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Immunochemical determination of human apolipoprotein B by laser nephelometry
405
Clinica Chimica Acta, 95 (1979) 405-408 @ Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 10109
IMMUNOCHEMICAL DETERMINATION BY LASER NEPHELOMETRY
OF HUMAN APOLIPOPROTEIN
C. FIEVET-DESREUMAUX, E. DEDONDER-DECOOPMAN, P. DEWAILLY and G. SEZILLE *
B
J.C. FRUCHART,
Laboratoire de Physiopathologie des Lipides (E. R.A. C.N.R.S. No. 070497)Xlinique Medicale Ge’ne’rale A, Facultk de Me’decine, Place Verdun, 59045 Lille Ce’dex (France) (Received
October
13th,
1978)
Summary The Hyland laser nephelometer PDQ system for the assay of apolipoprotein B (apo-B) in human serum is described. Within and between-batch precision, accuracy and reliability are discussed. This instrument represents an important development in the immunochemical assay of apo-B, and the speed, precision, and convenience of the methodology make such a system attractive. Quantitation of apo-B was assessed in normal and hyperlipaemic subjects. Comparisons were made with two other specific and sensitive immunological methods for quantifying apo-B: enzymeimmunoassay (EIA) and rocket immunoelectrophoresis (RIE). Results obtained by the three methods correlated very well.
Introduction Interest in apo-B which is the major protein moiety of low and very low density lipoproteins (LDL and VLDL) has led to the development of immunoassays; immunocrit [ 11, immunoprecipitation [ 21, gel immunodiffusion [ 31, rocket immunoelectrophoresis (RIE) [ 41, radioimmunoassay [ 51 and enzymeimmunoassay (EIA) 161. Quantitation of proteins by laser nephelometry [7] was based on the concept that monospecific antiserum reacts with a specific antigen; these antigen-
* To whom reprint
requests
should be addressed.
406
antibody complexes in a solution scatter an incident beam of light, the amount of light scattered being proportional to the concentration of antigen. The purpose of the present investigation was to use this methodology to measure human apo-B. Materials and methods Pure lipoprotein B (LPB) was isolated from pooled plasma by preparative ultracentrifugation [ 61 and the. protein concentration in the lipoprotein fractions were determined [ 81 with human serum albumin as a standard [9] ; a chromogenic correction factor 0.9 was subsequently used to correct values
[lOI * Pure LPB was then used to obtain rabbit antiserum [ 111 which produced a single immunoprecipitation peak when used with normal human plasma. Rocket immunoelectrophoresis was performed [12] with use of agarose (10 g/liter), containing 5 ml of antiserum to human apo-B per liter. Enzymeimmunoassay was carried out as described in an earlier paper [ 61. Laser nephelometry assays were carried out with the Hyland Laser Nephelowas diluted with phosphate-buffered isotonic meter POQTM. The immuneserum saline (phosphate, 10 mmol/l, pH 7.2) and filtered through a 0.4~pm (average pore diameter) filter; the samples and standards of LPB were diluted with filtered isotonic saline. 1 ml of the antiserum dilution (75-fold dilution) and 100 ~1 of antigen solution were used; after suitable incubation, the light scattering was read on the nephelometer. A regression of the relative percentage light scatter against the antigen concentration produced a linear curve (Fig. 1). Results Since it is known [ 131 that use of a polyethylene glycol phosphate buffered isotonic saline (PBS-PEG) for dilution of immuneserum decreased the solubility of high molecular weight antibody-antigen complexes, and that this resulted in increasing light scatter and detectability with relatively short incubation times, we tested the effect of this polymer on apo-B determination. The analysis of 32 sera with the same immuneserum diluted with PBS-PEG or PBS alone, compared with the reference method (RIE), shows a good correlation (r = 0.967) between both nephelometric measurements. Nevertheless, apo-B concentrations found in the absence of PEG (mean + S.D. = 3.15 ?r 1.17 g/l) are significantly
TABLE
I
COMPARISON
OF
NEPHELOMETRY
ENZYMEIMMUNOASSAY
(X2)
FOR
(Y)
WITH
ROCKET
APOLIPOPROTEIN
Number
of samples:
Mean
of
Y ? S.D.
1.96
? 1.02
Mean
of X,
r S.D.
(g/l)
1.93
k 0.79
Mean
of X2
k S.D.
