CLIN. CHEM. 22/9, 1465-1471 (1976)

CLINICALCHEMISTRY,Vol. 22, No. 9, 1976 1465

Use of Laser Nephelometry in the

Measurement of Serum Proteins

Carl D. Deaton, Kameron W. Maxwell, Richard S. Smith, and Robert L. Creveling

Measurement of immunoglobulins (Ig) and of complementcomponent C3 in human serum by automated and manualnephelometric techniques is tedious, and the effectivelinear range is too narrow. We describe a laser nephe-lometer/reagent system for measuring serum proteins bythe use of forward light scatter (which enhances the ratioof reaction/blank), electronic blank subtraction, laser light(632 nm), and electronic signal selection. We report dataestablishing the range of linearity for immunoglobulins IgO,IgA, 1gM,and complement C3 with this system, and cor-relations with results by a radial immunodiffusion. We alsocompared an electroimmunodiffusion system for quanti-tation of albumin and lgG in cerebrospinal fluid to ourtechnique. The nephelometric system described providesa rapid, accurate, precise, and objective way to measureimmunoglobulins and C3.

Actdftlonal Keyphrases: immunoglobulins #{149} complement C3#{149}radial immunodiffusion #{149} electroimmunodiffusion #{149}

percent relative light scatter #{149}multiple sclerosis #{149} cere-brospinal fluid

Measurement of immunological reactions by neph-elometry has been a method in which light scatter wasmeasured at an angle of 90#{176}(1). For immunologicalapplications this limits the sensitivity of the techniqueand does not allow sample background to be well dif-ferentiated from the specific immunological reactionbeing quantitated (2).

We have developed a nephelometer that incorporatesa laser as the light source and an optical system thatdetects forward light scatter (3). Therefore, detectionof light scatter from antigen/antibody complexes isimproved. The amount of signal from artifactual lightscatter (dust and larger contaminating particles) islimited by an electronic screening device. By incorpo-rating the principles of forward light scatter, we de-signed a cuvette chamber that accepts disposable roundcuvettes, obviating problems and cost of a flow-throughcuvette system or square cuvettes.

This instrument also has electronic memory capa-bility that enables automatic subtraction of light-scatterreadings attributable to the various blank constituents(buffer, antibody, and sample). The final digital readoutof percentage relative light scatter is proportional to theamount of antigen/antibody complexes present in thesample test cuvette, which obviates the subjectivity of

Hyland Division Travenol Laboratories, Inc., P. 0. Box 2214,3300Hyland Ave., Costa Mesa, Calif. 92628.

Received April 5, 1976; accepted July 6, 1976.

interpreting recorder traces and measuring precipitinzones. The use of a polymeric buffer in this system,which decreases the solubility of high-molecular-weightantibody/antigen complexes, has resulted in rapidmeasurement of serum proteins and an extended rangeof linearity. Therefore, it is possible to quantitate var-ious concentrations of serum proteins without addi-tional dilution or concentration of the specimen.

We describe here some of the applications of the lasernephelometer system for the quantitation of the im-munoglobulins (IgA, IgG, and IgM), complement C3,and albumin.

Materials and Methods

Instrumentation

The Laser Nephelometer “PDQ” (Hyland Labora-tories, Inc., Costa Mesa, Calif. 92626) contains a he-lium/neon laser light source emitting photons at 632.8nm, a photomultiplier tube (No. 931B with an S-4spectral response) mounted at an angle of detection of31#{176},electronic blanking capabilities for each constituent(buffer, antiserum and sample), and a signal selector,which differentiates light scatter attributed to largecontaminating particles.

Reagents

The “LAS-R” Nephelometric Reagent Test Kits

(Hyland) for IgA, IgG, IgM, complement C3, and al-bumin were used in the nephelometric procedures. Eachkit contains: monospecific goat antiserum diluted tocover an antigenic range from fourfold reference I toreference VI, a polymeric buffer (pH 7.0) at a concen-tration to produce precipitation of compounds of highermolecular weight than are normally present in serumor cerebrospinal fluid, and six multicomponent refer-ence sera (pooled delipidized human sera).

The reference value ranges are: IgA, 24 to 740 mg/100ml; IgG, 80 to 2700 mg/100 ml; 1gM, 7.5 to 235 mg/100ml; C3, 9.0 to 325 mg/100 ml; and albumin, 300 to 9420mg/100 ml. The kit also contains disposable 10 X 75 mmborosilicate cuvettes.

