determination of plasma renin activity by radioimmunoassay
TRANSCRIPT
Measurement of peripheral and renal venous reninactivity has achieved practical clinical importancein the evaluation of the role of the kidneys in hypertension and in the diagnosis of renovascular hypertension and of primary hyperaldosteronism (1—5).
The use of radioimmunoassay for the determination of plasma renin activity through the assay ofgenerated angiotensin I (6—8) has several advantages over the standard bioassay procedures currently in general use (9) . The radioimmunoassayprocedure facilitates the assay of a much larger numben of samples than can be handled for bioassay. Itobviates the necessity of maintaining a colony ofassay animals. The sensitivity and reproducibility ofthe method is greater. The volume of blood requiredfor analysis by radioimmunoassay is less than onefifth the volume required for bioassay.
Widespread application of immunoassay procedunes is limited partially by the lack of generalavailability of the necessary reagents from commercial sources including especially monospecificantisera and iodinated angiotensin I. Recently, tworadiopharmaceutical manufacturers, Squibb andSchwarz-Mann, have begun to market as test kitsthe angiotensin I immutopeT@' kit and the reninactivity radioimmunoassay kit, respectively. Since theinitial criteria in establishing an immunoassay forclinical use are quite stringent, careful quality controlis necessary to ensure accurate results. Such problemsas cross reactions of antisera and impurities of the‘251-angiotensin standard can lead to serious error.It is therefore essential that the reliability and reproducibility of any commercial kit of this nature becarefully assessed before introducing it into the laboratory for routine use.
The present report describes the results of a critical comparison of the results obtained through theuse of these two kits with the bioassay method of
Boucher, Ct a! (9) for the measurement of reninactivity in human plasma samples.
MATERIALS AND METHODS
The bioassay procedure is based on the methodoriginally developed by Boucher, et al (9), as modifled by Blaufox, et al ( 10) , and used extensively inthis laboratory during the last 6 years. Plasma incubation is carried out for 3 hr at pH 5.5 in thepresence of Dowex resin. The generated angiotensionis eluted from the resin and is assayed in the rat bya bioassay technique. The renin activity values obtamed using this procedure agree within 10% whencoded and assayed blind in two separate rats blockedwith pentolinium and atropine prior to assay againsta standard mixture of angiotensin II. Since theangiotensin I generated in the procedure is rapidlyconverted to angiotensin II in the rat, the resultsare expressed as ng A 11/100 ml/3 hr.
The preparation of reagents for the radioimmunoassay procedures followed with the two commercialkits differ slightly and are briefly described below.
Schwarz-Mann kit. Preparation of reagents for usewith the Schwarz-Mann kit requires the weekly preparation of tnisacetate buffer. For dilution of plasmasamples and 1251-angiotensin I, tnisacetate buffer contaming lysozyme must be prepared each day thetest is run. The barbital buffer is readily dilutedin water and Dextran-coated charcoal suspensionsare made using this buffer. A stock suspension isprepared and a working suspension is prepared by1—4dilution of the stock suspension. One milliliterof active 1251-angiotensin I is supplied frozen in
Received Mar. 27, 1972; revision accepted June 8, 1972.For reprints contact: Lakshman Rao Chervu, Dept. of
Radiology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, N.Y. 10461.
806 JOURNAL OF NUCLEAR MEDICINE
DETERMINATION OF PLASMA RENIN ACTIVITY BY
RADIOIMMUNOASSAY: COMPARISON OF RESULTS
FROM TWO COMMERCIAL KITS WITH BIOASSAY
1. Rao Chervu, Marc Lory, Theresa hang, Hyo Bok Lee, and M. Donald Blaufox
The Albert Einstein College of Medicine, Bronx, New York
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the presence of stabilizing material. After thawing,the activity is distributed into four aliquots of 250,.d in plastic tubes supplied with the kit containingadditional stabilizing material, and these are storedfrozen until used for dilution. At the time of dilutionof the activity for a test run, the contents of onetube are thawed, the stabilizer material is resuspended, and an aliquot from this lot is withdrawn anddiluted with tris buffer containing fresh lysozyme. Itis also recommended by the manufacturers that thestandard angiotensin I and the antisera be thawed foruse not more than two times.
Squibb kit. The preparation of reagents using theSquibb kit is simplified through the use of previously calibrated materials which are diluted to appropriate volumes. The trisacetate buffer solutioncan be stored up to 1 month. An aliquot of theactive 125I-angiotensin I preparation containing ascavenging resin for adsorption of any free radioiodide generated during storage is withdrawn anddiluted with the trisacetate buffer containing bovineserum albumin prepared freshly every week.
