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A non-competitive enzyme-linked immunosorbent assay for measuring human plasma apolipoprotein B levels
CIinicu Chimieu Acta, 1.51 (1985) 317-324 Elsevier
317
CCA 03296
Brief technical
note
A non-competitive enzyme-linked immunosorbent assay for measuring human plasma apolipoprotein B levels Richard
W. James *, Manuela
Dit$sionde Diabktologie,
Ferrer and Daniel Pometta
LQpurtement de MtLdecine, Kipitai
(Received
November
Cantonal de Gemhe, 121 I GE 4 (Switzerland1
9th, 1984; revision June 14th, 1985)
Key words: Apolipoprotein B; Low density lipoprotein; Enzyme-linked
immunosorbent
asmy
The need for precise measurements of plasma levels of apolipoprotein B-100 (apo B), the structural protein component of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) has been accentuated in recent years by suggestions that they may be superior to plasma or LDL cholesterol levels as an indicator for the risk of cardiovascular disease [1,2]. As pointed out in an excellent review by Rosseneu et al [3), quantification of apo B is fraught with numerous technical problems, and no single assay procedure has met with universal approval. The reference procedure, electroimmunoassay (EIA [4]), is not a practical proposition for routine clinical analyses. The merits of the enzyme-linked immunosorbent assay (ELISA) system have been advocated in many publications. Five ELISA systems for quantifying apo B have been described. Of these, two are based on competition assays [5,6] which appear less reproducible than the non-competitive ELISA, and require purified apo B or LDL. A third requires an LDL-enzyme complex, which somewhat precludes its use for routine analyses [7]. Fievet et al [8] have described a non-competitive ELISA using antibody-coated polystyrene balls, which has recently been extended to microtitre plates [9]. Given the advances in technology for automation of ELISA using such plates, this would appear to offer the most suitable technique for routine clinical analyses. Here we describe just such an approach. Materials and methods Preparatory of antigen A narrow density LDL fraction (d = 1.04-1.052 kg/l) was prepared by flotation ultracentrifugation of human plasma obtained from the Blood Transfusion Centre of the Geneva Cantonal Hospital. * To whom correspondence ~9-8981/~5/$03.30
should be addressed.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
The apo B protein content of LDL was measured by the Lowry method [IO] ah the tetramethylurea (TMU) precipitable fraction [ll]. A factor of 0.9 was emploved to correct for the difference in ~hromogeni~ity of BSA and ape B I-?].
Antiserum was raised in rabbits against LDL (d= 1.04-1.052 kg/l). The whole IgG fraction was obtained by precipitation with ammonium sulphate (277 g/l) and redissolved in phosphate buffered saline (PBS, phosphate buffer 10 mmol/l. pH 7.4. sodium chloride 137 mmol/l. potassium chloride 2.7 mmot/l. sodium azide 3.1 mmol/l). Monospecific anti-apo B antibodies were obtained by affinity chromatography as follows. An affinity column was prepared by covalently linking LDL (d = 1.04- 1.052 kg/l, 10 mg) to cyanogen bromide-activated Sepharose 4B (Pharmacia, Switzerland). The whole IgG fraction was passed twice down the column and. after extensive washing, specifically adsorbed antibodies were eluted with potassium thiocyanate (3 m&/l) in PBS. The antibodies were dialysed against ammonium bicarbonate, 50 ntmol/l, pH 8.0, then lyophilised. Preparation ofuntibody-enzyme conjugate Monospecific anti-apo B antibodies were conjugated to alkaline phosphatase (Sigma, USA) by the glutara~dehyde method. Briefly, antibodies (0.4 mg) were mixed with alkaline phosphatase (0.25 mg) and dialysed against PBS. The volume was made up to 1.0 ml and glutaraldehyde added to a final concentration of 2 ml/l. After thoroughly mixing, the reaction was allowed to continue for 2 h at room temperature. Subsequently the reaction mixture was dialysed at 4°C against PBS (2 x 500 mi) and Tris buffer (Tris 50 mmol/l. pH 8.0, magnesium chloride 1.0 mmol/l, 2 x 500 ml). The dialysate was diluted to 4 ml in the same buffer containing BSA (10 g/l) and azide (3.1 mmol/l) for storage at 4°C. Enzyme-linked immunosorhent assa_y The following protocol was adopted. Microt~tre piates (Mi~roelisa-plates. Dynatech, Switzerland) were coated during overnight incubation (4°C) with monospecific anti-apo B IgG (0.1 ml; 5 mg/l