“Immunoplate” III Radial Immunodiffusion TestKit for IgA, IgG, IgM, complement C3, and cerebro-spinal fluid, albumin, and IgG (Hyland) were used toobtain results by radial immunodiffusion. The IgG andIgM plates contained goat antiserum; the IgA and C3plates used contained horse antiserum. The three ref-erences supplied were prepared for a human serumpool.

1466 CLINICAL CHEMISTRY, Vol. 22, No. 9, 1976

Filters (0.4 m av pore diameter, 47 mm in diameter)and Filter Holder (47 mm diameter), both from Nu-clepore Corp., Pleasanton, Calif. 94566, were used tofilter the nephelometric reagents.

Electroimmunodiffusion plates were prepared (4)and “LAS.R” kit references II, III, and IV (Hyland) wereused to establish a reference curve.

A Precision Viewer (Hyland) was used to determineradial immunodiffusion zone diameters to the nearest0.1 mm.

Specimens

Serum specimens were collected from 60 apparentlyhealthy individuals, 18 to 60 years of age. Serum spec-imens were obtained from patients with diagnosed IgA,IgG, and 1gM monoclonal gammopathies. “Low level”sera were collected from patients with various diagnosedimmunological deficiencies that would be expected toresult in decreased concentrations of the protein beingassayed. Cerebrospinal fluid specimens were collectedfrom patients with normal concentrations of IgG andalbumin. Abnormal cerebrospinal fluid specimens wereobtained from patients with multiple sclerosis.

Blind samples used in our precision study were froma pool of delipidized and lyophilized human serum. Fivedifferent concentrations of protein were obtained byreconstituting each vial with distilled water or water andsaline, as follows:

(1) 0.3 ml of distilled water(2) 0.5 ml of distilled water(3) 1.0 ml of distilled water(4) 1.0 ml of distilled water plus 1.0 ml of saline (9 g

of NaCI per liter)(5) 1.0 ml of distilled water plus 2.0 ml of saline (9 g

of NaC1 per liter)Reconstituted vials were stored refrigerated (5 #{176}C)

until assayed. All other specimens were stored frozen(-20 #{176}C)unless otherwise noted.

Procedures

Nephelometry. For nephelometry, we diluted thevarious “LAS-R” kit antisera twofold with polymericbuffer. The working antiserum was filtered through a0.4 rn (av pore diameter) X 47 mm filter, to removeparticulates. Saline was filtered in the same manner. Foreach assay system, to a duplicate series of 10 X 75 mmdisposable cuvettes (for each reference and unknown),1 ml of the working antisera was added and labeledsample test. To a separate series of cuvettes (for eachreference and unknown blank) 1 ml of the previouslyfiltered saline was added. A saline blank and antiserumblank cuvette were prepared by pipetting into the cu-vettes 1 ml of saline and antiserum, respectively. Thenephelometer kit references and unknown specimenswere diluted 100-fold with the filtered saline, except forcerebrospinal fluid specimens, which were assayedundiluted. Reference and specimen dilutions were pi-petted into the sample blank and test cuvettes (25 /.LlforIgA and IgG, 100 Ml for 1gM and complement C3, and5 Mifor albumin). The cuvettes were sealed, their con-

tents mixed by inverting twice, and incubated for 1 h atroom temperature (25 #{176}C),except for cerebrospinalfluid assay tubes, which were incubated for 30 mm. Wemeasured the percentage relative light scatter fromthese cuvettes by placing the saline blank cuvette intothe cuvette well and “zeroing” the nephelometer in thebuffer position, with the photometer “blank-subtract”knob. The reference cuvette with the highest concen-tration of reactants was then placed in the instrumentand the upper reference point established by adjustingthe sensitivity (coarse and fine) to obtain a value ofabout 180% relative light scatter. The instrument modewas switched to “antiserum blank” and the antiserumblank cuvette was placed into the well. The computecycle was activated by pressing the “compute” button.At the end of a 15-s compute cycle the percentage rel-ative light scatter from the antiserum blank was sub-tracted from the results by adjusting the “antiserumblank” knob. The “sample blank” mode was activatedby placing reference I into the instrument, and thecompute cycle was activated. The percentage relativelight scatter attributed to the sample blank was auto-matically removed. The reference I test cuvette wasplaced in the instrument and read by pressing the “test”mode button. The displayed percentage relative lightscatter was recorded. The other sample blanks and testswere read in the same way. The displayed values areproportional to the concentration of the antigen con-centration within the sample. These values for the ref-erences were plotted on linear graph paper vs. the listedmg/100 ml, and a line of best-fit was drawn connectingthe points. The values for the unknown samples wereread from this curve and converted to mg/100 ml.

