lanthanum collection kit described by Pang et al., Life Sci. 42, 1935 (1988). The rat caudal artery strips were equilibrated in oxygenated (95% O ₂ , 5% CO _{2 in} KrebsHanseleit buffer for 2 hours. After equilibration, the strips were incubated in 30% Krebs plasma in the presence of 0.1 µCi of ⁴⁵ Ca. the tissues were rinsed in cold, Ca ²⁺ -free La ³⁺ solution, dried by rinsing, weighed and digested as described by Pang et al., Life Sci. 42, 1935 (1988). scintillation counter and is shown in Figure 6. The uptake of ⁴⁵ Ca follows a blood pressure rise curve and is dose dependent.

Example 3

Isolation of PHF by exclusion chromatography

Plasma from SHR, WKY, and SD rats was dialyzed against distilled water overnight (1000 molecular weight exclusions) and filtered using an Amicon ultrafiltration unit (5000 molecular weight exclusions) and chromatographed on a Bio-Gel Ρ-6 column. Eluted with 05 M ammonium acetate and the eluted fractions were measured at 280 nm using a Pharmacia UV photometer. Aliquots of 6.67 ml were collected. A fraction with a molecular weight of about 3500 Daltons was observed in SHR plasma, whereas the same was not observed in plasma derived from WKY or SD rats (Figure 1). The molecular weight markers used to determine the size of the column are ACTH (T.. T. 4541, 7), insulin B chain (T. T. T. 3496), insulin A chain (T. T. T. 2530) and a 13 amino acid synthetic peptide (T.. T. 1464).

The individual fraction from SHR rats was concentrated by freeze-drying and injected into SD rats catheterized as in Example 1. An increase in blood pressure is observed as a function of the duration of administration and the amount of plasma from SHR rats. The data are shown in Figure 7.

Example 4

Purification of PHF by HPLC

The individual fraction obtained in Example 3 was lyophilized and fractionated on a Brownlee RP-P (-8) reverse phase HPLC column using a gradient of 0.1% trifluoroacetic acid: acetonitrile and followed at 280 nm. Biological activity is found to be at the peak (see Figure 2a) which does not appear in the plasma sample obtained from WKY rats.

Example 5

Isolation from cell culture

Thyroid parathyroid glands were excised from SHR and WKY rats. The glands are cultured in Hank's medium by changing the medium daily. Collected media is dialyzed against distilled water and filtered in the same manner as used for plasma treatment. The fraction having a molecular weight of 1000 to 5000 is lyophilized and fractionated on a Brownlee RP-P (-8) reverse phase HPLC column using a gradient of 0.1% trifluoroacetic acid: acetonitrile and followed at 280 nm. An active fraction from SHR is eluted at the same site as plasma but no corresponding fraction is found in media from WKY rat cells. Figure 8a shows the elution profile of media from SHR cell lines, and Figure 8b shows media elution profiles from WKY rats.

Example 6

Separation of the culture medium by SDS-PAGE

SDS-PAGE mini lapgéleket, which are 15 to 18% containing Tris (pH 8.8), acrylamide / bis-acrylamide (Bio-Rad), tris (pH 8.3) and resuspended lyophilized (H ₂ O) cell culture media derived from SHR cultures and SD thyroid glands were prepared as described in Example 5. The gels were run for 1 hour at 200V (Bio-Rad Mini Protein II). The results are shown in Figures 10a-d.

Example 7

Separation of cell extracts by SDS-PAGE

Thyroid parathyroid cells from SHR and SD rats cultured in Hank's medium for 8 hours were isolated and homogenized in 50 mM acetic acid, centrifuged at 5000 x g, 10-18% acrylamide-bis-acrylamide sheet and developed for 1 hour at 200 V. The results are shown in Figures 9a-c.

Example 8

Isoelectric focusing

Analytical isoelectric focusing of the column fractions was performed using a BioRad Model 111 Mini-IEF analytical unit. This technique separates proteins by their isoelectric point and the results indicate the purity of the column fractions isolated according to Example 3. The procedure used is that described by Righettl in "Isoelectric Focusing Theory, Methodology and Applications," Elsevier Biomedical Press, Amsterdam (1983), modified by the "BioRad Model 111 Mini-IEF Cell Instruction Manual" (BioRad Laboratories, Richmond). , California). A thin plate of polyacrylamide gel containing ampholytes (pH 3-10) is poured onto a gel backing film. Samples purified from SHR plasma and thyroid parathyroid cell cultures were loaded onto the gel along with the standards, and the gel was directly applied to graphite electrodes. Proteins are focused for 60-90 minutes. The protein strips are stained with dye containing both Coomassie blue and Crocein red. The results are shown in Figure 17. An approx. A band at pH 6 appears in both plasma and culture medium.