in carbonate buffer, 50 mmol/l, pH 9.6). Remaining absorption sites were blocked with BSA (10 g/l in the same buffer; 37’C for 1 h) and the wells then washed with PBS containing Tween 20 (0.5 ml/l). The solid-phase antibody was subsequently incubated for 3 h with appropriately diluted standards or test solutions (0.1 ml in wash buffer containing BSA 10 g/l), washed thoroughly and incubated a further 2 h with antibody-enzyme conjugate (0.1 ml of a 1 : 100 dilution in same buffer). Bound conjugate was visualized by adding 0.1 ml of substrate solution ( p-nitrophenyl phosphate; 1 tablet for 5 ml of buffer, diethanolamine, 1 mol/l, pH 9.8 containing 0.5 mmol/l magnesium chloride). The reaction was allowed to continue for 30-45 min, giving a maximal absorption of 1.5 units. before being quenched with sodium hydroxide (3 mol/l, 0.05 ml). to 2.0 Ad,)5 All incubations were effected at room temperature with continual rotary agitation (Vari-Shaker, Dynatech, Switzerland). The absorbances of the reaction mixtures were read with the SLT 210 microelisa
319
reader (Kontron, Switzerland). All results were subjected to computer analysis by the four parameter logistic curve fitting programme developed by Rodbard et al [12]. Fractionation
of lipoproteins
Lipoprotein density subclasses were separated by sequential flotation ultracentrifugation at densities of 1.006, 1.063 and 1.21 kg/l respectively. Subfractionation of VLDL into large (Sf 400-60) and small (Sf 60-20) VLDL, and preparation of intermediate density lipoproteins (IDL, Sf 20-12) were performed as described by Lindgren et al [13]. Purification
of apolipoproteins
Apolipoproteins A-I and A-II were isolated from delipidized high density lipoproteins by gel filtration chromatography [14]. Apolipoproteins E and C were purified from delipidized VLDL on an affinity column of heparin-Sepharose [15]. Results Optimization
of assay conditions
Preliminary studies established a suitable concentration for coating of the microtitre wells with affinity-purified antibodies (5 mg/l), and for dilution of the antibody-enzyme conjugate (1 : 100). The latter has proven to be remarkably consistent over six different preparations of conjugate. Analysis of different incubation times and temperatures for absorption of the solid-phase antibody showed that 16 h at 4°C was sufficient, Parallelism of titration curves was maint~ned when precoated plates were analysed after storage for 3 weeks in PBS (4°C). Validation of ELISA
system
A typical calibration curve established with plasma is shown in Fig. 1. As little as 2.5 ng of apo B can be detected, with a working range of ld to 100 ng. When the specificity of the antibodies was controlled, no appreciable cross-reactivity with apolipoproteins A-I, A-II, C, E or with albumin (not shown) was observed (Fig. 1). Whole serum was shown to give a curve parallel to that obtained with purified LDL (Fig. 2A). With VLDL, no parallelism was at first apparent as the dose-response curve consistently gave a plateau below that observed for serum and LDL (Fig. 2A) and attempts to redress the situation by pretreatment of VLDL with Tween 20 (4 ml/l), sodium dodecyl sulphate (50 mmol/l), Triton X-100 (4ml/l) or Nonidet P-40 (4 ml/l) were unsuccessful. When the absorbance values were normalized to a plateau value of 1.0, parallelism of all three curves was evident (Fig. 2B). quantification
of apo~ipoprotein B in serum samples
A secondary standard of whole plasma was used for routine measurements of apo B. The secondary standard was calibrated using the primary standard (LDL d = 1.04-1.052 kg/l), the protein concentration of the latter being established by the Lowry method as described in the Methods section. The secondary standard was stored in aliguot at - 70°C in the presence of phenylmethylsulphonylfluo~de. No
Plasma Cnl)
r
1
t
101
-I
t 102
105
104
ADO B (ncl)
Fig. 1. Calibration curve developed with plasma for measuring plasma apo B levets. The secondary u-hole plasma standard was calibrated using the primary LDL (d = 1.04-1.052 kg/l) standard and processed by the ELISA procedure described in the Methods section. Each point is the average of 6 determinations +SD. To test the effects of other apolipoproteins. increasing concentrations of the purified apolipoproteins replaced plasma in the first. 3 h incubation period. The microtitre plates were then processed by the usual procedure. Tr.kpoE: O0. Apu C; A--A. Apo A-1; .A, Apo A-II.
