Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Renin is a key molecule produced and released by specialized juxtaglomerular cells in the kidney. Renin production and secretion is the rate-limiting step in a system of molecules whose primary function is to regulate blood pressure and salt and water balance.
• renin is secreted to rectify the initial assault.
• Hyperresponsiveness of the juxtaglomerular cells leads to pathologies such as hypertension and vascular sclerosis.
• Some success has been associated with intervention of renin enzymatic activity in renin hyperresponsive states.
• Inhibitors of angiotensin I conversion to angiotensin II as a consequence of renin enzymatic activity has been the primary therapeutic strategy. This approach has been successful mainly when blood levels of renin are very high and therefore blockable at the site of angiotensin I conversion.
• This invention provides a purified protein having an estimated molecular weight of about 55 to 65 kD under reducing conditions, or an active fragment thereof, wherein the protein binds renin and wherein the renin- binding activity can be deactivated in vitro in inside-out membrane vesicles incubated in Krebs-Ringer buffer with high potassium, by phospholipase K., lysolecithin, melittin, trypsin, calcium, barium and potassium chloride and wherein the renin-binding activity can be activated by magnesium and calmodulin.
• Also provided is a method of predicting an increased probability of a pathology associated with low renin syndrome in a subject comprising quantitating the amount of renin in a specimen from the subject, the presence of renin below about 1 ng/ml plasma per hour of incubation indicating the presence of a disease associated with the syndrome.
• the invention provides a method of predicting an increased probability of a pathology associated with low renin syndrome in a subject comprising quantitating the amount of renin in a specimen from the subject, the presence of renin below about 1 ng/ml plasma per hour"of incubation indicating the presence of a disease associated with the syndrome. Therefore, the invention provides the discovery that low renin is associated with a wide variety of pathologies.
• "low renin syndrome” as used herein means a low renin condition in a patient which is a prognosticator of a pathology which is associated with low renin.
• low renin means any amount of renin below about 1 ng/ml plasma per hour. However, the lower the renin, e.g. below about .5 ng/ml plasma per hour, the more predictive the occurrence of the syndrome and the resulting pathologies.
• a pathology associated with low renin syndrome means any pathology which a subject with low renin has in a greater frequency than a subject with normal renin.
• Normal renin is between about 1 ng/ml plasma per hour and about 5 ng/ml plasma per hour.
• pathologies associated with low renin syndrome are hypertension (with and.without nephropathy) and diabetes mellitus.
• pathologies associated with low renin syndrome include: renovascular hypertension (phase III) ; chronic salt excess; pheochromocytoma; pituitary deficiency; neuropathy with orthostatic hypertension; nephropathy with hypoaldosteronism; essential hypertension in blacks; Conn's syndrome; primary aldosteronis ; aldosterone-producing adenoma; idiopathic hyperaldosteronism; indeterminate hyperaldosteronism; glucocorticoid-remediable hyperaldosteronism; hyporeninemic hypoaldosteronism; congestive heart failure (iate stages) ; chronic stress; acromegaly; hyperthyroidism; genetic hypertension (late stages in rats) ; Cushing's syndrome; reduction of renal mass; syndrome of inappropriate ADH; cirrhosis (with ascites) ; Liddle's syndrome; licorice gluttony; vitamin D intoxication and hyper
• low blood levels of renin and renin secretory hyporesponsiveness have been documented in more than a few clinical disorders, and in every case the impairment has been localized to subcellular dysregulation of the renin secretory pathway. It is observed in the majority of blacks, particularly black hypertensives, diabetics, and diabetic hypertensives with obesity. It is this whole class of clinical disorders characterized by unmeasurably low blood renin levels and a profound renin secretory hyporesponsiveness, that has herein been termed low renin syndrome, for which there previously was no scientifically acceptable methodology for diagnosis or therapeutically reasonable regimen for treatment.