Comparative procedures. The active 125I-angiotensin received from both manufacturers was chromatographed on anion exchange resin paper to checkthe purity of the labeled compound. Blood samplesfor analysis were collected in ice cold stopperedtubes* containing disodium ethylenediamine tetraacetic acid (EDTA) . The tubes are tipped end toend gently to ensure that all of the EDTA is dissolved after adding 5 ml of blood and are thencentrifuged in the cold at 2°Cto separate the plasma.Simultaneously 20 ml of blood was collected intotubes containing ammoniated EDTA for use in thebioassay. One milliliter of plasma from each clinicalsample for radioimmunoassay is pipetted into a Falcon Plastics polystyrene tube ( 12 X 75 mm) andthen 10@ each of 8-hydroxyquinoline and dimercaprol are added (in the Schwarz-Mann kit, only2 @lof dimercaprol is recommended) . The mixtureis then separated into two 500-@J aliquots, and onetube is incubated at 4 °Cand one at 37 °Cfor 3 hr.After incubation, the samples are ready for immunoassay of the generated angiotensin I. If samples arenot assayed immediately, they are frozen and then
thawed at 4°Cfor assay at the time of carrying outthe series.
The protocol for the radioimmunoassay test procedure is slightly different for the two kits. With theSchwarz-Mann kit, 0.9 ml of tris buffer containinglysozyme is added to each tube, followed by theaddition of 5 @d(50 pg) , 10 @l( 100 pg) , 20 j@l
* Lavender top vacutainer tubes, Becton-Dickinson & Co.,Cat. No. 4770.
(200 pg), 30 @l(300 pg), 50 @d(500 pg), and70 @l(700 pg) of standard angiotensin I for calibration and 50-id plasma samples from the 4 and37°Cincubation for clinical samples. Fifty microliters of active angiotensin I solution is aliquottedinto each tube and finally 50 @lof angiotensin I antiserum is added to all of the tubes. In two tubescontaining tris buffer with lysozyme, only aliquotsof 50 @lof active angiotensin I are added and intwo others, buffer solution, 50 @Aof active angiotensin I and 50 @tlof antisera added—the first twoserving as controls and the second two as traceramounts of angiotensin only.
With the Squibb kit 1 ml of diluted active angiotensin I solution is added followed by addition of5 @l(50 pg), 10@ (100 pg), 20 @l(200 pg),30 @l(300 pg), 50 ,@l(500 pg), and 70 @l(700pg) of standard angiotensin I for calibration and50 ,@1of patientplasmasamplesincubatedat 4 and37°C. Two tubes containing only active angiotensinsolution serve as tracers. Fifty microliters of angiotensin antisera are added to all tubes.
All the tubes in each series prepared as above are
gently mixed, and incubated at 4°C for 24 hr, afterwhich 1 ml of charcoal suspension is added intoeach tube, genfly shaken, and centrifuged at 2,500—3,000 rpm for 5—10mm. The supernate from eachtube is decanted into similarly numbered tubes andboth the residue and supernate are counted in awell scintillation counter. The standards are generally done in triplicate, and the unknown plasmasamples for purposes of this study were analyzed in
::i@ 125iAngiotensin
60 -
—Free 1251
0 1 2 3 4 5 6 7 8 9 10 11RESINSTRIP(cm)
FIG. 1. Chromatographicseparationof angiotensinI and free“Isupplied with sample kit from each manufacturer. Each contains small amount of free radioiodide which remains at originin chromatographic system used. This represents potentially serioussource of error and should be checked frequently.
Squibb
—--Schwarz-Mann>-
>I-U4I—zuJU
w0@
50 -
40 -
30 -
20 -
10
Volume 13, Number 11 807
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CHERVU, LORY, LIANG, LEE, AND BLAUFOX
triplicate with an additional value obtained for asmaller aliquot of 10 @l.Aliquots were all measuredusing Oxford microliter pipettes except the standardand antisera which were dispensed using Hamiltonmicrosyringe pipettes with a repeating dispenserattachment.
A total of 40 plasma samples were obtained frompatients with a wide variety of sodium intake andpathology for comparison of the three test procedures: bioassay, Squibb immunoassay, and SchwarzMann immunoassay. The results of all three testswere obtained blind with coded samples. Statisticalcomparisons were carried out later after decoding.
RESULTS
The results of sample chromatograms as shownin Fig. 1 indicate that the radioiodide remains at thepoint of application in the system used and the active material moves with the solvent front. About3% free radioiodide is present in the active angiotensin I from both manufacturers.