loss of immunoreactivity has been observed over 6 months, A aliguot is thawed prior to an analysis and subsequently kept at 4°C for 2-3 weeks. It is not refrozen. Apo B in LDL samples from plasma with a range of serum cholesterol concentrations (3.98-14.37 mmol/l) were quantified by ELISA, EIA and by the Lowry method, after precipitation with TMU. Excellent correlations between the values were observed, as shown by the Pearson correlation coefficients (Table 1). The same was also true when apo B values measured by ELISA were compared to the LDL cholesterol and total cholesterol values of the same samples (Table I). Apo B levels measured in whole plasma by ELISA and EIA were also compared and, as shown in Table I and Fig. 3A, very good agreement between the values was observed. Interestingly, higher Pearson correlation coefficients were observed when the LDL and total cholesterol values of these samples were compared to apo B levels (Table I and Fig. 3B). Quantification of apo B (triplicate determinations per point) in a series of normolipidic subjects (age range 24-70 years) gave an average value of 0.92 & 0.22 g/l (n = 41). The intra-assay and interassay coefficients of variation were 5.2% (n = 23) and 7.9% (n = 12) respectively.
321
s
2 5
9
101
1
102
103
104 Plasma, LDL hi)
Plasma. LDL hl) I
I
101
102
I
103
1
104
1
I
105
101
I
1
102
I
103
104
I
105
VLDL (nl)
VLDL (nlf
Fig. 2. Effects of plasma, LDL and VLDL in the apo B ELISA system. Various dilutions (100 ~1) of whole plasma, LDL and VLDL (prepared by sequential ultracentrifugation) were incubated (3 h, room temperature) with microtitre wells precoated with anti apo B IgG. The wells were subsequently processed by the standard procedure. A, absolute absorbance; B, absorbance values normalized to a plateau value of VLDL. 0, plasma; n -A, LDL; * -----a, 1.0. 0 -
Analysis of the ELISA system for its ability to quantify apo B in VLDL subfractions and in intermediate density lipoproteins (IDL) showed that the majority (85%-88%) of the apoprotein in both VLDL subfractions from hyperlipidaemic sera was measured (Table II). Similar observations were made for small VLDL from normolipidaemic sera, although only 73% of apo B in large VLDL from the
TABLE
I
Pearson
correlation
Compared
function
Apo B, EIA Apo B, TMU Cholesterol, Cholesterol,
LDL total
coefficients
for plasma
and LDL concentrations
of apo B and cholesterol
ELISA apo B LDL
Plasma
0.98 0.98
0.76 -
0.99 0.96
0.83 0.81
Apo B levels were measured by ELISA either in LDL (n = 19) obtained by preparative ultracentrifugation or in whole plasma (n = 28). These values were then compared to apo B values measured in the same fractions by EIA or TMU precipitation (LDL only), and LDL and total plasma cholesterol. All Pearson correlation coefficients were significant at p = 0.001. The regression equation for the comparison of serum apo B measured by ELISA and EIA was y = 0.006.x +0.12, with r = 0.76.
32’
TABLE
II
Ape H in VLDL aubfrncttons and IIIlI?~l. I~__ Serum
Suhfracrlcrn
Normal Hyperlipidaemic
VLDL Sf 400-60
VLDL Sf 60-20
IDL Sf 20-12
73.2 x7.7
89.9 x4.7
95.5 95,s
_..
