Clin. Biochem. 11 (1) 28-31

(1978)

Evaluation of a Behring Laser-Nephelometer Prototype in the Measurement of IgG, IgA and IgM RI~JEAN DAIGNEAULT and DOROTHI~E LEMIEUX D6partement de Biochimie, H6pital Notre-Dame, 1560 est, rue Sherbrooke, Montr6al Canada H2L 4M1, and D6partement de Biochimie, Universit~ de Montr6al. (Accepted June 13, 1977)

CLBIA, 11 (1) 28-31 (1978) Clin. Biochcm.

Ddparteme~t de Biochimie, Hdpital Notre-Dame, 1560 est, r,e Sherbrooke, Montrdal, Canada H2L .~M1, and Ddpartement de Biochimie, Universitd de Mo~trdal. E V A ' L U A T I O N OF A B E H R I N G L A S E R - N E P H E L O M E T E R P R O T O T Y P E IN T H E M E A S U R E M E N T OF IgG, IgA AND lgM A helium-neon laser nephelometer prototype manufactured by Hoechst Behring Institut was evaluated and used in our laboratory during a two-month period for the measurement of IgG, IgA and IgM. Two methods of dilution were used to prepare standard curves. The best results were obtained when the patient serum was diluted one hundred fold (10-100 ~tl} and incubated for 15 rain at room temperature in a disposable plastic cuvette with the specific antiserum diluted five fold {100-300 gl). The intensity of the light scattered by the antigen-antibody complexes was then immediately read on the digital voltmeter. The working ranges were 186-5944 mg/dl for IgG, 33-1066 m g / d l for IgA and 17-545 mg/dl for IgM. Standard curves obtained for the three immunoglobulins on six different days could be superimposed perfectly demonstrating an excellent reproducibility. The coefficient of variation for lgG (n = 8) was 4.1% at 5800 mg/dl. The results (n = 37) produced by ~he laser nephelometer (y) correlated very well with those obtained by radial hnnaunodiffusion (x) for: IgG y r IgA y r Ig.{ y r

= 8.07 + 1.00 × = 0.997 = - 6.51 + 1.05 X = 0.9?6 = 3.35 + 0.98 X = 0.998.

M~EASUREMENT OF SERUM PROTEINS by m a n u a l or a u t o m a t e d n e p h e l o m e t r i c t e c h n i q u e s has g a i n e d p o p u l a r i t y in r e c e n t years. Most of t h e c o m m e r c i a l nephelo m e t e r s c u r r e n t l y used in clinical l a b o r a t o r i e s a r e d e s i g n e d to m e a s u r e t h e l i g h t s c a t t e r e d by t h e a n t i g e n - a n t i b o d y complexes in t h e visible r a n g e b e t w e e n 350 and 500 n m a t t h e f i x e d a n g l e o f 90 °. W e have e v a l u a t e d a m a n u a l l a s e r n e p h e l o m e t e r prototype during a two-month investigation period Request for reprints to Dr. P~jean Daigneault.

f o r the m e a s u r e m e n t of IgG, I g A and I g M in s e r u m . Th e i n s t r u m e n t e m i t t e d a n a r r o w m o n o c h r o m a t i c l i g h t beam (633 n m ) and m e a s u r e d t h e l i g h t scatt e r e d u n d e r a low a n g l e in t h e f o r w a r d d i r e c t i o n " ' . We r e p o r t h e r e i n o u r e x p e r i e n c e w i t h t h i s s y s t e m a nd c o r r e l a t e r e s u l t s of t h e l a s e r n e p h e l o m e t e r w i t h t hos e of r a d i a l i m m u n o d i f f u s i o n . MATERIALS AND METHODS Tl:e 3 mW helium-neon laser nephelometer manufactured by Hoechst Behring Institut, M a r g b u r g / L a h n , Germany, consisted of two parts: a voltage stabilizer and the main component containing the laser, the diaphragms, the cuvette compartment equipped with a metal lid that automatically cut off the laser beam when opened, the lense system and the photodiode. The result of the measurement was read on a digital meter calibrated from 0.00 to 11.50 volts. Serum specimens were obtained from hospitalized patients. They were either processed the same day or kept frozen at --20 C until assay. The standard serum (LS 176, Hoechst Behring In~titut), the serum samples and the anti-human IgG, IgA and IgM from rabbit (Hoechst Behring Institut) were diluted with isotonic saline and filtrated with a Millex disposable filter (0.22 urn, Millipore Corporation, Bedford, Mass., U.S.A.). All dilutions were prepared freshly eacla clay. A micro plastic disposable cuvette was assigned for each individual determination. The individual light scatter by the empty cuvette was recorded as the blank value. The standard or serum dilution was incubated with the antiserum dilution for 15 rain at room temperature in the pre-determined cuvette sealed with Parafilm to prevent dust contamination. The cuvette was then placed in the cuvette compartment of the laser system and the scattered light read on the digital meter a f t e r 15 sec. The blank value was subtracted from the sample value. The results in volts were plotted versus concentrations in mg/dl. Each sample was run in duplicate. The results obtained with the laser nephelometer were correlated with those of radial immunodiffusion using M-Partigen plates (Hoect:st Behring Institut). Statistical analyses and curve fitness studies between voltages in volts (y) and concentrations in mg/ dl (x) were computed with a Hewlett-Packard 65 programmable pocket calculator. RESULTS AND DISCUSSION

W e h a v e used t h r e e d i f f e r e n t a p p r o a c h e s to d i l u t e t h e s t a n d a r d s e r u m and t h e a n t i s e r u m f o r p r o d u c i n g s t a n d a r d cu r v es.