• the development of methodologies for diagnosing and the identification of dysfunctional molecules in the renin secretory cascade are novel developments in the context of low renin syndromes.
• End-organ damage is also associated with low renin syndrome.
• the usual end-organ damage precipitated by low renin syndrome is stroke (or generalized brain damage) , heart attack (or some sequelae of myocardial infarction) , and kidney failure (acute or chronic) .
• stroke or generalized brain damage
• heart attack or some sequelae of myocardial infarction
• kidney failure acute or chronic
• the invention also provides a method of treating a pathology associated with low renin syndrome comprising increasing the amount of renin secretion in hyporesponsive juxtaglomerular cells associated with the pathology.
• hyporesponsive means juxtaglomerular cells which substantially fail to secrete renin.
• any factor which acts on renin once secreted would fail to have an effect on hyporesponsive juxtaglomerular cells.
• hyporesponsive juxtaglomerular cells can be promoted to secrete renin.
• a compound which activates the transcription of the gene encoding renin could be administered to the subject. These compounds include, for example, cAMP and Furosemide. Alternatively, one could administer renin directly to the subject.
• One example of a method of screening compounds which increase renin transcription in hyporesponsive juxtaglomerular cells can comprise the steps of: (a) contacting a compound with a cell transformed with a vector containing a promoter region of the renin gene in proper orientation with a nucleic acid encoding a marker; and (b) detecting the presence of the marker protein secreted by the cell, an increase in marker protein indicating a compound which increases the amount of renin transcription.
• An alternative method of screening compounds which stimulates the renin secretion from hyporesponsive juxtaglomerular cells includes: (a) contacting a compound with a cell containing DNA encoding renin; and (b) detecting the presence of secreted renin, an increase in renin indicating a compound which increases the amount of secreted renin.
• a method of screening for compounds which affect the ability of renin- binding protein to transport renin from the cytosol to the plasma membrane is provided. The method comprises contacting a juxtaglomerular cell with a compound and determining the effect on the transport.
• the renin-secreting renal juxtaglomerular cell is the key rate-limiting component in low renin syndrome.
• the cell has a novel and unique pathway for exporting renin and its precursor, prorenin. This pathway involves participation of a potassium-chloride-hydrogen transporter, a hydrogen pump, a renin-binding protein, and a renin receptor. These molecules play a role in what is termed the divergence translocation secretory pathway observed in low renin syndromes. Divergence translocation differs from classical exocytic degranulation in that the plasma membrane plays a significant role in directly exporting renin by a mechanism involving a renin receptor.
• Divergence translocation also differs from constitutive vesiculation in that it is regulated by well-known cellular messengers.
• the potassium-chloride-hydrogen transporter and the hydrogen pump have been identified in the secretory granular membrane.
• the renin-binding protein has been identified exclusively in the cytosolic soluble space. It functions as a chaperone molecule for renin transport from granular storage to the plasma membrane.
• this invention provides that the secretion of renin is important in treating low renin syndrome.
• the invention also provides the discovery of a purified protein which binds renin and, therefore, is important to the ultimate secretion of renin.
• purified means the protein is sufficiently free of contaminants with which the protein normally occurs to distinguish the protein from the contaminants. It is known that this protein binds renin and facilitates the anchorage and transport of renin.
• the protein (sometimes referred to as the "renin receptor” herein) has an estimated molecular weight of about 55 ' to 65 kD under reducing conditions, and binds renin.
• the renin-binding activity can be deactivated, in vitro in inside-out membrane vesicles incubated in Krebs-Ringer buffer with high potassium, by phospholipase A ⁇ lysolecithin, melittin, trypsin, calcium, barium and potassium chloride and the renin-binding activity can be activated by magnesium and calmodulin.
• the protein sequence can be determined by molecular cloning and sequence analysis of a cDNA encoding for renin receptor.