The nephelometer kit reference I was diluted 12.5-fold with saline, producing a concentration eightfold thenormal (100-fold) reference I concentration. Lowerreference concentrations were made by serial twofolddilutions of the above reference dilution to 8192-fold(resulting in 14 different concentrations). These dilu-tions were assayed by the above procedure for eachantigen-IgA, IgG, 1gM, and complement C3-.to de-termine the range of linearity of the antiserum.

Nephelometry assays were conducted on the pre-viously collected normal, subnormal, and supranormalsera specimens by the above procedure, and for theseresults we calculated correlations with results by radialimmunodiffusion and electroimmunodiffusion. Speci-mens with antigen concentrations above reference Iwere diluted further than 100-fold and reassayed untiltwo consecutive dilutions resulted in values for per-centage relative light scatter that were on the referencecurve. Sera with concentrations below reference VI wereonly diluted 10-fold with saline and re-assayed to obtainvalues that fell on the reference curve. We assessedprecision by assaying the previously prepared lyophi-lized blind samples, within-run by 20 assays of eachconcentration of blind sample, with use of a duplicateseries of references, and day-to-day assays by recon-stituting the various concentrations of blind samplesand assaying them on each of several days.

-J

Fig. 2. lgG linearity on instrument sensitivity 2 (188-3000mg/100 ml)

gO vg/dI

‘C

-J

3,000 6,000 12,000

go mg/dI

18,ItOO 24000

Fig. 1. lgG linearity on instrument sensitivity 1 (3000-24000mg/100 ml)

Radial Immunodiffusion

CLINICALCHEMISTRY,Vol. 22, No. 9, 1976 1467

We used an end-point radial immunodiffusion systemto quantitate serum IgA, IgG, 1gM, and C3, and cere-brospinal fluid IgG and albumin in the previously de-scribed normal and abnormal specimens. Nephelometryreferences II, III, and IV were used with this system; thesquares of zone diameters were plotted vs. concentrationon linear graph paper, and the best-fit curve was drawn.

Specimens with above-normal concentrations wereappropriately diluted with saline so that zone diametersfell within the range of the reference curve. Cerebro-spinal fluid assays were done by diluting nephelometricreferences 100-fold in saline, but assaying the cerebro-spinal fluid undiluted.

Electroimmunodiffusion

Electroimmunodiffusion plates were prepared ac-cording to a modified Laurell technique (4). Nephe-lometer references II, III, and IV were diluted 100-foldwith saline and the appropriate values assigned. Ref-erenceprecipitin cone lengths were plotted on log-loggraph paper and a curve of best-fit was drawn alongthese points. Values for albumin and IgG in cerebro-spinal fluid from patients with multiple sclerosis wereobtained from this reference curve. Samples with pre-cipitin cones larger than that of reference II were dilutedwith saline and re-assayed.

ResultsThe range of linearity for the nephelometric reagents

was determined by plotting, on linear graph paper,percentagerelative light scatterof a series of referencedilutions vs. representative concentrations for IgG.Owing to the extended range of concentrations, multi-

ise

Sensitivity .1-

-

:I____ b

12e

1

graphs of the data obtained at the various instrumentsensitivities were used to demonstrate the linearity ofthe curve. The upper reference concentrations were readby using instrument sensitivity setting No. 1. IgG con-centrations that exceeded about 9000 mg/100 ml werein antigen excess (Figure 1). Below this value the rela-tionship was curvilinear. To expand the lower referencecurve, the instrument sensitivity switch was adjustedto No. 2 for the 188-3000 mg/100 ml range (Figure 2),to No. 5 for the 3-94 mg/100 ml concentration (Figure3).