(g/l)
2.12
k 1.13
Correlation
IMMUNOELECTROPHORESI9 B
31. (g/l)
coefficient:
Y vs. X1
(X2)
0.980
(0.907)
N.S. 1
(Xl)
AND
407
./’
i
APO
6 fms/‘lt
Fig. 1. Calibration curve for the assay of ape-Ei laser nephelometry.
higher (+ll%) and close to values obtained by RIE (mean = 3.25 g/l). All the subsequent experiments were done (incubation time = 45 min) in PBS buffer without PEG. The assay range of linearity, as determined with pure LP-B dilutions (Fig. 1) was 4 to 20 trig/l. The minimum detectable concentration lo.4 pg per assay) exceeds that required to measure the apo-B on plasma and a 200- to 400-fold dilution of plasma was made for routine study. Within-batch precision for plasma of normal (1 g/l) and increased (3 g/l) apo-B concentrations was, respectively, 3 and 4.6% (7.5% and 7.9% for day-today variance). Laser nephelometry, EIA and RIE were used to measure plasma apo-B in a group of normal and hyperlipaemic subjects (mean cholesterol + SD. = 2.2 f 0.9 g/l). Results obtained by the three methods (Table I) correlated well and the mean levels were closely similar. Discussion Specific and sensitive immunological methods for quantifying apo-B have been developed but many are not entirely satisfactory [6,14]. The results of this study suggest that laser nephelometry is a specific, precise and highly sensitive alternative method for measuring apo-B in plasma samples. Recently, Ballantyne et al. [ 151 reported a comparable study (Table II) and concluded that laser nephelometry gives higher results than other methods (automated immunoprecipi~tion or radioimmuno~say). With our experimental conditions, however, the values obtained were in good agreement with those measured by RIE which is the more accurate method for qu~tification of apo-B [12] and
408
TABLE
II
COMPARISON
BETWEEN
TWO NEPHELOMETRIC
Method a: according to Baiiantyne et al. 1151. PBS, phosphate-buffered isotonic saline.
METHODS Method
FOR
b: according
Method Dilution of plasma Dilution of immune serum in Recovery (mean) (S) Within-assay precision (C.V.) Between-assay precision (C.V.) Correlation coefficient (ape-B vs. cholesterol * Hyperchoiesterolemic sera (a2.8 g/i). a. b and C: at about 500, 2099 and 5900
mg/i.
in serum)
APO-B IN SERUM to us. PEG,
a
l/2000 PEG-PBS 76 4.2 a 2.5 b 7.1 a 0.75 (n = 87)
polyethylene
Method
giycol:
b
l/200 l/400 * PBS (l/75) 105 3.0 b 4.6 c 7.5 b 7.9 c 0.92 (n = 63)
reSPectiyeiY.
correlation with corresponding cholesterol concentrations was more satisfactory (Table II) than that described by Ballantyne et al.; moreover, the higher sample dilution used by these authors (l/2000) is a greater potential source of error. References 1 H&k&I, C.L., Fisk, R.T., Florsheim. W.H., Tachi, A., Goodman. J.R. and Carpenter. C.M. (1961) Am. J. Clin. Pathol. 35.222-226 2 Kahan, J. and Sundblad. L. (1967) in Automation in Analytical Chemistry, Technicon International Symposia, 1966. Vol. 2. pp. 361-364. Mediad Incorporated, New York 3 Lees, R.S. (1970) Science 169.493495 4 Kahan. J. and Sundblad. L. (1969) &and. J. Clin. Lab. Invest. 24.61-68 Ciin. Chim. Acta 70, 267-276 5 Bedford, D.K.. Shepherd. J. and Morgan, H.G. (1976) 6 Fruchart, J.C., Desreumaux, C., DewaiRy. P., Sezille, G.. Jaillard, J.. Cariier, Y.. Bout, D. and Capron. A. (1978) Clin. Chem. 24.455459 7 Ritchie, R.F. (1967) J. Lab. Chin. Med. 70.512-517 8 Lowry, O.H., Rosebrough. N.J., Farr, A.L. and Randail, R.J. (1951) J. Biol. Chem. 193.265-275 P.N., Whither, J.T., Warren, C., Bolton. C.H. and Hartog. M. (1976) Clin. Chim. Acta 71, 9 Durrington. 95-108 10 Curry. M.D., Gustafson, A., Alaupovic and MC Conathy, W.J. (1978) Clin. Chem. 24, 280-286 11 Vaitukaitis, J., Robbins, J.B.. Nieschlag, E. and Ross, G.T. (1971) J. Clin. Endocrinol. 33,988 12 Laurel& C.N. (1972) Stand. J. Clin. Lab. Invest. 29. Suppl. 124. 21-37 13 HeBsing, K. (1969) Biochem. J. 114.141-144 G.J., Simons, L.A., Williams. P.F. and Turtle, J.R. (1976) Atherosclerosis 21, 217-234 14 Bautovich, F.C., Williamson, J.. Shapiro, D.. Caslake, M.J. and Perry, B. (1978) Clin. Chem. 24.78815 Ballantyne, 792
Clinica Chimica Acta, 95 (1979) 405-408 @ Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 10109
IMMUNOCHEMICAL DETERMINATION BY LASER NEPHELOMETRY
OF HUMAN APOLIPOPROTEIN
C. FIEVET-DESREUMAUX, E. DEDONDER-DECOOPMAN, P. DEWAILLY and G. SEZILLE *
B
J.C. FRUCHART,
Laboratoire de Physiopathologie des Lipides (E. R.A. C.N.R.S. No. 070497)Xlinique Medicale Ge’ne’rale A, Facultk de Me’decine, Place Verdun, 59045 Lille Ce’dex (France) (Received
October
13th,
1978)
Summary The Hyland laser nephelometer PDQ system for the assay of apolipoprotein B (apo-B) in human serum is described. Within and between-batch precision, accuracy and reliability are discussed. This instrument represents an important development in the immunochemical assay of apo-B, and the speed, precision, and convenience of the methodology make such a system attractive. Quantitation of apo-B was assessed in normal and hyperlipaemic subjects. Comparisons were made with two other specific and sensitive immunological methods for quantifying apo-B: enzymeimmunoassay (EIA) and rocket immunoelectrophoresis (RIE). Results obtained by the three methods correlated very well.
