WO1994023301A1

WO1994023301A1 - Renin/angiotensin i diagnostic assay

Info

Publication number: WO1994023301A1
Application number: PCT/US1994/003595
Authority: WO
Inventors: R. Tyler Mccabe; Bryan R. Wilson; Christopher A. Rhodes
Original assignee: Pharmaceutical Discovery Corporation
Priority date: 1993-04-02
Filing date: 1994-04-01
Publication date: 1994-10-13
Also published as: AU6528994A

Abstract

A method and assay for the rapid, sensitive, accurate measurement of a component of the renin-angiotensin-aldosterone ('RAA') hormonal axis in a biological sample using spectrofluorometric measurement is disclosed. In the preferred embodiment a known quantity of a fluorescent labelled antibody immunoreactive with a specific RAA component is incubated with known volumes of the RAA component conjugated with an inert substrate and a biological sample. The amounts of bound and unbound fluorescent antibody are determined by spectrofluorometry, either after or during the incubation. The amount of RAA component in the sample is then quantified after construction of a standard curve based on spectrofluorometric measurements of fluorescent antibody bound to increasing amounts of RAA component in a second incubation mixture. As demonstrated by examples in which angiotensin is quantified, this method allows measurement of RAA components in picogram amounts in minutes and is useful for diagnosis of hypertension, renal disease, adrenal gland disease, and cardiovascular disorders.

Description

RENIN/ ANGIOTENSIN I DIAGNOSTIC ASSAY

This invention relates to a highly sensitive method for assaying components of the renin-angiotensin- aldosterone hormonal axis to aid in the diagnosis and treatment of hypertension, renal disease, adrenal gland disease, and cardiovascular disorders.

Background of the Invention

The renin-angiotensin-aldosterone ("RAA") hormonal axis contributes to the regulation of blood pressure, tissue perfusion, and sodium/potassium balance. The mechanism is initiated by a drop in arteriolar pressure, sodium intake, or blood volume, any of which stimulates the kidney to secrete renin into the blood. In the blood, renin cleaves the liver-derived plasma protein angiotensinogen to form angiotensin I, which is further cleaved by converting enzymes from the lungs to form angiotensin II. Angiotensin II directly raises blood pressure by arteriolar vasoconstriction and indirectly affects blood pressure and volume by causing sodium retention in the kidney and secretion of aldosterone from the adrenal cortex. Aldosterone then raises blood pressure and volume through action on the kidney distal tubule resulting in sodium retention coupled with potassium excretion.

Elevated levels of the major components of the RAA hormonal axis may lead to cardiovascular disorders such as hypertension and are excellent diagnostic indicators. Currently, to determine whether a patient is at risk for heart disease or high blood pressure, a health practitioner usually obtains a blood sample from the patient and submits it to an outside, site-licensed testing facility that identifies and measures the quantity of specific components of the blood, such as renin or angiotensin I.

Levels of components of the RAA hormonal axis can be measured by radioimmunoassay or bioassay. Assay methods and clinical uses are reviewed in Sealey, J.E., et al . _f "Hormone Assays: Renin, Aldosterone, Peripheral Vein, Renal Vein, and Urinary Assays," Hypertension: Pathophvsiology, Diagnosis, and Management, 1443-60, J.H. Laragh and B.M. Brenner, ed. , Raven Press, New York (1990) . A radioimmunoassay uses the competition between radiolabeled and unlabeled substances in an antigen- antibody reaction to determine the concentration of the unlabeled substance, which may be an antibody or a substance against which specific antibodies can be produced. A bioassay determines the activity of a component in a sample by comparing its effects on a live animal, cells or isolated tissue with those of a reference standard. The available assay methods are capable of detecting RAA components in the blood sample by radiometric or spectrophotometric analysis. However, radioimmunoassays and bioassays are plagued with the following problems, among others, that hinder effective diagnosis relating to disorders involving RAA components: (1) Many radioimmunoassays and bioassays are sensitive only to nanogram levels of peptide, whereas measurement of at least picogram levels of material are necessary for effective, reliable diagnosis. (2) Radioimmunoassay and bioassay systems with high non-specific binding that distorts measured values. (3) Typically, the use of radioactive testing reagents necessitates a site-licensed facility. (4) Radioimmunoassays and bioassays typically employ two antibodies, a primary and secondary antibody, instead of one, which increases incubation time as well as background noise in the diagnostic system. In general, these assays consist of passing a sample through an immobilized phase consisting of labelled antibody bound to immobilized antigen (for example, passing plasma through a column containing labelled antibody to bound angiotensin I conjugated onto activated CH Sepharose™ 4B) , separating the immobilized phase from the biological sample (plasma) , washing and stripping the labelled antibody from the immobilized phase, and determining how much label was bound prior to passage of the sample versus after passage of the sample. This type of displacement assay may also be useful for construction of standards. (5) Samples cannot be measured rapidly while the patient is in the health practitioner's facility but must be sent to an outside facility, which delays diagnosis. (6) The radioisotopes used in radioimmunoassays are associated with high cost, health risks, and disposal problems.

A simple, rapid, sensitive, highly selective assay for measuring RAA components in blood and screening individuals in the health practitioner's facility would result in quick, accurate diagnosis. It would also be useful to be able to measure more than one RAA component at a time to determine ratios of one to another for diagnosis of various disorders.

It is, therefore, an object of the present invention to provide a simple, rapid, sensitive method for measuring levels of RAA components in biological samples.

It is another object of the present invention to provide a method for measuring RAA components in biological samples at picogram levels.

It is a further object of the present invention to provide a method for measuring RAA components in biological samples that can be performed in a health practitioner's facility.

It is yet another object of the present invention to provide an assay for measurement of RAA components in biological samples.

