Method for quantitative determination of renin activity in blood employing phenyl methyl sulfonyl fluoride and polyethylene glycol

Abstract

A method for the quantitative determination of renin activity in blood utilizing the measurement of Angiotensin I by treating the blood with ethylenediaminetetraacetic acid (EDTA) and the plasma with phenylmethyl sulfonylfluoride (PMSF) at a preferred pH to ascertain the full range of concentration where PMSF is effective for accurate and quick determination. Also, a method for the quantitative determination of renin activity in blood utilizing the measurement of Angiotensin I by incubating the samples after antibody addition at room temperature (23° to 30° C.) for 1 to 2 hours and separating the free from the antibody bound species with polyethylene glycol after having treated the blood with ethylenediaminetetraacetic acid (EDTA) and the plasma with phenylmethyl sulfonylfluoride (PMSF) at a preferred pH. PMSF offering enough protection against Angiotensin I destruction at room temperature that in combination with polyethylene glycol at a preferred pH the time required for the radioimmunoassay determination is 1 to 2 hours at room temperature instead of 24±2 hours at 4° C. which takes with charcoal in the presence of other inhibitors and making the system less succeptible to time dependent errors.

What is claimed is: 
     
       1. In a method for measuring plasma renin activity in a sample of plasma comprising adding ethylene diamine tetraacetic acid, adding inhibitor to inhibit Angiotensin I destruction, incubating at about 37° C. to release Angiotensin I from the plasma sample, adding I 125  labeled Angiotensin I while adding antibody, incubating the labeled mixture sample and antibody and adding a material to separate free Angiotensin I from Angiotensin I bound to antibody prior to determining the amount of Angiotensin I that improvement consisting of: adding phenyl methyl sulfonyl fluoride as the inhibitor;   adjusting the pH from 5 to 7.5 after adding the inhibitor;   incubating the labeled mixture of sample and antibody for 1 to 2 hours at about 23° C. to 30° C. after the incubation at 37° C. to yield Angiotensin I for measurement;   separating the free Angiotensin I from the Angiotensin I bound to antibody with polyethylene glycol in 0.01 Molar Tris hydroxymethyl aminomethane at pH 7 in a concentration of 12% to 18% based on the total medium, in the presence of about 100 μl to 250 μl of serum or any equivalent material containing similar quantities of immunoglobulins to aid precipitation of immunoglobulins in the plasma; and,   said incubating and separating steps being carried out one after the other, allowing for the optimal yield of Angiotensin I to be obtained in the same day that the sample is received and minimizing time dependent errors occurring therein.   
     
     
       2. A method of measuring plasma renin activity in a plasma sample comprising adjusting the plasma pH from 5 to 7.5 in the presence of ethylenediaminetetraacetic acid and in the presence of between 0.013 and 13.2 mg phenylmethyl sulfonylfluoride per ml of plasma as inhibitor against Angiotensin I destruction, incubating at about 37° C. to release Angiotensin I from the plasma, adding I 125  labeled Angiotensin I while adding antibody to Angiotensin I, thereafter, incubating to allow reaction with antibody to Angiotensin I and adding a substance to separate the free Angiotensin I from the Angiotensin I bound to antibody prior to determining the amount of Angiotensin I, that improvement consisting of incubating for 1 to 2 hours at about 23° C. to 30° C. and separating free from bound Angiotensin I, using polyethylene glycol at a 12% to 18% final concentration in a pH range provided by 0.01 M tris hydroxymethylaminomethane at pH 7.0 or by distilled water at pH 5.6 to 6.5 in the presence of about 100 μl to 250 μl of serum or any equivalent material containing similar quantities of immunoglobulins and said incubating and separating steps being carried out one after the other allowing for the optimal yield of Angiotensin I to be obtained in the same day that the sample is received and minimizing time dependent errors occurring in the separation as compared to separations using solid adsorbent materials. .[.3. In a method for measuring plasma renin activity in a sample of plasma comprising adding ethylenediaminetetraacetic acid while adjusting the pH of said sample, adding inhibitor to prevent Angiotensin I destruction, incubating at about 37° C. to release Angiotensin I from the sample and thereafter determining the amount of Angiotensin I thus released that improvement consisting of adding as the inhibitor phenylmethyl sulfonylfluoride in a range of concentration between 0.013 mg/ml to 13.2 mg/ml of plasma sample while simultaneously adjusting the pH to 5 to 7.5..].