-
VLDL subfractions and IDL were isolated as described in Methods. The apo B content was measured by ELISA and as TMU-precipitable protein and resuk are expressed as percentages: ELISA apo BiTMU ape B x 100.The results are averaged for 4 normal and 7 Ilyp~rlipidaen~ic sera
200 2 d t.2
m
$
100
0
I
I
f
I
1 I
1 L
1
2
Apo B ELISA (g/l)
APO B ELISA (s/l)
Fig. 3. Comparison of apo B values measured in samples by ELISA with (A) apo B values measured by EIA and (B) total cholesterol levels. Twenty-eight samples with a range of apo B and cholesterol values were analysed. Apo B values measured by EIA are expressed in arbitrary units.
normolipidaemic sera was measured. serum was measured.
Over 95% of IDL apo B from both types of
Discussion
The criteria to be applied to an assay being considered for routine clinical analyses are precision, reproducibility, sensitivity and ease of application, the latter encompassing rapidity and the possibility of automation. The ELISA we have developed for apo B satisfies these criteria. The excellent correlations between plasma and LDL levels of apo B as measured by ELISA, EIA and TMU precipitation-the latter was not applied to plasma(Table I), testify to the precision of the ELISA. As corroborative evidence, there are also the excellent correlations between apo B levels measured by ELISA and the
323
total and LDL cholesterol concentrations (Table I). Intra- and interassay coefficients of 5.2% and 7.5% respectively indicate that the procedure displays the
of variation
necessary reproducibility. The sensitivity of the assay (working range lo-100 ng) is equivalent to that of radioimmunoassay. Its high sensitivity could lead to imprecision, given that dilutions of serum samples up to 2000-fold may be necessary. However the coefficients of variation affirm the reproducibility of the assay, if the usual laboratory care is taken when diluting the samples. Less sensitive assays such as EIA and radial immunodiffusion necessitate lower plasma dilutions. However their potential for automation is limited, which is not the case with the ELISA system. Immunonephelometry offers a rapid, inexpensive means of measuring apo B, but suffers from the requirement for a similar size of lipoprotein for sample and standard, and dyslipoproteinaemic samples can seriously affect the results [3]. As regards rapidity, the ELISA allows apo B levels to be measured in 6 h, with the potential for extensive automation of the process. We have chosen to use a secondary standard of whole plasma to generate the calibration curve, as suggested by Rosseneu et al 131. The parallelism of the calibration curves for whole plasma and the primary LDL standard (Fig. 2A), and the excellent correlations between apo B values measured by ELISA, EIA and TMU precipitation support this approach. With the ELISA we observed an average concentration of apo B in normolipidic subjects of 0.92 g/l. Values reported in the literature for a variety of methodologies range from 0.8 to 1.59 g/l, whilst radioimmunoassay, which is of equivalent sensitivity, gave values ranging from 0.81 to 0.94 g/l [3], within the range of our assay. Measuring the apo B content of VLDL is an acknowledged problem [3]. When we investigated this point by subfractionating hypertriglyceridaemic sera into large and small VLDL, the majority of apo B (85-S8%) was detected by the ELISA system (Table II). This level of detection is higher than that reported by Holmqvist [7]. Although Fievet et al [8] have reported equivalent reactivity of apo B from VLDL and LDL, the absence of data on this point in their publication means that comparisons cannot be made. TO recapitulate, a non-competitive ELISA has been developed for measuring human plasma apo B. It is based on a solid-phase antibody absorbed to the wells of microtitre plates. The assay is rapid, reproducible and sensitive and can be used both for experimental research and in routine clinical analyses. Acknowledgements We are very grateful to Lan Jornot and Dr. Blaise Martin for their help with the computer-based analyses mentioned in this report. We are also grateful to MarieClaude Brulhardt for the cholesterol analyses, Freda Ruinard for EIA measurements and Brigitte Wojtek for purification of apolipoproteins. The secretarial skills of Florence Kaempfen are also acknowledged. This study was supported by the Fonds National Suisse de la recherche scientifique, 3.808-0.83.