The calibration curve may be drawn in two ways:(A) by plotting the average percent bound values
against the standard amount of angiotensin I addedresulting in a curvilinear plot and (B) by plottingthe free/bound ratio against the standard whichresults in a straight line within the usual range ofvalues encountered. The concentration of angiotensin I in the 4 and 37°C incubated plasma samples is calculated by interpolation using these standard curves. The plasma renin activity values arethen calculated as angiotensin I generated per miiiliter per hour from the equation
renin activity (ng 37°—n @°) 20(ngAI/ml/hr) = 3
The renin activity values thus obtained are plottedin Figs. 2 and 3 against the corresponding bioassayvalues (expressed as ng A 11/100 ml/3 hr) for thesame plasma samples. In Fig. 4 are plotted the dataobtained from the samples analyzed with both thekits against one another. The calculated regressionlines are drawn with the inserts in the figures givingthe equations for the lines. The correlation coefficient, r, obtained with the Squibb kit and theSchwarz-Mann kit are 0.94 and 0.91, respectively.Between the two kits a correlation coefficient of 0.94is obtained. All three of these correlations are highlysignificant (p < 0.01).
As indicated above, the reagents supplied bySquibb are readily prepared by simple dilutionswithout any further weighings and the shelf life ofthe reagents is longer which may be advantageous.Thus for example, the trisacetate buffer with bovineserum albumin is made only once for use with the
Squibb kit with a shelf life of I month as againstthe trisacetate buffer to be weighed every week fordilution and pH adjustment and daily weighing oflysozyme for preparing the buffer needed for thetest procedure using the Schwarz-Mann kit. Theactive solution once made is stable for 1 week usingthe Squibb procedure which, unlike the SchwarzMann procedure, has to be made the day the testis run.
DISCUSSION
These results indicate that the two commerciallyavailable test kits for radioimmunoassay of anglo
FIG.2. Fortyvaluesof reninactivityobtainedfromanalysiswith Squibb kit reagents are plotted against corresponding bioessay values. There is good correlation over very wide range ofvalues. Regression equation is shown. Immunoassay is plotted asng/100 ml for ease of comparison. Overlapping values are plottedas one dot.
Immunoassay (Schwarz Mann):4.9 ÷0.23 BIOassayr:0.91n:25
1000 2000 3000 4000 5000 6000 1000 8000 9000 0,00011,0002,000RENINACTIVITY,ng/100m1I 3hr(BlO-ASSAY)
1600 -
1400 --C
E00
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1200
@ 1000
:@ 800
@ 6004
2000 3000 4000 5000 6000 1000RENINACTIVITY,ng/lOOmI/3 hr.
(BIO-ASSAY)
FIG. 3. Twenty-fivevaluesof renin activity obtained fromanalysis with Schwarz-Mann kit reagents are shown plotted againstcorresponding bioassay values. Regression equation is shown.
808 JOURNAL OF NUCLEAR MEDICINE
1600 - Immunoassay (Squibb) : 25.5 + 0.16 Bioassay
.- S@e 1400- rO.94
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r:0.94
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‘I
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400 600 800 10001200ACTIVITY, ng/lOOmI/hr.
(SQUIBB)
1400
DETERMINATION OF PLASMA RENIN ACTIVITY BY RADIOIMMUNOASSAY
tensin I are capable of providing accurate measurements of renin activity. The data correlate to ahigh degree of statistical significance with the wellestablished bioassay for renin activity. The majordifference between the two kits appears to be theease of performance rather than the reliability ofthe method. However, the user must constantly checkthe possible sources of error for variation amongtest lots.
In the Schwarz-Mann test procedure, repetitivepipetting of 0.9 ml of buffer is somewhat tedious,particularly when a large number of samples areto be assayed. Thus in the actual run, whereas theSquibb procedure involves four main pipetting steps,the Schwarz-Mann procedure has five such steps,and this, together with the above mentioned preparation of active reagent and buffer every day, constitutes a significant drawback for adapting the latterkit for assay of a large number of clinical samples.The 0.2 ml Biopette, available from Schwarz-Manntogether with the adapter kit for dispensing differentvolumes, is not of much use in aliquotting the different volumes necessary for the calibration curve,and in our opinion, the Oxford microliter samplersof different capacities with disposable tips are accu
SM(RIA):27.0+1.24 SQ(RIA)
rate and make it easier to dispense the desired volumes including 1 ml active solution. The recommended speed at which the Falcon Plastics disposablepolystyrene tubes can be centrifuged is below 3,000rpm and in our experience several are broken atany higher speed. This is contrary to the centrifuging speed of 5,000 rpm recommended in the SchwarzMann procedure for packing down the charcoal inthe tubes.