It is a still further object of the present invention to provide an assay for measurement of RAA components in biological samples that can be performed in minutes. ,

It is still another object of the present invention to provide an assay to be used in the diagnosis of hypertension and other cardiovascular disorders. summary of the Invention

A method and assay for the rapid, sensitive, accurate measurement of a component of the renin- angiotensin-aldosterone ("RAA") hormonal axis in a biological sample using spectrofluorometric measurement of a fluorescent labeled antibody to an RAA component, or fluorescent labeled RAA component, is disclosed. In contrast to previously used assays, there is no radioactivity, yet quantitative, sensitive results are obtained using fluorescent probes and standard curves constructed using the probe in the biological sample.

In the preferred embodiment, a known quantity of a fluorescent labelled antibody immunoreactive with a specific RAA component is incubated with known volumes of the RAA component conjugated with an inert substrate and a biological sample. The amounts of bound and unbound fluorescent antibody are determined by spectrofluorometry, either after or during the incubation. The amount of RAA component in the sample is then quantified after construction of a standard curve based on spectrofluorometric measurements of fluorescent antibody bound to increasing amounts of RAA component in a second incubation mixture. In another embodiment, plasma renin activity (PRA) is measured using fluorescently labeled angiotensin I, which is the RAA component. This is performed after generation of plasma angiotensin I from renin using an enzyme kinetic assay.

As demonstrated by examples in which angiotensin I is quantified, this method allows measurement of RAA components in picogram amounts in minutes and is useful for diagnosis of hypertension, renal disease, adrenal gland disease, and cardiovascular disorders.

Brief Description of the Drawings

Figure 1 is a graph of spectrofluorometric measurement of plasma angiotensin I (ng/ml) versus radioimmunoassay measurement, using the method of Sealey et al., Hypertension: Pathophysiology Diagnosis, and Management, 1990; Sealey, Clin. Che . , (1991), of plasma angiotensin I (ng/ml) .

Figure 2 is a graph showing a standard curve of spectrofluorometric (B/B₀) /1-(B/B₀) versus angiotensin I (ng) , where B represents specific counts per second (CPS) for the known standard quantities of angiotensin I, and B₀ is cps in the absence of angiotensin I.

Detailed Description of the Invention

The method and spectrofluorometric assay described below can be used to measure in minutes, with accuracy and sensitivity, a component of the renin-angiotensin- aldosterone ("RAA") hormonal axis in a biological sample, including, for example, whole blood, serum, plasma or urine, using a fluorescent label bound to an antibody immunoreactive with the specific RAA component to be measured or an RAA component, and spectrofluorometric technology.

In the preferred embodiment, the method uses disposable columns that contain immobilized angiotensin I along with active sites. Samples of 0.1, 0.25, 0.5, 0.75 and 1 ml of plasma are passed through a rack of disposable columns. Columns are washed with a blocking agent to inactivate unused sites for angiotensin I. A constant concentration of fluorescent antibody is passed over the columns. The eluent is collected and the fluorescent content measured. A ten to one hundred fold increase in the amount of plasma assayed compensates for not carrying out the time consuming step of angiotensin I generation. In order to calibrate the activity of the fluorescent antibody the column matrix has a fixed amount of angiotensin I pre-attached. Each time the assay is run, the column is standardized by passing buffer and blocking agent over the column, followed by the assay volume of fluorescent antibody to be used for the standard. Alternatively, fluorescent labelled RAA component is introduced into a well, which contains a plasma sample, of a 96 well plate that has been coated with polyclonal antibody raised against angiotensin I. The plates are then evaluated for their free labelled RAA component.

In a second embodiment, a radioactive method described in Clin. Chem. 37/10(B) 1811-1819 (1991), is modified to use fluorescent labeled antibody, and results in sensitive, quantitative measurement of the RAA component in the absence of radioactivity. The amount of angiotensin I is evaluated by placing 10 μl of sample in an assay tube followed by 200 μl of labelled angiotensin I. Labelled angiotensin I is added to empty tubes for the measurement of nonspecific binding. 20 μl of antibody is added to all tubes. The tubes are then incubated for between two and 18 hours. Two ml of polyethyleneglycol reagent is added to all tubes except the control tube and the tubes are centrifuged. Pellets are redissolved and measured spectrofluorometrically.

RAA components and antibodies thereto.

The RAA components that can be measured in a biological sample include angiotensin I, renin, angiotensin II, angiotensinogen, and aldosterone, or any other components to which antibodies can be raised. Antibodies to most components are commercially available or can be generated using standard immunization procedures. The immunoglobulin fraction (IgG fraction) is isolated from the immunized animal using Fast Protein, Peptide and Polynucleotide Liquid Chromatography (FPLC) , as described below with reference to the purification of polyclonal IgG raised against angiotensin I. The isolation of the affinity pure fraction is the same for components other than angiotensin I with the exception of which RAA component is conjugated within the column. The RAA component should exhibit specific and reversible binding affinity along with a chemically modifiable group which can bind to the mixture. The RAA components are available from commercial sources including Sigma Chemical Co. (St. Louis, MO), Peninsula (Belmont, CA) , and Bachem/Cal (Torrance, CA) .

For use as a reagent in the assay, a known amount of an RAA component is provided. In general, the RAA components are found in the blood in amounts for PRA in normal subjects (male) of 2.4 ± 0.2 (female) 2.3 ± 0.2 ng/ml hr^"1; Prorenin (male) 17.5 ± 1.4 (female) 14.0 ± 1.3 ng/ l hr^"1; and Renin substrate (male) 1615 ± 40 (female) 1750 ± 60 ng/ml^"1 hr^"1, as reported by Sealey et al., 1990. Angiotensin I in the standard assay typically ranges between 0.01 ng and 100 ng ml^"1.