• a complementary DNA encoding for a renin receptor protein can be isolated from human kidney cDNA library by immunological screening of in vitro translation products from the cDNAs. The procedure is similar to that conventionally used for other renin- binding protein family (Inoue et al., J. Biol. Chem.
• double-stranded cDNA can be synthesized from human kidney poly(A)* RNA (4ug) and the Eco Rl adaptor ligated to the cDNA ends.
• the double- stranded cDNA can be sized fractionated on a 1% agarose gel. DNA of more than 1000 bp can be recovered from the gel and ligated into the Eco Rl site of the pGEM-4Z plas id vector, which usually contains the promoter for SPG RNA polymerase and ulti cloning sites.
• the resultant plasmids can be used to transform E coli strain HB101 and sublibraries formed of transformants.
• the recombinant plasmid of each sublibrary can be isolated from respective subcultures and the RNAs transcribed in vitro from the template DNA, and then the RNAs translated in a reticulocyte lysate translation system including [ 35 S]methionine.
• the translation products can be immunoprecipitated with anti-renin receptor antiserum and then subjected to SDS-polyacrylamide gel electrophoresis.
• the cDNA clone can be digested with a variety of restriction enzymes to obtain particular DNA fragments for subcloning into MI3 phage vectors mpl ⁇ and mpl9.
• the nucleotide sequences can be determined by the dideoxy " chain-termination method. Nucleotide sequence and corresponding amino acid sequence and homology analyses can be performed with GenBenk, Swiss protein data bank, and the National Biomedical Research Foundation's protein data bank with the aid of conventional software.
• the invention also includes fragments of the renin receptor which contain the portion of the renin receptor which binds renin.
• the region critical for binding can be determined by standard methods (Bjork an et al.. Nature 329:512-518 (1987); Cunningham et al.. Science 254:821-825 (1991); Young, Protein Engineering 5:117-119 (1992); Fernandez-Botran, FASEB J. 5:2567-2574 (1991)). Briefly, renin binding domain of the renin receptor can be determined by established crystallization, size exclusion chromatography and calorimetry. Crystals of the complex between renin and the renin receptor (cytosolic domain) can be grown by vapor phase diffusion.
• the composition of the crystals can be analyzed by dissociation in 0.1% trifluroacetic acid and chromatography under denaturing conditions.
• the components can be eluded isocratically with a linear acetonitrile (10%) gradient at a flow rate of 1 ml/min.
• the complex can be prepared by adding a slight excess of renin receptor, and purifying the complex over a sephadex G75-120 size exclusion column equilibrated in 10 mM tris (pH 8.0) and 100 mM NaCl.
• the invention also provides the nucleotide sequence encoding the renin receptor.
• the sequence encoding renin receptor can be determined by standard procedures. For example, the amino terminal sequence of the renin receptor can be determined and a corresponding nucleotide sequence can be deduced. This nucleotide sequence can then be used to make a probe to hybridize sequences from a gene library.
• antibodies can be made by standard procedures (Harlow and Lane, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988) . Briefly, purified renin receptor can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly or spleen cells can be obtained from the animal. The cells are then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen DNA libraries for cells secreting the renin receptor.
• the probe and/or antibodies described above or the probes deduced from the isolated nucleic acid sequence can be used in similar methods to detect the protein or gene from any other animal, e.g. humans.
• the invention also includes any variations in the renin receptor, or active fragments of the receptor, that exist in the human renin receptor.
• the invention also provides the renin receptor labeled with a detectable moiety.
• This moiety could, for example, include a radioactive label or an enzyme for use in an ELISA.
• the renin receptor could be labeled by any of a number of methods known in the art (P. Michael Conn (Ed.) Neuropeptide Technology: Gene Expression and Neuropeptide Receptors, Academic Press, New York (1991)).
• physicochemical characterization studies can utilize covalent labeling of the membrane-bound form of the receptor with [ 125 I]renin prior to detergent extraction and analysis by established procedures.