The slopes of 0. 16 and 0.23 for the regressionlines drawn in Fig. 2 and 3 may be compared withthe value of I .8 given by Cohen, Ct al (8) for asimilar comparison of radioimmunoassay with bioassay. In the latter work, incubation of plasma isdone at a pH of 5.5 at which value, generation ofangiotensin I, it is reported, is approximately threetimes that at pH 7.5 (the incubation pH in the present study) . Cohen, et al considered a pH of 5.5 asthe optimum plasma incubation pH value with ashorter time of incubation for the plasma, particularly when excess substrate is not assured. Thisapproach was not considered very suitable for routine assay using the kits on all plasma samples.However, in those clinical cases where renin activity levels are suspected to be abnormally low,as in primary aldosteronism, plasma incubation atpH 5.5 is perhaps warranted.
SUMMARY
The availability of the radioimmunoassay kits formeasurement of renin activity levels through thegeneration of angiotensin I may prove to be of aidin the clinical evaluation of renal diseases in a number of medical institutions throughout the country.The present study has established that a high degreeof correlation exists between the well establishedbioassay method and the radioimmunoassay resultsobtained using the kits. The comparative ease andaccuracy with which the protocol for the radioimmunoassay procedure is followed has led to our useof immunoassay in favor of bioassay as a routineprocedure.
ACKNOWLEDGMENT
This work was supported in part by NIH Grant No. HL11984.
REFERENCES
1. STOCKIGTJR. COLLINS RD. BIGLIERI EG: Determination of plasma renin concentration by angiotensin I immunoassay. Diagnostic import of precise measurement ofsubnormal renin in hyperaldosteronism. Circ Res (SupplII)28,29:175—191,1971
2. BULL MB, HILLMAN RS, CANNON PJ, et al: Reninand aldosterone secretion in man as influenced by changesin electrolyte balance and blood volume. Circ Res 27 : 953—960, 1970
C
:@1600N
a-C
ULI) 1200
1000
800
600
400
200
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FIG. 4. Valuesobtainedfromduplicateanalysisof 24 plasmasamples with both commercial kits are shown. Schwarz-Mann kityields slightly higher values than Squibb kit, but relationshipbetween two is linear over wide range of values.
Volume 13, Number 11 809
200RENIN
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CHERVU, LORY, LIANG, LEE, AND BLAUFOX
3. KOTCHEN TA, LYTrON B, Mop@ow LB, et al : Angiotensin and aldosterone in renovascular hypertension. ArchIntern Med (Chicago) 125: 265—272,1970
4. BLAUFOXMD, FROMOWITZA, LEE HB, et al: Renalblood flow and renin activity in renal venous blood inessential hypertension. Circ Res 27: 913—920, 1970
5. PAWSEY CG, VANDOGEN R, GORDON RD: Renalvenous renin ratio in the diagnosis of renovascular hypertension. Measurement during active secretion of renin.Med I A ustralia, I : I21—126, 1971
6. HABER E, KOERNERT, PAGELB. et al: Applicationof a radioimmunoassay for angiotensin I to the physiologicmeasurements of plasma renin activity in normal humansubjects.I C/inEndocr29: 1349—1355,1969
7. GIESEJ, JORGENSENM, NIELSENMD, et al : Plasmarenin concentration measured by use of radioimmunoassayfor angiotensin I. Scan I C/in Lab Invest 26: 355—367,I970
8. COHEN EL, GRIM CE, CONN JW, et al: Accurateand rapid measurement of plasma renin activity by radioimmunoassay.I Lab ClinMed 77: 1025—1038,1971
9. BOUCHERR, VEYRATR, D'CHAMPLAINJ, et al: Newprocedures for measurements of human plasma angiotensinand renin activity levels. I Canad Med Ass 90: 194—201,I964
10. BLAUFOX MD, BIRBARI A, HICKLER RB, et al:
Peripheral plasma renin activity in renal homotranspiantrecipients. Ne;s' Eng I Med 275: 1165—1168, 1966
810 JOURNAL OF NUCLEAR MEDICINE
SOUTHWESTERN CHAPTER
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Send abstract and two copies:
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1972;13:806-810.J Nucl Med. L. Rao Chervu, Marc Lory, Theresa Liang, Hyo Bok Lee and M. Donald Blaufox from Two Commercial Kits with BioassayDetermination of Plasma Renin Activity by Radioimmunoassay: Comparison of Results
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