Antibodies to the RAA component are selected based on their immunoreactive specificity for the RAA component to be measured. The antibodies are purified from immunoglobulin obtained from commercial sources such as Berkeley Antibody Company, Berkeley, California, using procedures known to one skilled in the art, such as affinity chromatography.

Fluorescent labels and methods of conjugation.

As used herein, the labelled antibody or labelled RAA component is referred to jointly as "RAA Reactive Component" .

The RAA reactive component is bound to a fluorescent label. The criteria for selecting an appropriate fluorescent label are that the components of the reaction media (i.e., buffer composition, temperature) do not interfere with the emission properties of the label. Fluorescent labels are selected for various reasons, including the reactivity of the compound, e.g., amine reactive or sulfhydryl reactive; its excitation and emission wavelengths, and other reasons such as the ability to quench the emission upon binding of the compound. Examples of suitable fluorescent labels include fluorescein, 7-chloro-4-nitrobenzo-oxo-l,3-diazole ("NBD chloride") , rhodamine, Texas red, dichlorothrazinylamino fluorescein ("DTAF"), tetramethyl rhodamine isothiocyanate ("TRITC") , isothiocyanate ("FITC") , other fluorescein and rhodamine derivatives, as well as additional commercially available fluorescent moieties from sources such as Molecular Probes (Eugene, OR) , Research Organics (Cincinnati, OH) , and Aldrich Chemical Co. (Milwaukee, WI) or Pierce Chemical Co. (Rockford, IL) . The preferred label is DTAF for labeling antibodies, based on its label- antibody conjugate stability.

Methods for binding the fluorescent label to the selected antibody or RAA component are known to those skilled in the art and are described in detail in the Examples below.

For example, in one method for preparation of the fluorescent antibody, the fluorescent label and the antibody are each dissolved in an appropriate buffer and then dialyzed together to produce the conjugate, which is then purified to remove unbound label by procedures known in the art, such as dialysis, gel filtration, or hydroxylamine treatment.

In an example of one method for preparation of the fluorescent RAA component, such as angiotensin I, sufficient concentrations of the label and RAA component are incubated together at a selected temperature and for an appropriate period of time to allow formation of the conjugate. The reaction is monitored by thin layer chromatography for consumption of starting material. The conjugate in the incubation mixture is then concentrated, for example, by lyophilization to remove water, and the conjugate is purified by procedures known in the art, including thin layer chromatography, HPLC, recrystallization, FPLC, and LC. The ratio of label to RAA reactive components in the labeled conjugate is calculated based on the ratio of label and compound concentrations (expressed in moles/liter) , by procedures known in the art, which are determined by using ultraviolet and visible spectrophotometry.

Inert substrates and methods of binding RAA components thereto.

A known amount of the RAA component to be measured is bound to an inert substrate, resulting in solid phase (immobilized) component. The criteria for selecting an appropriate inert substrate are the ability to bind antigen and chemical stability of matrix. Examples of suitable inert substrates are activated CH-Sepharose™ 4B and Biorad™ immunobead reagent, using the manufacturers' procedures. These inert substrates are available from various commercial sources, for example Pharmacia Fine Chemicals (Piscataway, N.J.). The preferred inert substrate is CH-Sepharose™ 4B, which is suitable for use with all compounds.

Binding of the inert substrate to the RAA component to be measured is accomplished by methods known in the art. In general, the inert substrate is exposed to the dissolved protein in a basic environment to couple the protein to the substrate, usually over a period of between two hours and 24 hours. The excess active groups are blocked at the end of the incubation. The matrix is then washed to remove unbound protein.

Methods for incubating.

In the preferred method for incubation, the labelled reactive component, immobilized component and biological sample mixtures are incubated in microcentrifuge tubes or other suitable containers. For example, when a fluorescent antibody is used, the control incubation mixture for measurement of background fluorescence contains known amounts of fluorescent labelled RAA reactive components, control biological sample (defined as containing no RAA component) or egg albumin, and buffer and is measured spectrofluorometrically against "blank" incubation mixtures (defined as containing control biological sample and buffer but no fluorescent reactive component) . The excitation and emission wavelengths are determined empirically for a given labelled RAA component preparation. These wavelengths are then used for the measurements. The second control mixture, placed in a series of microcentrifuge tubes and used for construction of a standard fluorescence curve, contains known amounts of labelled RAA component, control biological sample, and immobilized component, such as angiotensin I bound to CH- Sepharose™ 4B, in all tubes, and increasing known amounts of free angiotensin I in the series of tubes. The sample incubation mixture contains the same known amounts of labelled reactive component, immobilized component, in the same amount as the control mixture, and a known amount of biological sample, such as blood, plasma, or serum, to be tested.

The mixtures are incubated with shaking at an appropriate temperature, such as room temperature (25°C) and for an appropriate time (30-90 minutes) to react the labelled reaction compound, RAA component to be measured and immobilized component.

Methods for removal of conjugate that is not complexed.

Unbound labelled reactive component is removed in the supernatant after centrifugation of the mixtures. Fluorescence of unbound labelled reactive component is measured spectrofluorometrically. The amount of RAA component in the test sample is calculated from the standard fluorescence curve constructed from the control incubation mixtures. Methods of measuring fluorescence.

The excitation and emission spectra of the fluorescent label are measured using commercially available instrumentation, for example, a SPEX Fluorolog Model 1680 0.22-m Double Spectrometer with DM3000 software (SPEX Industries, Edison, N.J.). Quantitation of fluorescent label bound to the selected compound is accomplished by creating a standard line relating fluorescence intensity values to known amounts of RAA component, such as angiotensin I.

Summary of Assay.