The total range of curvilinearity for the IgG antise-rum was observed to be from 3 to about 9000 mg/100 ml.Similar assays were conducted for IgA, 1gM, and C3,with the following results: IgA, 21-2800 mg/100 ml; 1gM,7-1200 mg/100 ml; and C3, 8-1200 mg/100 ml.

We did nephelometric and radial immunodiffusionassays of sera from 70 normal and IgA-deficient sera aswell as sera from patients with IgA gammopathies. Acorrelation coefficientof 0.960 was obtained. Figure 4shows the results. The radial immunodiffusion tech-nique gaveslightly higher values for the IgA myelomasamplesthan those obtained by nephelometry (slope,1.192). To observe the correlation from specimens with“normal” antigenic characteristics, we constructed ascatter graph for results from the normal and deficientsamples, omitting the elevated; the slope decreased to1.06 (Figure 5).

Sixty-nine sera from normal, deficient, and patientswith IgG gammopathies were assayed for IgG by usingboth nephelometry and radial immunodiffusion. Thecorrelation coefficient was 0.988, the slope 0.984. Ascatter graph (Figure 6) shows these results. Again,when the results from the paraproteinemias were ex-cluded, the slope was 1.039. Similar assays with 1gM

4

-i

I

go ing/di

Fig. 3. lgG linearity on instrument sensitivity 5 (3-94 mg/lOUml)

NEPHELO,IETRY (mg/div 100)

a

‘4

I’

-I4

Fig. 5. Nephelometry and radial immunodiffusion assay com-pared, for 54 samples with deficient and normal concentrationsof IgA

§‘C

z0(.‘1

00z

-J

0

Fig. 6. Nephelometry and radial immunodiffusion assay com-pared, 69 samples with deficient, normal, and above-normalconcentrations of lgG

§

‘4

5’

I

-J4

NEPHELOMETRY (mg/dI v 1000)

Fig. 4. Nephelometry and radial immunodiffusion assay com-pared, for 70 samples with deficient, normal, and above-normalconcentrations of IgA

3 5NEPHELOMETRY (mg/div 1000)

1468 CLINICAL CHEMISTRY, Vol. 22, No. 9, 1976

gave correlation coefficients of 0.985 and a slope of 1.697(Figure 7).

As previously explained, by excluding the results from1gM myelomas, the slope changes to 0.814 (Figure 8),which again indicates that higher readings were ob-tained by radial immunodiffusion than by nephelom-etry for paraproteinemia specimens. Complement C3assays on 90 samples gave a correlation coefficient of0.9696, with a slope of 1.181 (Figure 9).

Three groups of specimens, consisting of 60 normalsera, were assayed by three individual laboratories bythe nephelometric technique. Table 1 lists the valuesand range (±2 SD) for each protein specificity. A series

of blind samples at different concentrations, preparedfrom a pool of normal human serum, was assayed bynephelometry for IgA, IgG, 1gM, and C3. Run 1 con-sisted of a single run of 20 replicate assays of each con-centration, to ascertain within-run precision. Run 2consisted of 55 assays of each of the freshly reconsti-tuted and diluted blind samples, for day-to-day preci-sion. Table 2 shows the results.

Table 3 lists albumin and IgG values determined bynephelometry and radial immunodiffusion for 12 nor-mal samples of cerebrospinal fluid. Nine individualspecimens and one pool of cerebrospinal fluid frompatients with multiple sclerosis were also assayed for

gM (N-b)

SLOPE 1.697V-INTERCEPT I-) 96.2

IFig. 9. Nephelometry and radial immunodiffesion assay com-pared, for 90 samples with deficient and normal concentrationsof complement C3

3 5 I 7

NEPHELOMETRY (mg/dIv 1000)

Fig. 7. Nephelometry and radial immunodiffusion assay com-pared, for 70 samples with deficient, normal,and above-normalconcentrations of 1gM

NEPHELOMETRY I,.,g/dII

Table 1. Results for Assay of Normal Serum byThree Individual Laboratories (n = 60/Laboratory)

Mean ±2 SD

Laboratory mg/100 mla

U’

U.U.

0

-I40.4

Fig. 8. Nephelometry and radial immunodiffusion assay com-pared, for 56 samples with deficient and normal concentrationsof 1gM

these proteins by nephelometry and electroimmuno-diffusion (Table 4).

NEPHELOMETRV (mg/dI v 100)

CLINICAL CHEMISTRY, Vol. 22, No. 9, 1976 1469

§‘C

E

20‘4I’.