Introduction Interest in apo-B which is the major protein moiety of low and very low density lipoproteins (LDL and VLDL) has led to the development of immunoassays; immunocrit [ 11, immunoprecipitation [ 21, gel immunodiffusion [ 31, rocket immunoelectrophoresis (RIE) [ 41, radioimmunoassay [ 51 and enzymeimmunoassay (EIA) 161. Quantitation of proteins by laser nephelometry [7] was based on the concept that monospecific antiserum reacts with a specific antigen; these antigen-
* To whom reprint
requests
should be addressed.
406
antibody complexes in a solution scatter an incident beam of light, the amount of light scattered being proportional to the concentration of antigen. The purpose of the present investigation was to use this methodology to measure human apo-B. Materials and methods Pure lipoprotein B (LPB) was isolated from pooled plasma by preparative ultracentrifugation [ 61 and the. protein concentration in the lipoprotein fractions were determined [ 81 with human serum albumin as a standard [9] ; a chromogenic correction factor 0.9 was subsequently used to correct values
[lOI * Pure LPB was then used to obtain rabbit antiserum [ 111 which produced a single immunoprecipitation peak when used with normal human plasma. Rocket immunoelectrophoresis was performed [12] with use of agarose (10 g/liter), containing 5 ml of antiserum to human apo-B per liter. Enzymeimmunoassay was carried out as described in an earlier paper [ 61. Laser nephelometry assays were carried out with the Hyland Laser Nephelowas diluted with phosphate-buffered isotonic meter POQTM. The immuneserum saline (phosphate, 10 mmol/l, pH 7.2) and filtered through a 0.4~pm (average pore diameter) filter; the samples and standards of LPB were diluted with filtered isotonic saline. 1 ml of the antiserum dilution (75-fold dilution) and 100 ~1 of antigen solution were used; after suitable incubation, the light scattering was read on the nephelometer. A regression of the relative percentage light scatter against the antigen concentration produced a linear curve (Fig. 1). Results Since it is known [ 131 that use of a polyethylene glycol phosphate buffered isotonic saline (PBS-PEG) for dilution of immuneserum decreased the solubility of high molecular weight antibody-antigen complexes, and that this resulted in increasing light scatter and detectability with relatively short incubation times, we tested the effect of this polymer on apo-B determination. The analysis of 32 sera with the same immuneserum diluted with PBS-PEG or PBS alone, compared with the reference method (RIE), shows a good correlation (r = 0.967) between both nephelometric measurements. Nevertheless, apo-B concentrations found in the absence of PEG (mean + S.D. = 3.15 ?r 1.17 g/l) are significantly
TABLE
I
COMPARISON
OF
NEPHELOMETRY
ENZYMEIMMUNOASSAY
(X2)
FOR
(Y)
WITH
ROCKET
APOLIPOPROTEIN
Number
of samples:
Mean
of
Y ? S.D.
1.96
? 1.02
Mean
of X,
r S.D.
(g/l)
1.93
k 0.79
Mean
of X2
k S.D.