In the preferred embodiment, which is described in detail in the following example below, the renin/angiotensin I spectrofluorometric assay method focuses on measurement of angiotensin I and includes the following steps: (1) Antibody immunoreactive with angiotensin I, i.e., anti-angiotensin I, is purified from commercially available immunoglobulin G and conjugated with a fluorescent label, such as dichlorotriazinylamino fluorescein ("DTAF") or tetramethyl rhodamine isothiocyanate ("TRITC") . The amounts of label and antibody in the conjugate are determined by spectrophotometric measurement of visible and ultraviolet absorption. (2) Angiotensin I (Bachem/Cal, Torrence, CA.) is conjugated to an inert substrate, such as activated CH- Sepharose™ 4B. (3) A known amount of the angiotensin I- substrate conjugate is incubated with known amounts of the fluorescent antibody and biological test sample. (4) After incubation, the reaction mixture is centrifuged to separate the pellet containing fluorescent antibody bound to the angiotensin I in the test sample and to the angiotensin I-substrate conjugate, from the supernatant, containing the unbound fluorescent antibody. The emission intensities of the bound and unbound fluorescent antibody are determined by spectrofluorometric measurement. The centrifugation step can be omitted and the incubation reaction monitored directly in the spectrofluorometer. This alternative step is possible when the fluorescent antibody has the ability to change spectral properties or emission intensity (spectral shift or quench, DNBC, and NBD, respectively) on complexing with the RAA component, allowing not only measurement of the amount of the antibody-RAA component complex, but also measurement of the kinetic rate of formation in real time, which obviates separation of bound from unbound fluorescent antibody using a centrifugation step. The result is a more rapid and convenient assay. (5) The amount of angiotensin I in the test sample is then quantified, based on comparison of the spectrofluorometric values to a standard straight line correlation graph constructed from a spectrofluorometric measurements of fluorescent anti-angiotensin I bound to increasing amounts of angiotensin I in a series of control samples. (6) Fluorescence measurement is used to confirm that the attachment of the fluorescent label to the antibody did not alter the antibody/antigen binding affinity, as described by (McCabe et al. , FASEB J.. 4:2934-2940 (1990), the teachings of which are incorporated herein as representative of methods now known to those skilled in the art.

Variations on the steps outlined above are described below. For example, one can label the RAA component, such as angiotensin I peptide, with a fluorescent probe, such as 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole ("NBD chloride"), and conjugate the purified antibody to an inert substrate, such as activated CH- Sepharose™ 4B. One would then measure the fluorescence intensity of the labeled angiotensin I rather than the labeled RAA component (as used herein, either the RAA component protein or antibody to the protein) . The principles and end result would be the same, with only the components of the assay system differing slightly.

Fluorescently labelled angiotensin I would be displaced by unlabelled angiotensin I found in the plasma. The greater the amount of angiotensin I in the plasma, the lower the value of the measured fluorescent intensity.

The present invention is further illustrated by the following non-limiting examples.

Example l: Preparation of anti-angiotensin I labeled with DTAF and TRITC for measurement of angiotensin I in biological samples.

1. Preparation of protein sample

The IgG fraction of polyclonal antibodies was extracted from whole rabbit serum (Berkeley Antibody Company (BABCO) , Berkeley, CA) by FPLC by passing the IgG fraction in a 1 M Tris buffer pH 8 over an XK16/20 column (Pharmacia) containing Protein A Sepharose Fast Flow™ in 100 mM Tris. The IgG fraction is stripped from the column using low pH, 50 mM Na citrate pH 3.5. Fractions are collected from the column utilizing a UV-1 monitor and a FRAC 100™ fraction collector. The pH of the fractions are raised to physiologic values (in the range of pH 7-8) upon collection. The non-specific IgG content is then passed to another XK16/20 column containing activated CH Sepharose 4B™ conjugated to angiotensin I. The specific IgG are eluted from the column in similar fashion as to that of the non-specific IgG fraction. The specific antibody undergoes gel filtration and buffer exchange through a Sephacryl S-100™ column.

Specific polyclonal anti-angiotensin I antibodies were further purified by i munoaffinity chromatography using angiotensin I (10 mg/ml, Bachem/Cal, Torrance, CA) conjugated to activated CH-Sepharose™ 4B (Pharmacia Fine Chemicals, Piscataway, N.J.). Fractions were evaluated by dot blot analysis for the detection of peptide antibody. Specific antibodies were diluted with 50 mM sodium phosphate, 27.6 mM maleic acid, 3 mM EDTA, 1.4 mM phenyl ethyl sulfonyl-fluoride, 0.01% neomycin sulfate, 0.9% NaCl, pH 7.8.

In the alternative, polyclonal anti-angiotensin I antibody (Berkeley Antibody Company (BABCO) ) was prepared at a concentration of approximately 14 mg/ml in 0.1 M sodium phosphate buffer (pH 7.4) based on the absorbance at 280 nm.

The following method used in preparation of the protein sample and conjugation with dichlorotriazinylamino fluorescein ("DTAF") or tetramethyl rhodamine isothiocyanate ("TRITC") is known to those skilled in the art and is described in Research Organics, Inc., Catalog, p. 186 (1992) .

Polyclonal anti-angiotensin I was brought to a concentration of 2 mg/ml by dilution of 1 ml aliquots with

6 ml 0.05 M borate buffer containing 0.04 M sodium chloride (pH 9.3). Two 7 ml samples of the protein were placed in dialysis membranes and dialyzed against 1000 ml of buffer at 4°C for a period of eight hours. The dialysis buffer was replaced four times during this period for each sample. Each 7 ml sample was divided in three portions and placed in separate dialysis membranes for conjugation, resulting in six samples, each about 2.3 ml in volume.

