Title:
Method and apparatus for angiotensin I determination
United States Patent 3899298


Abstract:
Apparatus for determination of Angiotensin I comprises a vial of 125 I Angiotensin I containing anion exchange resin, a vial of Angiotensin I standard, at least one vial of Angiotensin I Antiserum, and the following reagents: tris(hydroxymethyl)aminomethane, powdered charcoal, barbital buffer mixture, 8-hydroxyquinoline sulfate, bovine serum albumin powder, and Dimercaprol solution. The method involves the radioimmunoassay of Angiotensin I generated by the action of renin in incubated plasma samples. Antibody to Angiotensin I is employed as the specific antibody, and 125 I Angiotensin is employed as the labeled antigen, and synthetic Angiotensin I is employed as a reference standard. Powdered charcoal is used to separate free Angiotensin I from antibody-bound Angiotensin I.



Inventors:
SZCZESNIAK RAYMOND A
Application Number:
05/242956
Publication Date:
08/12/1975
Filing Date:
04/11/1972
Assignee:
E. R. SQUIBB & SONS, INC.
Primary Class:
Other Classes:
206/568, 206/569, 435/7.1, 435/23, 435/810, 436/538, 436/804, 436/808, 930/40
International Classes:
G01N33/539; (IPC1-7): G01N33/16
Field of Search:
23/23B,253TP,253R 424
View Patent Images:
US Patent References:



Other References:

Chemical Abstracts, 75: 137064s, (1971)..
Primary Examiner:
Serwin R. E.
Attorney, Agent or Firm:
Levinson, Lawrence Smith Merle Rodney Burton S. J.
Claims:
What is claimed is

1. Apparatus for determination of Angiotensin I comprising a carrier being compartmented to receive at least three vials and to maintain said vials in close confinement, a first vial comprising an acetic acid solution containing about 10 picograms of Angiotensin I per microliter, a second vial of Angiotensin I antiserum, and a third vial comprising an aqueous solution of 125 I Angiotensin I having a total activity of about 20 microcuries, and having therein a strip of paper, said paper impregnated with an anion exchange resin.

2. Apparatus in accordance with claim 1 which contains a plurality of vials of Angiotensin I antiserum.

3. Apparatus in accordance with claim 2 which contains 5 vials of Angiotensin I antiserum.

Description:
BACKGROUND OF THE INVENTION

Renin is an enzyme that is synthesized, stored, and released from granules contained in the juxtaglomerular apparatus of the kidney. It is generally accepted that the major physiological role of renin is to act as the primary stimulus for maintenance of body sodium balance. Plasma renin activity is increased when there is a decreased renal perfusion pressure, and when there is a decreased delivery of sodium and water to the distal tubule. Renin acts on substrate, angiotensinogen, an α-2-globulin produced by the liver, to form a decapeptide, Angiotensin I. Angiotensin I is biologically inactive, and is converted to the biologically active octapeptide, Angiotensin II, in the pulmonary circulation. Angiotensin II acts as a highly potent vasopressor, and stimulates the adrenal gland to produce aldosterone. Aldosterone promotes the reabsorption of sodium by the distal tubule, and when secreted in excessive amounts, results in hypertension.

Plasma renin assays have been found to be a useful adjunct in determining whether hypertension is due to primary aldosteronism or to renal vascular disease. The hypertensive patient that has plasma renin levels suppressed below normal, and has a high aldosterone secretion rate will usually be found to have adrenal hyperplasia or an adenoma of the zona glomerulosa of the adrenal gland. In patients with renal artery stenosis, the renal venous blood from the ischemic kidney may be found to have higher plasma renin levels than renal venous blood from the unaffected kidney.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a kit and a method for the determination of Angiotensin I. Another object is to provide means for determining whether hypertension is due to primary aldosteronism or to renal vascular disease. A further object is to provide a kit and a method for the radioimmunoassay of Angiotensin I. These and other objects of the present invention will be apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawing is a perspective view of the kit according to the present invention with the top removed for viewing of the contents.

DETAILED DESCRIPTION

Measurement of body constituents by the technique of radioimmunoassay offers a bioanalytical tool that combines the extreme sensitivity of radioisotope methodology with the extreme specificity of immunological techniques. The procedure requires a specific antibody, a radiolabeled antigen, a pure sample of the antigen to serve as a reference standard, and a means of separation of free antigen from antibody-bound antigen. The procedure follows the basic principle of saturation analysis, where there is competition between labeled and unlabeled antigen for a fixed number of antibody binding sites. As the concentration of unlabeled synthetic antigen (the substance actually being measured) increases, less of the added radiolabeled antigen will be bound to the antibody. When equilibrium has been reached in the antigen-antibody reaction, the free and bound components of the mixture are separated, and the relative amounts of each are determined by measuring the radioactivity of the separated components. The absolute quantity of unlabeled antigen in the sample being analyzed is determined by comparing the assay results to a standard curve prepared with known amounts of the unlabeled antigen.