324
References 1 Avagaro P. Bittolo Bon G. Cazzolato G. Quince GB. Belussi F. Plasma levels of apolipoprotem A-l and apolipoprotein B in human atherosclerosis. Artery 1978; 4: 3855394. 2 De Backer G. Rosseneu M. Deslypere JP. Discriminative value of lipids and apoproteins in coronary heart disease. Atherosclerosis 1982; 42: 197-203. 3 Rosseneu M, Vercaemst R. Steinberg KK. Cooper GR. Some considerations of methodology and standardization of apolipoprotein B immunoassays. Clin Chem 1983: 29: 427-433 4 Laurel1 CB, McKay EJ. Electroimmunoassay. Meth Enrymol 1981: 73: 3399369 5 Fruchart JC. Desreumaux C. Dewailly P et al. Enzyme immunoassay of human apolipoprotem B, the major protein moiety in low-denaltyand very-low-density lipoproteins. Clin Chem 1978: 24: 4555459. 6 Vander Heiden GL. Saase EA. Yorde DE, Madiedo G, Barboriak JJ. Examination of a competitive enzyme-linked immunoassay (CELIA) technique and a laser nephelometric immunoassay technique for the measurement of apolipoprotein B. Clin Chim Acta 1983: 135: 209-218. 7 Holmqvist L. Quantitation of human serum apolipoprotein B by enzyme immunoassay. Clin Chim Acta 1982; 121: 3277336. 8 Fievet C, Koffigan M. Quvry D. Marcovina S. Moschetto Y. Fruchart JC. Noncompetitive enzymelinked immunoassay for apolipoprotein B in serum. Clin Chem 1984; 30: 988100. 9 Fruchart JC. Fievet C. Ouvry D et al. Enzyme-linked immunoassay on microtitre plates for human apolipoprotein B. Ric Clin Lab 1984; 14: 5699574. 10 Lowry OH. Rosebrough NJ. Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951: 193: 2655275. 11 Kane JP. Sata T. Hamilton RL, Have1 RJ. Apoprotein composition of very low density lipoproteins of human serum. J Clin Invest 1975: 56: 1622-1634. 12 Rodbard D, Hutt D. Statistical analysis of radioimmunoaasays and immunoradiometrtc (labeled antibody) assays. In: RIA and related procedures in medicine. Vienna: International Atomic Energy Agency. 1974: 1655192. 13 Lindgren FJ. Jensen LG. Hatch FT. The isolation and quantitative analysis of serum lipoproteins. In: Nelson GJ. ed. Blood lipids and lipoproteins: quantitation. composition, and metabolism. New York: Wiley, 1972: 181-274. 14 Steinberg KK. Cooper GR, Gratser SR, Rosseneu M. Some constderations of methodology and standardization of apolipoprotein A-I immunoassays. Clin Chem 1983; 29: 415-426. 15 Shelburne FA, Quarfordt SH. The interaction of heparin with an apoprotein of human very low density lipoprotein. J C’lin Invest (1977): 60: 944-950.
317
CCA 03296
Brief technical
note
A non-competitive enzyme-linked immunosorbent assay for measuring human plasma apolipoprotein B levels Richard
W. James *, Manuela
Dit$sionde Diabktologie,
Ferrer and Daniel Pometta
LQpurtement de MtLdecine, Kipitai
(Received
November
Cantonal de Gemhe, 121 I GE 4 (Switzerland1
9th, 1984; revision June 14th, 1985)
Key words: Apolipoprotein B; Low density lipoprotein; Enzyme-linked
immunosorbent
asmy
The need for precise measurements of plasma levels of apolipoprotein B-100 (apo B), the structural protein component of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) has been accentuated in recent years by suggestions that they may be superior to plasma or LDL cholesterol levels as an indicator for the risk of cardiovascular disease [1,2]. As pointed out in an excellent review by Rosseneu et al [3), quantification of apo B is fraught with numerous technical problems, and no single assay procedure has met with universal approval. The reference procedure, electroimmunoassay (EIA [4]), is not a practical proposition for routine clinical analyses. The merits of the enzyme-linked immunosorbent assay (ELISA) system have been advocated in many publications. Five ELISA systems for quantifying apo B have been described. Of these, two are based on competition assays [5,6] which appear less reproducible than the non-competitive ELISA, and require purified apo B or LDL. A third requires an LDL-enzyme complex, which somewhat precludes its use for routine analyses [7]. Fievet et al [8] have described a non-competitive ELISA using antibody-coated polystyrene balls, which has recently been extended to microtitre plates [9]. Given the advances in technology for automation of ELISA using such plates, this would appear to offer the most suitable technique for routine clinical analyses. Here we describe just such an approach. Materials and methods Preparatory of antigen A narrow density LDL fraction (d = 1.04-1.052 kg/l) was prepared by flotation ultracentrifugation of human plasma obtained from the Blood Transfusion Centre of the Geneva Cantonal Hospital. * To whom correspondence ~9-8981/~5/$03.30
should be addressed.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
The apo B protein content of LDL was measured by the Lowry method [IO] ah the tetramethylurea (TMU) precipitable fraction [ll]. A factor of 0.9 was emploved to correct for the difference in ~hromogeni~ity of BSA and ape B I-?].