EVALUATION

OF A BEHRING

LASER-NEPHELOMETER

TABLE

29

PROTOTYPE

1

M E T H O D OF DILUTION 1

Dilution of Antiserum

Dilution of Standard 176

Volume of diluted Standard 176 (uD

Dilution of Patient Serum

Volume of diluted Anti.cerum (ul)

5.88 2.94

1:200 1:400

IgG

IgA

1:5

1:800

1:500

50

I00

1.47

1:1600 1:3200

0.74 0.37

1:32 1:64 1:128 1:256

6.59 3.29 1:5

1:80

50

100

1:8

IgM

Concentration of diluted Standard 176 (mg/dl)

1:16 1:32 1:64 1:128

1:5

1:30

50

lO0

1.65

0.82 13.50 6.75 3.37 1.69 0.84

TABLE 2 METHOD OFDILUTION I I I

Dilution of Antiserum

Dilution of Standard 176

IgG

IgA

1:20 1:40 1:80 1:160 1:320 1:649

1:5

Dilution of Patient Serum

1:101

Volume of diluted Standard 176 lul,

10

Volume of Antiserum lul~

Concentration of dilute:l Standard 176

300

58.85 29.43 14.71 7.36 3,68

1.84

1:20 1:40 1:80 1:160 1:320 1:640

1:5

1:101

100

250

5.,10 2.70

1:20

IgM

10.55 5.28 2.64 1.32 0.66 0.33

1:40 1:80 I : 160

1.35

1:5

1:1(0

1:320 1:640

In the f i r s t method of d i l u t i o n I Table 11, the t h r e e a n t i s e r a are diluted five fold a n d the same volume of d i l u t e d s t a n d a r d (50 ul) a n d a n t i s e r u m (100 ul) are m i x e d together. The p a t i e n t s e r u m is d i l u t e d 500, 80 a n d 30 fold respectively for IgG, I g A a n d IgM. F i g u r e 1 shows six s t a n d a r d c u r v e s o b t a i n e d on six d i f f e r e n t days f o r I g A u s i n g t h i s apl)roach. T h e r e p r o d u c i b i l i t y is n o t very good. S i m i l a r r e s u l t s were o b t a i n e d for IgG a n d IgM. TAELE 3 T MEASUREMENT RANGES FOR THE "I'HREE IMMLNOGLCBLLINS

Method of dilution

Dilution factor

Range (mg,'dl I

IgG

I III

500 1C1

185 -2940 186 - 5944

IgA

I III

80 1Ol

66 - 527 33 - 1066

IgM

I Ill

39 101

25 - 405 17 - 545

103

100

0.68 0.34 0.17

A second mode of d i l u t i o n gave r e s u l t s identical to those in F i g u r e 1 and will n o t be described h e r e i n . The t h i r d m e t h o d of d i l u t i o n is described in Table 2. I n this approach, d i l u t i o n s of s t a n d a r d serum, a n t i s e r u m a n d p a t i e n t s e r u m are i d e n t i c a l for the t h r e e i m m u n o g l o b u l i n s . Volumes of diluted s t a n d a r d 176 and of a n t i s e r u m d i l u t i o n v a r y respectively f r o m 10 to 100 ul and f r o m 100 to 300 ul. S u p e r i m p o s e d s t a n d a r d curves for IgG o b t a i n e d on 6 d i f f e r e n t days are p r e s e n t e d in F i g u r e 2. E q u a l l y r e p r o d u c i b l e restilts were o b t a i n e d for I g A a n d IgM. T h i s t h i r d mode of d i l u t i o n should be p r e f e r r e d to the f i r s t mode of d i l u t i o n for several r e a s o n s : b e t t e r r e p r o d u c i b i l i t y , g r e a t e r s i m p l i c i t y for the user a n d the same p a t i e n t s e r u m d i l u t i o n available for t h e t h r e e immunoglobulin measurements. Furthermore, this mode of d i l u t i o n gives a w i d e r r a n g e of m e a s u r e m e n t for all t h r e e i m m u n o g l o b u l i n s as d e m o n s t r a t e d in T a b l e 3. The r a n g e of m e a s u r e m e n t in m g / d l were 186-5944 for IgG, 33-1066 for I g A a n d 17-545 f o r IgM. These values r e p r e s e n t w o r k i n g r a n g e s obt a i n e d when d i l u t i n g the s t a n d a r d s e r u m LS 176