• Bifunctional N- hydroxysuccinimide (NHS) reagents have been used to cross ⁇ link proteins of the renin family to their receptor (P.
• the membrane preparation can be pelleted and washed thoroughly by resuspension in cold phosphate buffer and recovered by centrifugation.
• Cross-linking can be performed in phosphate buffer containing 1-5 mM NHS reagent (added as a 50 mM solution freshly prepared in DMSO) , incubated for 30 mins at 10*C.
• the labeled renin receptor could then be utilized to bind renin to detect or quantitate the amount of renin in a sample from a subject.
• the renin receptor could be used to bind renin and the presence of binding could be detected in another manner.
• the renin receptor could be bound to a solid support; (b) the sample containing renin could then be passed through the support under binding conditions; and (c) a labeled antibody could then be used to detect or quantitate the amount of renin captured by the renin receptor.
• a labeled antibody could then be used to detect or quantitate the amount of renin captured by the renin receptor.
• Such methods are well- known in the art and are generally described in Harlow and Lane, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988.
• the invention provides a method of detecting renin comprising contacting a ligand selected from the group consisting of the protein of claim 1 and an antibody with a specimen containing renin and detecting the presence of binding between the protein and renin.
• the invention also provides a method of detecting prorenin comprising contacting an antibody specifically reactive with prorenin with a specimen containing prorenin and detecting the- presence of binding between the antibody and prorenin.
• An antibody specifically reactive with prorenin is also provided.
• the invention also provides vectors and hosts for expression of the renin receptor.
• Suitable hosts include, for example, the adenovirus-transformed human kidney cell line 293.
• Suitable vectors can be constructed using the cDNA cloning sequence described below.
• the renin receptor cDNA clone, inserted into the polylinker of pSP65, can be excised on a BamHIII restriction fragment and the 3' protruding ends made blunt by treatment with the Klenow fragment of E. coli polymerase.
• the plasmid can be constructed by replacing the human protein C coding sequence in pLPC-hd vector with the blunt ended renin receptor cDNA sequence at the plasmid Bcl.l site.
• These host vector systems can be used to produce the renin receptor by recombinant methods.
• Juxtaglomerular (JG) cells were isolated from male Sprague-Dawley rats (100-150g) and human kidneys by the following technique. Kidneys were quickly excised and placed into a beaker of cold (4*C) isotonic Krebs-Ringer buffer (KRB) of the following composition (mM) : 118 NaCl, 2.5 CaCl 2 , 1.2 MgS0 4 , 25 NaHC0 3 , 1.2 KH 2 P0 4 , 11.1 glucose, 1.5% bovine serum albumin (BSA) , pH 7.4. The renal capsule was carefully removed and 2-3 cortical slices (0.5 mm thick) per side per kidney were excised with a manually-operated kidney microtome.
• KRB isotonic Krebs-Ringer buffer
• the slices were placed in a small beaker (25 ml) containing 15 ml isotonic KRB, washed, weighed and harvested to yield 6g tissue slices, and then transferred to a prechilled plastic petri dish on an iceplate and minced into pieces (about lxlxl mm) using a single-edge razor.
• the minced tissue was- washed with 30 ml KRB and resuspended in 30 ml fresh KRB supplemented with 0.1% collagenase (Type III or IV, Sigma) and 100 mg/ml DNAse (Type I, Sigma) equilibrated for 20 min at 37"C.
• the tissue was resuspended in fresh enzyme media (30 ml) and incubated in a shaker water bath for two successive 45 min periods.
• the resulting cell digest at the end of the first period was poured through a 20 ⁇ m nylon mesh; the undigested tissue was returned to 30 ml KRB with enzymes and incubated in the water bath for the second period.
• the cell filtrate was diluted with 10 ml enzyme-free KRB and centrifuged at 250 x g for 5 min.