Antiserum was raised in rabbits against LDL (d= 1.04-1.052 kg/l). The whole IgG fraction was obtained by precipitation with ammonium sulphate (277 g/l) and redissolved in phosphate buffered saline (PBS, phosphate buffer 10 mmol/l. pH 7.4. sodium chloride 137 mmol/l. potassium chloride 2.7 mmot/l. sodium azide 3.1 mmol/l). Monospecific anti-apo B antibodies were obtained by affinity chromatography as follows. An affinity column was prepared by covalently linking LDL (d = 1.04- 1.052 kg/l, 10 mg) to cyanogen bromide-activated Sepharose 4B (Pharmacia, Switzerland). The whole IgG fraction was passed twice down the column and. after extensive washing, specifically adsorbed antibodies were eluted with potassium thiocyanate (3 m&/l) in PBS. The antibodies were dialysed against ammonium bicarbonate, 50 ntmol/l, pH 8.0, then lyophilised. Preparation ofuntibody-enzyme conjugate Monospecific anti-apo B antibodies were conjugated to alkaline phosphatase (Sigma, USA) by the glutara~dehyde method. Briefly, antibodies (0.4 mg) were mixed with alkaline phosphatase (0.25 mg) and dialysed against PBS. The volume was made up to 1.0 ml and glutaraldehyde added to a final concentration of 2 ml/l. After thoroughly mixing, the reaction was allowed to continue for 2 h at room temperature. Subsequently the reaction mixture was dialysed at 4°C against PBS (2 x 500 mi) and Tris buffer (Tris 50 mmol/l. pH 8.0, magnesium chloride 1.0 mmol/l, 2 x 500 ml). The dialysate was diluted to 4 ml in the same buffer containing BSA (10 g/l) and azide (3.1 mmol/l) for storage at 4°C. Enzyme-linked immunosorhent assa_y The following protocol was adopted. Microt~tre piates (Mi~roelisa-plates. Dynatech, Switzerland) were coated during overnight incubation (4°C) with monospecific anti-apo B IgG (0.1 ml; 5 mg/l in carbonate buffer, 50 mmol/l, pH 9.6). Remaining absorption sites were blocked with BSA (10 g/l in the same buffer; 37’C for 1 h) and the wells then washed with PBS containing Tween 20 (0.5 ml/l). The solid-phase antibody was subsequently incubated for 3 h with appropriately diluted standards or test solutions (0.1 ml in wash buffer containing BSA 10 g/l), washed thoroughly and incubated a further 2 h with antibody-enzyme conjugate (0.1 ml of a 1 : 100 dilution in same buffer). Bound conjugate was visualized by adding 0.1 ml of substrate solution ( p-nitrophenyl phosphate; 1 tablet for 5 ml of buffer, diethanolamine, 1 mol/l, pH 9.8 containing 0.5 mmol/l magnesium chloride). The reaction was allowed to continue for 30-45 min, giving a maximal absorption of 1.5 units. before being quenched with sodium hydroxide (3 mol/l, 0.05 ml). to 2.0 Ad,)5 All incubations were effected at room temperature with continual rotary agitation (Vari-Shaker, Dynatech, Switzerland). The absorbances of the reaction mixtures were read with the SLT 210 microelisa
319
reader (Kontron, Switzerland). All results were subjected to computer analysis by the four parameter logistic curve fitting programme developed by Rodbard et al [12]. Fractionation
of lipoproteins
Lipoprotein density subclasses were separated by sequential flotation ultracentrifugation at densities of 1.006, 1.063 and 1.21 kg/l respectively. Subfractionation of VLDL into large (Sf 400-60) and small (Sf 60-20) VLDL, and preparation of intermediate density lipoproteins (IDL, Sf 20-12) were performed as described by Lindgren et al [13]. Purification
of apolipoproteins
Apolipoproteins A-I and A-II were isolated from delipidized high density lipoproteins by gel filtration chromatography [14]. Apolipoproteins E and C were purified from delipidized VLDL on an affinity column of heparin-Sepharose [15]. Results Optimization