I’

002

-J.40.4

Discussion

IgA

2

3

/gG

2

3

1gM

23

C3

2

3

271259

303

1089

9451073

138153162

166159140

97-445109-409109-497

647-1531447-1443

630-1515

26-25627-279

9-315

100-232105-213

92-188

Molecular light scattering has been used to estimatethe molecular weights of biological materials (5) and todetect antigens by immunological reactions (6).Nephelometric techniques in which 90#{176}light scatter ismeasured have been used to quantitate proteins inserum and cerebrospinal fluid (7, 8). However, it hasbeen known for some time that it is possible to detectgreater total light scatter by observing the scatter at aforward angle (9, 10). The advantages obtained in doingso include increased sensitivity and an increased dif-

ference between the sample blank and immunologicalreaction in percentage relative light scatter ratios. Withthese advantages in mind, we have developed a nephe-lometer that measures forward light scatter, for en-hanced detection of antigen/antibody complexes. Thisnephelometer includes a laser, which provides an in-tense source of illumination that is highly collimatedand coherent. The reagent system used with this in-strument incorporates a polymeric buffer, which en-hances the insolubilization of antigen/antibody com-

Table 2. Within-Run and Day-to-Day Precision for Nephelometry of IgA, lgG, 1gM, and Complement C3IgA lgG 1gM C3

no. Mean,rng/lOOmI CV,% Mean,mg/lOOmI CV,% Mean,mg/lOOml CV,% Mean,mg/lOOrnl CV,%

Within-run (n = 20)1 754 1.5 2201 3.6 243 5.3 222 5.02 355 3.5 1325 3.4 164 4.0 140 5.03 240 4.4 740 4.0 93 4.4 70 3.54 129 4.2 400 4.7 46 4.6 28 5.35 84 6.9 274 7.2 28 3.9 24 3.6

Average - 4.1 - 4.6 - 4.4 - 4.5

1 760 6.1 2278Day-to-day (n = 55)

5.2 244 5.6 224 8.02 494 7.8 1374 5.7 170 6.4 138 8.83 240 7.7 730 5.6 94 7.3 67 5.24 130 7.5 395 5.5 46 5.2 31 9.55 85 9.9 276 6.2 30 11.4 23 6.9

Average - 7.8 - 5.6 - 7.2 - 7.7

Aib. lgG Aib.

mg/i 00 ml

lgG Patientno. mg/i 00 ml

Table 3. lgG and Albumin in 12 Normal HumanCerebrospinal Fluids, as Measuredby Radial

Immunodiffusion and Nephelometry

Table 4. lgG and Albumin in Cerebrospinal Fluidfrom Patients with Multiple Sclerosis,as Measured

by Electroimmunodiffusion and NephelometryRID Nephelometry EID Nephelometry

AIb. lgG Alb. lgG

1 21.0 15.0 18.8 13.511.2 1.9 12.0 0.920.9 2.4 22.5 1.6 2 30.4 12.4 32.0 7.015.2 1.4 18.8 1.1 3 24.8 13.0 22.5 12.715.3 1.8 17.5 0.9 4 22.6 8.4 23.7 8.218.0 4.4 18.5 3.8 4a 18.2 3.2 18.5 3.3

8.7 1.5 10.2 1.1 5 12.2 12.6 10.0 10.319.5 1.8 21.0 1.2 6 27.0 6.0 28.8 6.5

8.7 1.2 11.0 0.6 7 22.5 3.5 24.8 5.214.5 1.8 16.3 1.4 8 34.2 8.0 33.0 7.415.3 3.0 17.1 2.4 9 97.2 26.8 85.5 25.912.5 2.7 14.5 1.7 lOb 24.0 7.0 25.7 7.920.9 2.0 23.0 1.6

a Treated multiple sclerosis.

1470 CLINICAL CHEMISTRY, Vol. 22, No. 9, 1976

plexes, in turn increasing light scatter and detectabilitywithin relatively short incubation times. Clearly, lasernephelometry is a sensitive, precise, and accurate

technique for quantitating proteins in human serumand cerebrospinal fluid. The results obtained from thelinearity studies demonstrate the versatility of thisprocedure.