(g/l)
2.12
k 1.13
Correlation
IMMUNOELECTROPHORESI9 B
31. (g/l)
coefficient:
Y vs. X1
(X2)
0.980
(0.907)
N.S. 1
(Xl)
AND
407
./’
i
APO
6 fms/‘lt
Fig. 1. Calibration curve for the assay of ape-Ei laser nephelometry.
higher (+ll%) and close to values obtained by RIE (mean = 3.25 g/l). All the subsequent experiments were done (incubation time = 45 min) in PBS buffer without PEG. The assay range of linearity, as determined with pure LP-B dilutions (Fig. 1) was 4 to 20 trig/l. The minimum detectable concentration lo.4 pg per assay) exceeds that required to measure the apo-B on plasma and a 200- to 400-fold dilution of plasma was made for routine study. Within-batch precision for plasma of normal (1 g/l) and increased (3 g/l) apo-B concentrations was, respectively, 3 and 4.6% (7.5% and 7.9% for day-today variance). Laser nephelometry, EIA and RIE were used to measure plasma apo-B in a group of normal and hyperlipaemic subjects (mean cholesterol + SD. = 2.2 f 0.9 g/l). Results obtained by the three methods (Table I) correlated well and the mean levels were closely similar. Discussion Specific and sensitive immunological methods for quantifying apo-B have been developed but many are not entirely satisfactory [6,14]. The results of this study suggest that laser nephelometry is a specific, precise and highly sensitive alternative method for measuring apo-B in plasma samples. Recently, Ballantyne et al. [ 151 reported a comparable study (Table II) and concluded that laser nephelometry gives higher results than other methods (automated immunoprecipi~tion or radioimmuno~say). With our experimental conditions, however, the values obtained were in good agreement with those measured by RIE which is the more accurate method for qu~tification of apo-B [12] and
408
TABLE
II
COMPARISON
BETWEEN
TWO NEPHELOMETRIC
Method a: according to Baiiantyne et al. 1151. PBS, phosphate-buffered isotonic saline.
METHODS Method
FOR
b: according
Method Dilution of plasma Dilution of immune serum in Recovery (mean) (S) Within-assay precision (C.V.) Between-assay precision (C.V.) Correlation coefficient (ape-B vs. cholesterol * Hyperchoiesterolemic sera (a2.8 g/i). a. b and C: at about 500, 2099 and 5900
mg/i.
in serum)
APO-B IN SERUM to us. PEG,
a
l/2000 PEG-PBS 76 4.2 a 2.5 b 7.1 a 0.75 (n = 87)
polyethylene
Method
giycol:
b
l/200 l/400 * PBS (l/75) 105 3.0 b 4.6 c 7.5 b 7.9 c 0.92 (n = 63)
reSPectiyeiY.
correlation with corresponding cholesterol concentrations was more satisfactory (Table II) than that described by Ballantyne et al.; moreover, the higher sample dilution used by these authors (l/2000) is a greater potential source of error. References 1 H&k&I, C.L., Fisk, R.T., Florsheim. W.H., Tachi, A., Goodman. J.R. and Carpenter. C.M. (1961) Am. J. Clin. Pathol. 35.222-226 2 Kahan, J. and Sundblad. L. (1967) in Automation in Analytical Chemistry, Technicon International Symposia, 1966. Vol. 2. pp. 361-364. Mediad Incorporated, New York 3 Lees, R.S. (1970) Science 169.493495 4 Kahan. J. and Sundblad. L. (1969) &and. J. Clin. Lab. Invest. 24.61-68 Ciin. Chim. Acta 70, 267-276 5 Bedford, D.K.. Shepherd. J. and Morgan, H.G. (1976) 6 Fruchart, J.C., Desreumaux, C., DewaiRy. P., Sezille, G.. Jaillard, J.. Cariier, Y.. Bout, D. and Capron. A. (1978) Clin. Chem. 24.455459 7 Ritchie, R.F. (1967) J. Lab. Chin. Med. 70.512-517 8 Lowry, O.H., Rosebrough. N.J., Farr, A.L. and Randail, R.J. (1951) J. Biol. Chem. 193.265-275 P.N., Whither, J.T., Warren, C., Bolton. C.H. and Hartog. M. (1976) Clin. Chim. Acta 71, 9 Durrington. 95-108 10 Curry. M.D., Gustafson, A., Alaupovic and MC Conathy, W.J. (1978) Clin. Chem. 24, 280-286 11 Vaitukaitis, J., Robbins, J.B.. Nieschlag, E. and Ross, G.T. (1971) J. Clin. Endocrinol. 33,988 12 Laurel& C.N. (1972) Stand. J. Clin. Lab. Invest. 29. Suppl. 124. 21-37 13 HeBsing, K. (1969) Biochem. J. 114.141-144 G.J., Simons, L.A., Williams. P.F. and Turtle, J.R. (1976) Atherosclerosis 21, 217-234 14 Bautovich, F.C., Williamson, J.. Shapiro, D.. Caslake, M.J. and Perry, B. (1978) Clin. Chem. 24.78815 Ballantyne, 792