2. Preparation of fluorescent label solutions used in conjugation

TRITC (Research Organics, Inc., Cleveland, OH) was dissolved in dimethylsulfoxide (3 mg in 0.2 ml) and added to 100 ml of buffer with rapid stirring at room temperature. DTAF (Research Organics, Cleveland, OH) was dissolved in buffer (3 mg in 100 ml) . Three TRITC and three DTAF label solutions were prepared and stored at 4°C in the dark until use to prevent decomposition of the labels. 3. Protein-label conjugation

The label was conjugated to the protein by dialysis of the protein samples against aqueous solutions of the labels.

Dialysis was carried out in membrane tubing (Spectrum Medical Industries, Inc., Los Angeles) with a molecular weight cut-off of 3500 g/mole. This allowed low molecular weight label and buffer to diffuse in and out of the membrane, which retained the high molecular weight protein.

Three dialysis bags, each containing 2.33 ml protein solution, were placed in three separate DTAF solutions, 5.28 x 10^'5 M, 1.06 x 10^ M, and 5.28 x 10"⁵ M, respectively. The other three protein samples were placed in three separate TRITC solutions (approximately 0.3 mg/ml - 0.6 mg/ml) . The solutions (samples 1-6) were stirred overnight at 4°C in the dark.

4. Conjugate purification

Each of the three protein-label conjugate samples for each label was subjected to a different purification procedure: dialysis, gel filtration, or hydroxylamine treatment followed by gel filtration. Dialysis removes unbound label or any low molecular weight (less than 3500 g/mol) impurities and changes the buffer. Gel filtration differentially elutes unbound label and conjugate from an ion-exchange resin. Treatment with hydroxylamine removes unstable covalent linkages between protein and label.

Samples 1 and 4 were dialyzed against 0.1 M phosphate buffer (pH 7.4) for 24 hours at 4°C in the dark. The one liter dialysis solutions were changed several times during this period.

Samples 3 and 6 were treated with 0.1 M hydroxylamine hydrochloride by transferring the volume remaining in the dialysis membranes (approximately 2 ml) to 5.0 ml graduated test tubes, adding 0.25 ml 1.0 M hydroxylamine in 0.05 M borate buffer (resulting in pH 5.4), and bringing the volume to 2.5 ml with borate buffer. These samples were kept at 4°C for two hours until purification.

Samples 3 and 6 (after hydroxylamine treatment) and samples 2 and 5 were subjected to gel filtration (2.5 ml Sephadex™ G-25 columns, Pharmacia Fine Chemicals, Piscataway, N.J.) to change the buffer to saline. When necessary, the protein volumes were diluted to 2.5 ml with 0.1 M phosphate buffer (pH 7.4). Before sample addition, the Sephadex™ columns were equilibrated by passing 25 ml phosphate buffer (pH 7.4) through the column. The 2.5 ml samples were added to the top of each of four columns. The first fraction (approximately 2.5 ml) was collected until the sample volume had eluted just to the top of the column. The protein was removed from the column by adding 3.5 ml 0.1 M phosphate buffer (pH 7.4) to the top and collecting until this volume reached the top of the column bed volume.

5. Quantitation of label/protein ratio

The label to protein ratio ("1/p") in the conjugate was calculated from the ratio of the label and protein concentrations. The label concentration in the conjugate was approximated by using the extinction coefficient of the free label at its wavelength maximum. The protein concentration was approximated from the absorbance at 280 nm using a correction factor for the label contribution to the absorbance at 280 nm; this procedure is known to those skilled in the art and is described in Brinkley, M. , "A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens, and Cross-linking Reagents," 3(1) Bioconiu ate Chem. 2-13 (1992) . The absorbance at 280 nm is a standard method for determining protein concentration.

The properties of free label needed in the calculations are the wavelength maximum, the extinction coefficient, and the ratio of the label absorbance at 280 nm to that at the maximum wavelength. These values were determined for DTAF and TRITC and are shown in Table 1.

TABLE l: Spectrophotometric Properties of Fluorescent

Labels

Wavelength Extinction Absorbance (280 nm) / Maximum coefficient Absorbance Max. (nm) _ (M^cm¹)

DTAF 495 36,629 0.309

TRITC 550.09 31,509 0.332

The extinction coefficient of the protein at 280 nm and the molecular weight used in the calculation were those for human immunoglobulin G (molecular weight 153,000 g/mol, extinction coefficient 212,000 M'cm^"1) . Practical Handbook of Biochemistry and Molecular Biology 265-268, G.D. Fasman, ed. , CRC Press, Boca Raton, Fla. (1989).

The data required from each protein sample was the absorbance at the label wavelength maximum (A^) and the absorbance at 280 nm (A₂g_0nm) • The label concentration was calculated directly from the absorbance at its wavelength maximum. The protein concentration was calculated from the absorbance at 280 nm minus the label contribution at

280 nm by using the correction factor according to the method set forth in Brinkley, 3(1) Bioconiuqate Chem 2-13

(1992) . The data and results are shown in Table 2.

TABLE 2: Spectrophotometric Properties of Protein - Fluorescently Labelled Conjugates

Sample A(280nm) "(Max.) Label Cone. Protein Label/ (M-¹) Cone. (K^l) Protein

1 1, .0568 0.4841 1.322X10^"5 4.279X10^"6 3.088

2 1. ,4986 0.7344 2.005X10-⁵ 5.998X10^"6 3.342

3 1. ,4948 0.7238 1.976X10^'5 5.996X10^"6 3.296

4 0. ,2968 0.0643 2.041X10-⁶ 1.299xl0^~6 1.571

5 0. ,6958 0.1404 4.456X10-⁶ 3.062X10^"6 1.455

6 0. ,8230 0.1494 4.742X10^"6 3.648X10-⁶ 1.300 6. Evaluation of anti-angiotensin I antibodies for binding activity.