Since purified renin is not available, the direct radio immunoassay of renin is not yet possible. In lieu of a direct renin radioimmunoassay, a methos has been developed for the estimation of plasma renin activity through the radioimmunoassay of Angiotensin I generated by the action of renin in incubated plasma samples. The radioimmunoassay for Angiotensin I and its application to the determination of plasma renin activity is of great value in studying physiological processes associated with hypertension and important in the clinical evaluation of hypertensive disease. The measurement of plasma renin activity by radioimmunoassay has several advantages over the standard bioassay procedures. The simplicity, specificity, and rapidity with which the radioimmunoassay can be performed remove the obstacles to routine clinical determination of plasma renin activity that were associated with the more complex bioassay procedure.

The use of 125 I rather than 131 I considerably lengthens the shelf-life of the radioiodinated Angiotensin I employed in the test, and reduces radiation exposure to laboratory personnel. The half-life of 125 I is 60 days.

The apparatus for testing for Angiotensin I according to the present invention comprises a chassis having a plurality of receptacles for receiving vessels containing Angiotensin I antiserum, Angiotensin I standard, and 125 I Angiotensin I. Preferably the chassis contains a plurality of vessels containing Angiotensin I antiserum.

Angiotensin I is a decapeptide having the following amino acid sequence:

L-asp-L-Arg-L-Val-L-Tyr-L-Ile-L-His-L-Pro-L-Phe-L-His-L-Leu.

This decapeptide is prepared synthetically and is commercially available. The Angiotensin I standard is an acetic acid solution containing about 10 picograms of Angiotensin I per microliter. The acetic acid may have a concentration of from about 0.0001 mole to about 1.0 mole. The 125 I Angiotension I consists of an aqueous solution of 125 I labeled Angiotensin I having a total activity of about 20 μCi. Preferably, this solution contains some acetic acid. A strip of paper about 2.54 cm2 in area impregnated with anion exchange resin (WB-2 resin paper) is also present in the vial containing the 125 I labeled Angiotensin I to scavenge iodide ion released from the 125 I Antiotensin I and thus maintain a low level of free iodide ion.

The Angiotensin I antiserum is obtained from the blood serum of rabbits which have been injected with Angiotensin I poly-L-lysine conjugate.

In carrying out the radioimmunoassay of the present invention, a number of test reagents are required. These reagents are as follows:

1. Tris acetate buffer [tris(hydroxymethyl)aminomethane], 7.26 g.

2. Charcoal, powdered, 3.75 g.

3. Barbital buffer mixture: sodium barbital, 0.883 g; sodium chloride, 4.59 g; sodium acetate trihydrate, 0.582 g.

4. 8-Hydroxyquinoline, 660 mg.

5. Dimercaprol solution, 2 ml. (contains 200 mg. dimercaprol and 400 mg. benzyl benzoate in peanut oil)

6. Bovine serum albumin fraction V powder, 1.5 g.

While bovine serum albumin is preferred, any other albumin may be used. Examples of other albumins are egg albumin, mycogen of muscle, lactalbumin of milk, legumelin of peas and leucosin of wheat. In lieu of albumin, a protein having a molecular weight of from about 75,000 to about 4,000,000 may be used.

As shown in the drawing, the kit consists of a bottom member 10 formed of shock-resistant material, e.g., polystyrene foam, having a plurality of receptacles 12-18 and a top closure 11. Receptacle 12 contains a vial 19 of 125 I Angiotensin I, receptacle 13 contains a vial 20 of Angiotensin I standard, and receptacles 14-18 each contain, respectively, vials 21-25 of Angiotensin I antiserum.

In carrying out the test procedure according to the present invention, it is necessary to first prepare the test reagent.

PREPARATION OF TEST REAGENTS

1. Tris Acetate Buffer: Transfer the entire contents (7.26 g) of the vial containing Tris(hydroxymethyl)aminomethane (TRIS) to a suitable container and add 600 ml of distilled water. Adjust the solution to pH 9.0 ± 0.5 using a few drops (.about.0.4 ml) of glacial acetic acid. Store final solution at 2°-8°C.

2. Tris Acetate Buffer with BSA: For each milliliter of final solution desired, add 2.5 milligrams of the bovine serum albumin powder that is supplied with the kit. This solution can be stored up to one month when kept at 2°-8°C.

3. Charcoal Suspension: Transfer the entire contents of the vial labeled "Barbital Buffer Mixture" (6.055 g) to a suitable container and add 600 ml of distilled water. Adjust the solution to pH 9.0 ± 0.5 by adding 0.2 N HCl (.about.1 ml). The 0.2 N HCl can be prepared by diluting 2 ml of concentrated HCl to 125 ml with distilled water.