• the cell pellet was resuspended in 5 ml fresh KRB on ice. Both 45 min-washed cell digests were pooled, counted with a hemocytometer, and diluted to give a final cell count of 5-7 x 10 6 cells/ml KRB.
• the cell suspension was then mixed with standard iso-osmotic Percoll to form a range of 30-60% Percoll solutions and centrifuged in polycarbonate centrifuge tubes at 16,000 x g for 30 min in a fixed angle rotor (Ti 60, Beckman) which resulted in a self-formed Percoll gradient optimized for minimal aggregation and maximal band/gradient capacity.
• the 45% Percoll gradient was found to be most suitable for the isolation of a JG cell enriched fraction.
• the gradient formed was calibrated with density marker beads (Pharmacia) having a density range from 1.016 to 1.139 g/ml.
• each Percoll fraction (or band) was removed following ultracentrifugation and washed free of Percoll with 10 volumes of enzyme-free KRB and centrifuged at 2,300 x g for 10 min. The resulting supernatant was assayed for lactate dehydrogenase. This assay was repeated after subsequent washing and incubating of cells as a means of monitoring cell integrity throughout the isolation and experimental procedure. The cell pellet was resuspended in 5 ml KRB and a cell count done and cell viability assayed by monitoring trypan blue exclusion. The remaining cell suspension was sonicated and assayed for renin and tubular contamination.
• the tubular marker enzyme, ⁇ -glutamyl transpeptidase (GGT) was chosen because it has been reported to be virtually absent from the renal vasculature and highly concentrated in the proximal tubule brush border and basal labyrinth.
• a sample of the fraction with the lowest tubular marker enzyme activity and highest renin activity was reserved for renin and protein assays, whereas the remainder was washed with 10 ml KRB : 1 ml sample and gently pelleted at 2,300 x g for 10 min and the pellet used for further experimentation as enriched JG cells.
• Viability after sedimentation ranged from 87% to 97% in the F3 and F4 fractions from 5 trials. Dead or degenerating cells were consistently found in the F2 fraction (d. 1.04 g/ml) along with cell debris and greater than 65% of the GGT-specific activity. An additional 25% of the GGT specific activity was found in the Fl fraction.
• Phase contrast microscopy of cells obtained from the F3 fraction revealed relatively small cells (5 to 10 ⁇ m in diameter) with phase contrast optics, although it was not possible to differentiate renin-containing from non-renin-containing cells by this technique. Tubular cell contamination of the F3 fraction was minimal ( ⁇ 4%) as evidenced by the GGT specific activity.
• Electron micrographs of the same fractions clearly demonstrate the absence of brush borders or distinct microvilli, suggesting further that these cells were not proximal tubule cells. Granules as well as several large vacuoles containing electron-dense material were visible in the cytoplasm.
• Rough ER and Golgi, granules, and plasma membrane vesicles (PMVs) were isolated as follows.
• the JG cell suspension was homogenized in a Waring blender for 20s at low speed and the resulting homogenate diluted to give a 6% (w/v) homogenate in KRB supplemented with 50 mM K* " , pH 7.4 or in the same KRB used to isolate the cell- enriched fraction.
• This diluted homogenate was centrifuged for 10 min at 4 ⁇ C at 1,400 x g in a Sorvall RC-3 refrigerated centrifuge (r av 14 cm) and the resulting pellet brought to a 6% (w/v) suspension with fresh KRB or modified KRB (KRB + 50 mM K*) .
• a volume of this suspension was mixed with Percoll to yield a 12% final Percoll concentration using a 30 ml syringe fitted with a #18 gauge hypodermic needle.
• the resulting washed membrane vesicles were resuspended in a buffer"of the following composition (mM) : sucrose, 250; triethanolamine-HOAc, 50; pH 7.5 ((EtOH) 3 N * -HOAc) ; dithiothreitol, 1.
• An ice-cold salt solution (1.5 mM KDAc and 15 mM Mg (0Ac) 2 ) was slowly added to washed membrane vesicles in a ratio of 10 ml solution: 20 ml membrane vesicles. The mixture was incubated on ice for 15 min.