The fluorescent conjugates were evaluated using radioimmunoassay according to the method of Sealey et al., (1990) ; Sealey, (1991) . A binding affinity for anti- angiotensin I labeled with fluorescent label was obtained which was comparable to untreated anti-angiotensin I antibody.

Preservatives, such as Tween 80™ at a concentration of 0.001%, can also be added to the aqueous buffer in which the conjugates are stored, to stabilize the antibodies. Alternatively, the conjugates can be lyophilized and stored as a powder. The best conjugate purification method is dialysis or hydroxylamine treatment followed by gel filtration.

Example 2: Conjugation of anti-angiotensin I antibody with DTAF.

This method is a modification of the procedure used for conjugation of DTAF with antibody in Example 1. Three samples of label were prepared differently to optimize 1/p. Sample A was identical to that used in Example 1. Sample B was conjugated in a label solution twice as concentrated as in Example 1. Sample C was at a lower pH for the conjugation.

1. Label and buffer preparation.

The following buffer and label solutions were prepared and stored in the dark at 4°C: (1) buffer A, 0.05 M sodium borate, 0.04 M sodium chloride, pH 9.3; (2) buffer B, 0.05 M sodium bicarbonate, pH 8.2; (3) buffer C, 0.1 M phosphate, 0.2% neomycin, 0.01% sodium azide; (4) label solution A, 6 mg DTAF dissolved in 200 ml borate buffer A resulting in a label concentration of 5.28xl0^"5 M; (5) label solution B, 12 mg DTAF dissolved in 200 ml borate buffer A resulting in a label concentration of 1.06x10^ M; and (6) label solution C, 6 mg DTAF dissolved in 200 ml bicarbonate buffer B resulting in a label concentration of 5.28xl0^"5 M. 2. Protein preparation

Three anti-angiotensin I antibody samples (approximately 2 mg/ml) were prepared and placed in dialysis membranes (molecular weight cut-off 3500 g/mol) as follows: Samples A and B, l ml protein aliquot added to 6 ml borate buffer A; and Sample C, 2 ml protein aliquot added to 6 ml sodium bicarbonate buffer B.

These samples were stored in the dark at 4°C until used.

3. Conjugation

The protein samples were placed in the label solutions and stirred overnight in the dark at 4°C as follows: The dialysis membranes were placed near the top of the 200 ml label solutions where they were not disturbed by the stir bar. The volume of the protein sample was carefully maintained below the top of the label solution. Sample A was placed in label solution A. Sample B was placed in label Solution B. Sample C was placed in label solution C.

4. Conjugate purification

Dialysis was chosen as the most effective and simplest purification method based on the results of Example 1. Each dialysis membrane was dialyzed against 1000 ml phosphate buffer C in the dark at 4°C. The buffer was changed four times during the dialysis, resulting in dialysis against the first 1000 ml of phosphate buffer for 2 hours, the second volume for 12 hours, the third volume for 30 hours, and the fourth volume for 24 hours. The dialysis buffer was changed more frequently at the beginning when the largest amount of unbound label was removed from the sample. The yield after dialysis for sample A was 5.2 ml, sample B 6.25 ml, and sample C 4.8 ml. These samples were separated into 0.5 ml aliquots and stored in the freezer after snap freezing on dry ice. 5. Determination of label/protein ratio

The label to protein ratios were determined using the absorption properties of the conjugates as described in Example l. The samples were diluted five times with phosphate buffer to bring the protein absorbance (280 nm) to about 0.5 absorbance units. Some of the samples contained particulate matter, which was removed by centrifugation. The results are shown in Table 3.

TABLE 3: Protein < Conceritrations in Sampile.

Protein ι Cone. Protein Cone, Label/Protein

Sample (M-¹) (microαram/ml)

A 1.12X10^'5 1706.6 3.61

B 9.71X10-⁶ 1485.8 5.01

C 1.08X10^"5 1646.5 3.59

Samples A and C had identical label/protein ratios (3.6) , indicating that conjugation can be carried out with DTAF and anti-angiotensin I antibody at pH 8.2 rather than pH 9.3. This was important for the scale-up, since less of the protein and DTAF were degraded at the lower pH. Sample B had a higher label/protein ratio (5.01), which was expected because of the larger concentration of DTAF (twice that used in samples A and C) in the conjugation step. In comparison to the results of Example 1, these ratios were slightly higher (3.6 in contrast to approximately 3.3). This was probably due to the larger volume of label solution used in Example 2 than in Example 1 (200 ml versus 100 ml) . The amount of protein remained the same, but the quantity of label to which the protein was exposed was doubled, although the label concentration was the same. In scaling up this procedure, the preferred way to attain higher label/protein ratios is by increasing the concentration of the label used in the conjugation. Example 3. Preparation of angiotensin I labeled with

7-chloro-4-nitrobenzo-2-oxo-l,3-diazole ("NBD chloride") .

This example demonstrates the attachment of the fluorescent label to the antigen or analyte rather than to the antibody. In this procedure, a one:two molar ratio of NBD chloride to angiotensin I was used to ensure that only the most reactive amino group was conjugated with NBD chloride.

Angiotensin I (0.0078 g, Aldrich Chemical Co., Milwaukee, WI) and NBD chloride (0.0006 g, 0.5 equivalents, Aldrich) were added to a 10 ml round-bottomed flask (14/20, single neck) with a stir bar. Distilled water (3 ml) and 10% sodium bicarbonate solution (85 microliters) were added to the flask. The flask was capped and stirred at 23°C for 24 hours. At the start of the reaction, the angiotensin I was soluble, but the yellow NBD chloride solids floated on top of the aqueous solution. As the reaction proceeded, the solids dissolved and the aqueous solution turned orange. After 24 hours, thin layer chromatography (TLC; silica, 4:1:1 1- butanol/acetic acid/water) showed that NBD chloride had been consumed (Retention Factor (R,-) 0.9, fluorescent spot), producing a new major fluorescent spot at R_f 0.4 with minor fluorescent spots at R_f 0.9 and 0.75. Some unconsumed angiotensin I was still present (R_f 0.2, iodine active) .