To prepare the charcoal suspension, add the entire contents (3.75 g) of the vial containing powdered charcoal to the above solution and stir vigorously for several minutes. Store the suspension at 2°-8°C. and stir vigorously before removing aliquots for use. 4. 8-Hydroxyquinoline Solution: Transfer the entire contents (660 mg) of the vial containing 8-hydroxyquinoline sulfate to a suitable container and bring to a volume of 10 ml with distilled water. Store in the dark. 5. Dimercaprol Solution: No further processing of this reagent is required before use. Shake well before use. 6. Diluted 125 I Angiotensin I: Dilute a portion of the 125 I Angiotensin I supplied with the kit with the Tris Acetate Buffer with BSA (prepared in Step 2 above) so that the final solution has a radioactivity concentration of approximately 5000 counts per minute per milliliter. Approximately 0.2 ml of the radioactive solution diluted to 100 ml will give the desired .about. 5000 cpm/ml. The required volume will vary with the efficiency and discriminator settings on the counting equipment used. The diluted 125 I Angiotensin I should be stored at 2°-8°C. and prepared fresh each week.

COLLECTION OF BLOOD SAMPLES FOR ANALYSIS

Blood should be collected in a cold tube containing EDTA. The blood samples should be stored under refrigeration, and centrifuged in the cold to collect the plasma. Plasma samples should be frozen until used.

INCUBATION OF PLASMA SAMPLES TO GENERATE ANGIOTENSIN I

To 1.0 ml of plasma, collected in the manner described above, add 10 microliters (μl.) of 8-Hydroxyquinoline Solution and 10 μl. of Dimercaprol Solution. Transfer 0.5 ml of the above mixture to another test tube and incubate at 4°C. for 3 hours. Incubate the original test tube at 37°C. for 3 hours. The sample incubated at 4°C. will reflect circulating levels of Angiotensin I, while the sample incubated at 37°C. will reflect the quantity of Angiotensin I generated through the action of renin in the plasma sample. The net quantity of Angiotensin I generated at 37°C. is calculated by subtracting the Angiotensin I level in the sample. If the incubated samples are not used immediately in the radioimmunoassay procedure described below, the samples should be frozen until used.

RADIOIMMUNOASSAY TEST PROCEDURE

The procedure described below is based on performance of duplicate analyses on all samples assayed. It is recommended that all assays be conducted in plastic test tubes.

1. Mark a series of plastic test tubes with the numbers 1 through 16. The first 12 tubes are required for preparation of the standard curve, and the remaining four tubes are required for one clinical sample. Four additional tubes should be used for each additional clinical sample to be assayed.

2. Add 1.0 ml of diluted 125 I Angiotensin I to each tube.

3. Add the following quantities of Angiotensin I (AI) Standard to the respective tubes: Tubes 3 and 4----5 μl.; tubes 5 and 6----10 μl.; tubes 7 and 8----20 μl.; tubes 9 and 10----30 μl.; tubes 11 and 12----50 μl.

4. Add 50 μl. of the clinical sample incubated at 4°C. to tubes 13 and 14.

5. Add 50 μl. of the clinical sample incubated at 37°C. to tubes 15 and 16.

The composition of the 16 tubes is summarized below:

AI Content Tube No. Sample Added (μμg) ______________________________________ 1 and 2 None 0 3 and 4 AI Std. (5 μl.) 50 5 and 6 AI Std. (10 μl.) 100 7 and 8 AI Std. (20 μl.) 200 9 and 10 AI Std. (30 μl.) 300 11 and 12 AI Std. (50 μl.) 500 13 and 14 Clin. Sample 4°C. (50 μl.) unknown 15 and 16 Clin. Sample 37°C. (50 μl.) unknown ______________________________________

6. Add 50 μl. of Angiotensin I Antiserum to all tubes, mix gently, and incubate in refrigerator (4°C.) for 24 ± 2 hours.

7. At the end of the incubation period add 1.0 ml of the charcoal suspension to all tubes. Mix gently and then centrifuge for 2-3 minutes at 3000-4000 rpm.

8. Decant the supernatant from each tube into similarly numbered tubes.

9. Measure the radioactivity in all tubes (i.e., tubes containing charcoal residue and tubes containing supernatant) by counting in a standard well-type gamma scintillation counter (discriminator settings 20-50 Kev). Subtract background cpm and record net cpm for each tube.

10. Calculate the % Bound (AI bound to antibody) for each of the original tubes using the following formula, and record results. ##EQU1## For example: Tube No. 1 Supernatant----2400 cpm

Tube No. 1 Charcoal ----2600 cpm ##EQU2##

11. Calculate the average % Bound values for the duplicate samples and record average values.

12. Prepare a Standard Curve by plotting the average % Bound values against the quantity of Angiotensin I Standard added to the respective tubes (i.e., tubes 1 through 12).

13. Determine the quantity (μμg) of Angiotensin I in the 4°C. sample and the 37°C. sample by referring to the standard curve prepared above. The % Bound for each sample will correspond to the specific quantity of Angiotensin I contained in the sample.

14. Calculate plasma renin activity (PRA) in terms of nanograms of Angiotensin I generated per ml of plasma per hour of incubation at 37°C. as follows: ##EQU3## For example: 37°C. Sample contains 160 μμg AI

4°c. sample contains 10 μμg AI ##EQU4##

While the foregoing specification has indicated that the anion exchange resin is in the vial containing the 125 I labeled Angiotensin I, it is to be understood that the anion exchange resin need not be present in this vial but be present in a separate container in the kit, in which case the person performing the test may add it to the vial containing the 125 I Angiotensin I.