A potential problem in serum immunoglobulinmeasurement is the formation of soluble immunecomplexes in the presence of antigen excess. Therefore,samples with extremely above-normal concentrationsof antigen must be diluted and re-assayed until linearitywith dilution can be established within the range of the

reference curve. However, the quantitation of myelomaproteins is subject to error because of the unique anti-genic mosaic of these proteins, the method of stan-dardization, and the nature of the immunizing antigen.The correlation studies show that samples with ex-tremely supranormal amounts of antigen must be di-

luted for measurement by radial immunodiffusion,which may alter the diffusion characteristics of theseproteins in agar gel.

When normal sera were assayed by three separatelaboratories by laser nephelometry, there were onlyslight differences in the mean values. The coefficientsof variation for the five concentrations of pooled serawere relatively constant in magnitude, indicating an-tigen concentration not to be a source of substantivevariance. The panel of normal cerebrospinal fluidspecimens assayed for IgG by nephelometry and radialimmunodiffusion gave means of 1.5 and 2.2 mg/100 ml,respectively. The albumin assays for the same systems

gave values of 16.9 and 15.1 mg/100 ml. For IgG in ce-rebrospinal fluid from patients with multiple sclerosiswe found mean values of 10.8 mg/100 ml by the nephe-lometric method, 12.5 mg/100 ml by electroimmuno-

diffusion. Values for albumin were 34.8 and 35.1 mg/100ml, respectively, for these same samples.

CLINICAL CHEMISTRY, Vol. 22, No. 9, 1976 1471

As indicated from the results, laser nephelometry

provided acceptable correlation with the various com-

parison methods, but had the advantage of an extendedrange.

Precision was acceptable for the various proteinspecificities and concentrations assayed. Hence, webelieve the laser nephelometric system offers the clinicallaboratory a rapid, precise, objective means to assessproteins in sera and cerebrospinal fluid.

We thank Dr. Wallace W. Tourtellotte and staff for electroimmu-nodiffusion studies performed in his laboratory, Mr. Gregory Batemanand Mr. Robert Hughes for technical assistance, and Dr. MichaelCaputo for clinical results.

References1. Killingsworth, L. M., and Savory, J., Manual nephelometricmethods for immunochemical determination of immunoglobulins IgG,IgA, 1gM in human serum. Clin. Chem. 18, 335 (1972).

2. Buffone, G. J., Savory, J., and Hermans, J., Evaluation of kineticlight scattering as an approach to the measurement of specific proteinswith the centrifugal analyzer. II. Theoretical considerations. Clin.Chem. 21, 1735 (1975).

3. Buffone, G. J., Savory, J., Cross, R. E., and Hammond, J. E.,Evaluation of kinetic light scattering as an approach to the mea-surement of specific proteins with the centrifugal analyzer. I. Meth-odology. Clin. Chem. 21, 1731 (1975).

4. Tourtellotte, W. W., Tavolato, B., Parker, J. A., and Comiso, P.,Cerebrospinal fluid electroimmunodiffusion. Arch. Neurol. (Chicago)25, 345 (1971).

5. Dubin,, S. B., Lunacek, J. H., and Benedek, G. B., Observation ofthe spectrum of light scattered by solutions of biological macromol-ecules. Proc. Nat. Acad. Sci. USA 57,1164 (1967).

6. Pope, C. G., and Healey, M., A photoelectric study of reactionsbetween diptheria toxin and antitoxin. Br. J. Exp. Pathol. 19, 397(1938).

7. Killingsworth, L. M., and Savory, J., Nephelometric studies of theprecipitin reaction: A model system for specific protein measure-ments. Clin. Chern. 19, 403 (1973).

8. Markowitz, H., and Tschida, A. R., Automated quantitative im-munochemical analysis of human immunoglobulins. Clin. Chem. 18,1364 (1972).

9. Marrack, J. R., and Richards, C. B., Light.scattering studies of theformation of aggregates in mixtures of antigen and antibody. Im-munology 20, 1019 (1971).

10. Buffone, G. J., Savory, J., and Cross, R. E., Use of a laser modifiedcentrifugal analyzer for kinetic measurement of serum IgG. Clin.Chern. 20, 1320 (1974).

11. Gitlin, D., and Edelhoch, H., A study of the reaction betweenhuman serum albumin and its homologous equine antibody throughthe media of light scattering. J. Immunol. 66,67 (1951).