• the membrane vesicles were sedi ented at 120,000 xg for lhr through a cushion of 0.5 M sucrose in a buffer (B) of the following composition (mM) : (EtOH) 3 N*HOAc, 50; KOAc, 500; Mg(OAc) 2 5; dithiothreitol, 1.
• the resulting pellet of salt-extracted membrane vesicles was suspended in 20 ml of buffer B containing 250 mM sucrose: The supernatant, not including the cushion, was centrifuged at 200,000 xg for 3.5 to deplete it of ribosomes and bound proteins.
• This purified membrane vesicles preparation (ER, granules and PMVs) was then solubilized and renin receptor extracted, purified and characterized.
• Renin receptor was isolated and characterized as follows. The solubilized fractions purified above served as starting material. This renin receptor preparation was applied to a pepstatin-aminohexyl-agarose column (1 7 cm) which binds renin, but had no affinity for renin receptor. The column, which was pre-equilibrated with 10 ml pyrophosphate buffer containing 100 mM NaCl (pH 6.5), was washed with the same buffer following the sample application. Fractions #1-10 which passed through the column were used as crude renin receptor preparation. This preparation, after further purification, was used to develop antibodies to renin receptor and to characterize the molecule further.
• sheep were injected subcutaneously at multiple sites with 1 ml of an emulsion containing 100 ⁇ g of the purified human renin receptor and Freund's complete adjuvant.
• the same sample was injected subcutaneously at 4 and 8 weeks after the first injection and anti-renin receptor raised by conventional methods. Molecular weight was estimated by conventional methods.
• renin entrapment in three cases of low renin syndrome pituitary deficiency (Hypox) , diabetes mellitus, and thyroid deficiency (Thyrox). Renin content ( ⁇ g/ml.h/mg protein) in freshly isolated JG cells from normal, Thyrox, Hypox, and diabetic kidneys was 2.17 ⁇ 0.10, 2.86 ⁇ 0.30, 13.66 ⁇ 1.30, and 4.33 ⁇ 0.10, respectively. All three models of low renin syndrome stored a significantly higher cellular renin than normal kidneys (p ⁇ 0.05 for Thyrox and diabetes and p ⁇ 0.01 for Hypox).
• the assay system is a direct radiolabelled immunological system with the use of a solid-phase coupled ligand for renin, purified renin, renin receptor and sheep anti-renin receptor antibodies, and biotinylated rabbit anti-sheep renin receptor antibody with labelled strep- avidin as the radioactive isotope. Renin is first bound to the solid-phase coupled high-affinity ligand plated on 96 well polyvinyl plates. The solid-phase is then washed after a specific incubation period (lhr) .
• Renin receptor is then added, incubated (lhr) , and the above step repeated with subsequent additions of sheep renin receptor antibody (lhr) , rabbit anti-sheep antibody (lhr) and radiolabelled strep-avidin (lhr) .
• Each addition binds avidly to the solid matrix.
• the uptake of labelled strep- avidin is directly correlated to the amount of renin receptor in the test solution.
• the assay system has the disadvantage of being a multi-stage incubation and washing procedure.
• the assay system has a number of advantages over currently used assay systems for renin and its components, (l) it measures the molecular species of renin receptor directly. (2) It measures the molecular species of renin directly, providing known -amounts of renin receptor is added to the assay system. (3) With known amounts of renin or renin receptor a single (or duplicate) assay plate can detect both renin and renin receptor in pg quantities. This is particularly significant for renin because the values of significance in low renin syndromes are in the pg range, whereas current indirect assay methods can only detect ng quantities. (4) The amount of both the ligand and labelled isotope added is less critical.

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• 1993
  • 1993-05-14 WO PCT/US1993/004640 patent/WO1993023433A1/en active Application Filing
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