Example 9

Production of polyclonal antibodies

Male Balb / C mice were immunized with partially purified PHF (20-30% purity). The amount of PHF in the formulation is estimated from the absorbance at 210 nm. Peptide samples, each containing about 10 µg of PHF, are bound to an aminophenylthioether (APT) derivatized paper disk by diazo bonds according to the procedure described by Viamontes et al. Immun. Meth. 94: 13-17 (1986)]. An aliquot of 0.3 ml of a 10 mg / ml solution of NaNO _{3 in} double-distilled water is added to a 10 ml aliquot of 1.2 N HCl. An aliquot of 0.3 mL of NaNO ₃ / HCl solution was placed on top of each 6 mm APT disks and placed in a dish and kept at 4 ° C for 30 minutes on a shaker. The discs were washed with cold distilled water and then dried with 0.2 M Na acetate (pH 4). The derivatized paper disks are then soaked in Freund's complete adjuvant and implanted ip. After two weeks, the anti-PHF titer was determined by ELISA and the mice were re-inoculated (booster) as described above, except that the disks were implanted in Freund's incomplete adjuvant. The titer is checked by ELISA one week later. Mice are vaccinated every two weeks, and samples are also examined using the ELISA procedure described above.

Plates were coated with 500 ng / ml PHF solution in Tris buffer (pH 9.0) at 100 μΐ per well, stored overnight and washed with phosphate buffered saline Tween 20 and three times with PBS. washed. The plates are then covered with 0.2% gelatin in PBS, incubated for 30 minutes at 37 ° C, and then washed three times with PBS-Tween 207.

EN 205 859 B threads. Immunized serum from mice (100 μΐ / well, 1% gelatin (PBS-Tween 20) in serial dilutions depending on the procedure and expected results) was added as described above and the plates were incubated for 1 hour at room temperature and wash three times with PBS-Tween 20. Horseradish peroxidase (1: 2000 in 1% gelatin in PBS-Tween 20) was added at 100 μΐ / well, incubated for 1 hour at room temperature, and the plates were washed with PBS-Tween 20. A mixture of 10 ml of citric acid (0.05 mol / L, pH 4), 100 μΐ of 2,21-azinobis (3-ethylbenzthiazolinesulfonic acid) and 40 μΐ of hydrogen peroxide was added at 100 μΐ / well, followed by 1 hour, after incubation at room temperature, read at 405 nm (Titer-Tek Multi Scan). The results are shown in Table 1 and Figures 11 and 12, respectively.

Table 1 Mouse 1

dilution control NMS ** * 4 weeks * 6 weeks * 8 weeks 8 weeks * (5ng / well) 30 0.019 0.141 0.216 0.557 0.674 60 0,013 0,138 0,145 0.549 .499 120 0 0,038 0,075 0.506 0.492 240 0 0.031 0,030 .306 0.483 480 0 0,021 0 0.111 0.242 960 0 0 0 0,012 0.071 1920 0 0 0 0 0,024 3840 0 0 0 0 0

Table 2 Mouse 2

dilution control NMS ** * 4 weeks * 6 weeks * 8 weeks 8 weeks * (5ng / well) 30 0.019 0.144 0.272 0.662 .526 60 0,013 0.118 0.243 0.692 0.581 120 0 0.041 0.103 0.681 0.733 240 0 0,035 0,085 0.279 0.410 480 0 0.031 0,051 0.091 0.222 960 0 0 0 0 0,104 1920 0 0 0 0 0,026 3840 0 0 0 0 0

Note: * 50 ng / well.

** Normal mouse serum

Claims (6)

PATENT CLAIMS CLAIMS 1. A method for producing a substantially pure parathyroid hypertension factor by isolating and processing the plasma of a hypertensive mammal comprising: - removing components with a molecular weight of less than 1000 by dialysis against separated plasma from distilled water; - ultrafiltration to separate plasma components with a molecular weight of less than 5000; concentrating the resulting solution by lyophilization; fractionating the material on a molecular sieve column eluting with dilute ammonium acetate solution; - further purifying the parathyroid high blood pressure fraction fraction by lyophilization and, if desired, the resulting product by reverse phase high performance liquid chromatography. 2. A method for detecting the presence of parathyroid hypertension, characterized in that: a) collecting serum containing polyclonal antibodies raised against PHF by injecting a mammalian parathyroid hypertension (PHF) solution; and screening the serum sample suspected of containing PHF by immunoassay using polyclonal antibodies; obsession b) isolating antibody secreting B lymphocytes from the immunized vertebrate by injecting PHF into the vertebrate; antibody-secreting B lymphocytes are fused with myeloma cells to form hybridomas; selecting and cloning hybridomas that select for PHF antibodies; culturing hybridomas that select for cloned PHF antibodies and isolating the selected monoclonal antibodies; and screening the serum sample suspected of containing PHF by immunoassay using monoclonal antibodies. 3. The method of claim 2, wherein the immunoassay is an enzyme-linked immunoassay. 4. The method of claim 2, wherein the immunoassay is an enzyme-linked immunosorbent method. 5. The method of claim 2, wherein the immunoassay is an immunoprecipitation method. 6. A kit for detecting the presence of hypothyroidism in a mammal, comprising: (a) an anti-hypertensive antibody to a solid phase bound parathyroid gland; an enzyme-labeled secondary antibody against parathyroid hypertension; a substrate for the signaling enzyme on the secondary antibody; and standard solutions of parathyroid hypertension; obsession b) anti-hypertensive antibody to the parathyroid gland; a solid phase to which said antibody binds; and standard solutions of parathyroid hypertension.