30

DAIGNEAULT volts

IgA

9

BY D I L U T I O N

AND LEMIEUX volts 10

[

IgO BY DILUTION Ill 9

8

/

7

B

6

7

5

6

4

5

3

2

1

0

i

~

i

I

3 4 mg/dl x 102

I

i

i

i

5

6

7 i

o

Fi47. 1 Sta'ndard c~crves for I g A obtai~ed on six differant days using method of dilution I. -

i

2

3

4

5

6

-

mg dl x 10'

Fig. 2 Standard curves for IgG obtained on six d~fferent days using method of dih~tion HI. -

-

volts 10

9

8

7

5400

6

10 4800

5

•

4200

,4 3600

3 3000

2 240

1

0

i

I

2

i

I

I

i

3

4

5

6

mg/dl

x I0

y

:

8 977 ° ! 00 •

r

:

0 997

!!i;'

•"

3

Fig. 3 - - Typical standard curve for IgG assayed by the laser nephelemeter. Each standard was analysed in duplicate and each point represenSs the mean of the voltage measurement,

60O 600

1200

1800

24'00

30'00

36'00 RID

.t 2'00

4g'00

54'00

6000

Fig. ~ - - Correlation of concentrations of IgG in serum between laser nephelometry and radial immunodiffu, swn.

E V A L U A T I O N OF A B E H R I N G L A S E R - N E P H E L O M E T E R P R O T O T Y P E

31

900000 IoA

700-

( •

• •

1206

600

1050

500.

900

•

m

750,

400[1 . 0:! 5 X.5.51. !ir:1910.996

300-

600 .... 3 35 . 0 0 8 .

~

o

o

~ '

•

n

37

200-

0

090

J7

300-

100-

150100

200

300

400

500R I O 600

"/O0

OO0 900

Fig. 5 - - Correlation o.f c o n c e n t r a t i o n s o f I g A in s e r u m b e t w e e n laser nephelometr!/ and radial i m m u n o d i f f u s i o n .

according to the manufacturer's recommendations. T h e s e d i l u t i o n s did n o t e x p l o i t t h e full c a p a b i l i t i e s o f t h e i n s t r u m e n t . T h e values should t h e r e f o r e be considered as conservative especially for IgM. Rarely did we m e a s u r e v o l t a g e s g r e a t e r t h a n 10 f o r I g G a n d I g A , a n d 6 f o r I g M . No a t t e m p t s w e r e m a d e f r o m o u r p a r t to e x t e n d t h e s e r a n g e s . F i g u r e 3 shows a t y p i c a l s t a n d a r d curve o b t a i n e d by m e t h o d of d i l u t i o n I I I f o r IgG. T h e r e l a t i o n s h i p b e t w e e n v o l t a g e s and c o n c e n t r a t i o n s w a s p e r f e c t l y c o r r e l a t e d (r = 0.999) w i t h t h o g e n e r a l e q u a t i o n y = a + b x + cx 2 + dx "~ + ex ~ w h e r e y is t h e v o l t a g e in volts a n d x t h e p r o t e i n c o n c e n t r a t i o n in m g / d l . The c o e f f i c i e n t of v a r i a t i o n f o r I g G (n = 8) w a s 4.1C,~ a t 5800 m g / d l . T h i r t y - s e v e n s e r a w e r e a n a l y s e d f o r IgG, I g A a n d IgM concentration with the laser system using method of d i l u t i o n I I I a n d w i t h r a d i a l i m m u n o d i f f u s i o n . V e r y good c o r r e l a t i o n w a s f o u n d f o r I g G (r = 0.997, F i g u r e 4), I g A ( r = 0.996, F i g u r e 5) a n d I g M (r = 0.998, F i g u r e 6). I n conclusion, t h e l a s e r n e p h e l o m e t e r p r o d u c e d p r e c i s e a n d r e p r o d u c i b l e r e s u l t s over an e x t e n d e d r a n g e ' . T h e r e s u l t s c o r r e l a t e d v e r y well w i t h t h o s e o f r a d i a l i m m u n o d i f f u s i o n . I n o u r opinion, l a s e r n e p h e l ometry has a great potential for protein measurement in biological f l u i d s .

' The digital voltmeter on the commercial version of this instrument is calibrated from 0.00 to 19.99 volts. This should f u r t h e r increase the workable ranges for the measurement of proteins.

RID

Fig. 6 - - C o r r e l a t i o n o f c o n c e n t r a t i o n s of l g M in s e r u m between, laser n e p h e l o m e t r y and radial i m m u n o d i f f u s i o n .

ACKNOWLP_J)GEMENT

We thank Francine Prud'homme for radial immunodiffusion measurement and Ga4tan Thibault for establishing the relationship between voltages and concentrations. We are indebted to Dr. F. E. Hoyer and the Behring I n s t i t u t for the loan of the laser nephelometer and greatly appreciated the courteous collaboration of the people from Hoechst in Germany and Canada. REFEREN

CES

1. Sieber, A. and Gross, J. (1975). P r o t . Biol. F l u i d s . 23, 295-298.