The reaction mixture was lyophilized to remove the water. The results of a repeated TLC after lyophilization were identical to those of the untreated reaction mixture.

Purification was carried out using preparative TLC on 250 micron silica plates in 4:1:1 1-butanol/acetic acid/water. The orange solid was dissolved in 2.5 ml methanol, and the flask was rinsed with an additional 0.5 ml methanol. The methanol solution was placed on the thin layer plate and developed over a period of about four hours. The TLC plate was left standing in air overnight in the hood to allow evaporation of the solvents. Fluorescent material at R_f 0.4 was scraped from the glass plate and extracted three times with 0.5 ml 20% aqueous acetic acid (centrifuging to remove solids at each extraction) . This procedure solubilized essentially all of the yellow color in the sample. The aqueous acetic acid extracts were centrifuged to remove suspended material, then evaporated to a film under a jet of nitrogen gas. The yellow residue was redissolved in 0.2 ml 20% acetic acid and applied in a strip covering approximately the central 4 inches of a silica gel G plate (plastic backing, about 1.5 inches from the bottom of the plate) . The strip was applied in this position on the plate to allow slow movement of the solvent when it reached the sample, thus ensuring clean development of the bands and efficient separation.

The sample was concentrated into a tight band by chromatographing 20% aqueous acetic acid into the sample band from both sides. The plate was thoroughly air dried, then developed in 4:1:1 n-butanol/acetic acid/water. The development was allowed to proceed overnight and went all the way to the top of the plate. Two fluorescent bands appeared on the plate, one at about a quarter of the way up the plate, and the other almost at the solvent front.

The plate was air-dried. The chromatographic layer was scored all the way across on either side of the lower fluorescent band (cut with a scalpel to give a clean-cut edge) , and then a strip was scraped away on the distal side of the clean cut.

The chromatogram was turned at right angles to the orientation in which it was originally developed and processed again in 20% aqueous acetic acid. This step was repeated several times, until the yellow color between the scored lines was concentrated into about 1-2 square centimeters of the silica layer. After complete air drying of the layer, the yellow zone was scraped into a small test tube and stirred vigorously with 0.5 ml water. Only a small amount of the yellow color was extracted into the water, and the suspension was filtered through a glass wool plug in a Pasteur pipet. The solid was caught on the filter and washed with another 0.2 ml water. The filter cake was then washed into a fresh tube with 1 ml of 20% acetic acid in water, added in small portions and allowed to pass through the filter-cake slowly. The yellow color eluted from the filter-cake by the time about half of the acetic acid solution was through the filter. Both the water wash and the 20% acetic acid wash were separately evaporated to films under a jet of nitrogen.

Almost all of the yellow color was in the acetic acid and was redissolved in about 0.2 ml of 50% acetic acid. A few microliters of this were taken for TLC and the remainder of the solution was transferred to a weighed (microbalance) microcentrifuge tube and thoroughly evaporated to dryness under a stream of nitrogen gas. Reweighing of the preweighed tube gave a net weight of 0.38 mg material recovered from the thin layer plate.

Assuming only one NBD molecule per angiotensin I, the molecular weight is 1456.79 g/mol.

Example 4: Preparation of solid phase (immobilized) antigen using activated CH-Sepharose™ 4B.

Activated CH-Sepharose™ 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) was washed and reswelled with 1 mM HC1 (1 g lyophilized Sepharose™ in 200 ml of 1 mM HC1, several changes) and then combined with 5 mg/ml angiotensin I in 0.1 M NaHC0₃, 0.5 M NaCl, pH 8.0, by mixing on a rotating table for 8 hours at 4°C.

The affinity gel was washed with 0.1 M Tris - HC1, pH 8, to block excess active groups and then with 0.1 M NaHC03, 0.5 M NaCl, pH 8.0, followed by 0.05 M Tris, 0.5 M NaCl, pH 8.0, and 0.05 M sodium acetate, 0.5 M NaCl, pH 4. Solid phase antigen in suitable storage buffer was stored at 4°C.

Example 5: Fluorescent immunoassay for determination of plasma angiotensin levels.

The antibody capture assay included the following steps. All incubation mixture aliquots were placed in 1.5 ml microcentrifuge tubes. (1) For measurement of fluorescence background associated with use of the fluorescent antibody, the control incubation mixture 1 contained 1.0 ml fluorescent antibody diluted with 0.05 M phosphate/0.9% NaCl; 100 μl control plasma consisted of Sigma coagulation control level 1 (catalog # 7916) which was diluted 1:2 with phosphate buffer; and made 3 mM EDTA, 1.2 mM PMSF, 0.0276 M maleic acid and 0.1% Neomycin pH 7.4, or egg albumin, and 100 μl buffer, and was measured spectrofluorometrically against "blank" tubes of incubation mixture 1 (defined as containing control plasma and buffer but no fluorescent antibody) . For construction of a standard straight correlation line, incubation mixture 2 contained 1.0 ml diluted fluorescent antibody, 100 μl control plasma or egg albumin, and 20 μl of 0.1% immobilized RAA component, such as angiotensin I bound to CH-Sepharose™ 4B, in each of a series of tubes, and increasing amounts of free angiotensin I (Bachem/Cal, Torrance, CA) (0.01 to 10 ng) in the series of tubes. Incubation mixture 3 contained the same amounts of fluorescent antibody and immobilized RAA component as in the second mixture, but not control plasma, and 100 μl plasma test sample. The incubation mixtures were incubated at room temperature for 90 min on a shaker table. After incubation, the assay tubes were centrifuged at 18,000 x g for 30 min. A 1 ml aliquot of test sample was removed and transferred to a cuvette.

Fluorescence was measured using a SPEX Fluorolog Model 1680 0.22-m Double Spectrometer with DM3000 software (SPEX Industries, Edison, N.J.). The amount of RAA component in the test sample was calculated from the standard fluorescence curve constructed from incubation mixture 2.

Figure 1, which is a graph showing the measurement of plasma angiotensin I by the spectrofluorometric assay method versus the radioimmunoassay measurement of plasma angiotensin I, illustrates that there is approximately a 94% correlation between the values achieved by the two methods. (Angiotensin I correlation data, log (X)=RIA, log (Y)=spectrofluorometric assay-PDC. Least squares linear regression. Slope = .7873424 +/- .1196724. 1/slope = 1.270095. Y intercept = .212471 +/- .1365423 (Y when X=0) . X intercept = -.2698584 (X when Y=0) . N = 8. r = 0.9372 (correlation coefficient), r squared = 0.8783. The SD of residuals from the line (Sy.x) = .1598272. P value = 0.0006 (two tailed). As shown, the slope is very significantly different than zero.)

Figure 2, which ia a graph showing a standard curve of spectrofluorometric (B/B₀)/1-(B/B₀) versus angiotensin I (ng) , where B represents specific counts per second (CPS) for the known standard quantities of angiotensin I, and B₀ is cps in the absence of angiotensin I, illustrates that there is approximately a 92% correlation between angiotensin I concentration and spectrofluorometric measurement of varying amounts of angiotensin I in the assay. (Least squares linear regression. Slope = .040946 +/- 8.568E-03. 1/slope = 24.4224. Y intercept = .6946056 +/- .01049378 (Y when X=0) . X intercept = -16.96394 (X when Y=0) . N = 4. r = 0.9589 (correlation coefficient). r squared = 0.9195. The SD of residuals from the line (Sy.x) = .01915894. P value = 0.0411 (two tailed). The slope is significantly different than zero.)

Claims

We claim:

1. A method for assaying a component of the renin-angiotensin-aldosterone hormonal axis (RAA) in a biological sample, comprising the steps of:

(a) providing a known amount of a compound bound to a fluorescent label, wherein the compound is selected from the group consisting of antibodies immunoreactive with the specific RAA component to be assayed and the RAA component to be measured;

(b) providing a protein selected from the group consisting of the RAA component to be measured, when the labeled compound is an antibody to the RAA component, and antibody to the RAA component to be measured, when the labeled compound is the RAA component, and binding the protein to an inert substrate;

(c) providing a known volume of a biological sample;

(d) incubating a mixture of the fluorescent labelled compound, the protein immobilized to inert substrate, and the biological sample;

(e) determining the amounts of bound and unbound fluorescent labelled compound in the mixture by spectrofluorometry; and

(f) calculating the amount of the specific renin- angiotensin-aldosterone component in the biological sample by comparison with standard curves constructed using the same procedure with known quantities of the RAA component to be assayed.

2. The method of claim 1 wherein the fluorescent labelled compound is the RAA component to be assayed and the protein immobilized to the inert substrate is antibody to the RAA component.

3. The method of claim 1 wherein the fluorescent labeled compound is an antibody immunoreactive with the specific RAA component to be assayed and the protein immobilized to the inert substrate is the RAA component.

4. The method of claim 1 further comprising the step of monitoring the incubation reaction during the assay by spectrofluorometry.

5. The method of claim 1 wherein the renin- angiotensin-aldosterone component to be assayed is selected from the group consisting of angiotensin I, renin, angiotensinogen, aldosterone, and angiotensin II.

6. The method of claim 1 wherein the fluorescent label is selected from the group consisting of fluorescein, 7-chloro-4-nitrobenzo-oxo-l, 3-diazole, rhodamine, Texas red, dichlorothrazinyla ino fluorescein, tetramethyl rhodamine isothiocyanate, and other fluorescein and rhodamine derivatives.

7. The method of claim 1 wherein the biological sample is selected from the group consisting of whole blood, plasma, serum and urine.

8. The method of claim 1 wherein the mixture is incubated for a period of time between thirty and ninety minutes.

9. An assay for determining the level of a component of the renin-angiotensin-aldosterone hormonal axis in a biological sample comprising in the assay:

(a) a known amount of a compound bound to a fluorescent label, wherein the compound is selected from the group consisting of antibodies immunoreactive with the specific RAA component to be assayed and the RAA component to be measured;

(b) a protein selected from the group consisting of the RAA component to be measured, when the labeled compound is an antibody to the RAA component, and antibody to the RAA component to be measured, when the labeled compound is the RAA component, wherein the protein is bound to an inert substrate; and

(c) a known amount of biological sample containing the component to be measured.

10. The assay of claim 9 further comprising standard calibration samples of the renin-angiotensin- aldosterone component for calculating the amounts of bound and unbound fluorescent antibody in the assay.

11. The assay of claim 9 wherein the renin- angiotensin-aldosterone component to be measured is selected from the group consisting of angiotensin I, renin, angiotensinogen, aldosterone, and angiotensin II.

12. The assay of claim 9 wherein the fluorescent label is selected from the group consisting of fluorescein, 7-chloro-4-nitrobenzo-oxo-l,3-diazole, rhodamine, Texas red, dichlorothrazinyla ino fluorescein, tetramethyl rhodamine isothiocyanate, and other fluorescein and rhodamine derivatives.

13. The assay of claim 9 wherein the biological sample is selected from the group consisting of whole blood, plasma, serum and urine.