of assay conditions
Preliminary studies established a suitable concentration for coating of the microtitre wells with affinity-purified antibodies (5 mg/l), and for dilution of the antibody-enzyme conjugate (1 : 100). The latter has proven to be remarkably consistent over six different preparations of conjugate. Analysis of different incubation times and temperatures for absorption of the solid-phase antibody showed that 16 h at 4°C was sufficient, Parallelism of titration curves was maint~ned when precoated plates were analysed after storage for 3 weeks in PBS (4°C). Validation of ELISA
system
A typical calibration curve established with plasma is shown in Fig. 1. As little as 2.5 ng of apo B can be detected, with a working range of ld to 100 ng. When the specificity of the antibodies was controlled, no appreciable cross-reactivity with apolipoproteins A-I, A-II, C, E or with albumin (not shown) was observed (Fig. 1). Whole serum was shown to give a curve parallel to that obtained with purified LDL (Fig. 2A). With VLDL, no parallelism was at first apparent as the dose-response curve consistently gave a plateau below that observed for serum and LDL (Fig. 2A) and attempts to redress the situation by pretreatment of VLDL with Tween 20 (4 ml/l), sodium dodecyl sulphate (50 mmol/l), Triton X-100 (4ml/l) or Nonidet P-40 (4 ml/l) were unsuccessful. When the absorbance values were normalized to a plateau value of 1.0, parallelism of all three curves was evident (Fig. 2B). quantification
of apo~ipoprotein B in serum samples
A secondary standard of whole plasma was used for routine measurements of apo B. The secondary standard was calibrated using the primary standard (LDL d = 1.04-1.052 kg/l), the protein concentration of the latter being established by the Lowry method as described in the Methods section. The secondary standard was stored in aliguot at - 70°C in the presence of phenylmethylsulphonylfluo~de. No
Plasma Cnl)
r
1
t
101
-I
t 102
105
104
ADO B (ncl)
Fig. 1. Calibration curve developed with plasma for measuring plasma apo B levets. The secondary u-hole plasma standard was calibrated using the primary LDL (d = 1.04-1.052 kg/l) standard and processed by the ELISA procedure described in the Methods section. Each point is the average of 6 determinations +SD. To test the effects of other apolipoproteins. increasing concentrations of the purified apolipoproteins replaced plasma in the first. 3 h incubation period. The microtitre plates were then processed by the usual procedure. Tr.kpoE: O0. Apu C; A--A. Apo A-1; .A, Apo A-II.
loss of immunoreactivity has been observed over 6 months, A aliguot is thawed prior to an analysis and subsequently kept at 4°C for 2-3 weeks. It is not refrozen. Apo B in LDL samples from plasma with a range of serum cholesterol concentrations (3.98-14.37 mmol/l) were quantified by ELISA, EIA and by the Lowry method, after precipitation with TMU. Excellent correlations between the values were observed, as shown by the Pearson correlation coefficients (Table 1). The same was also true when apo B values measured by ELISA were compared to the LDL cholesterol and total cholesterol values of the same samples (Table I). Apo B levels measured in whole plasma by ELISA and EIA were also compared and, as shown in Table I and Fig. 3A, very good agreement between the values was observed. Interestingly, higher Pearson correlation coefficients were observed when the LDL and total cholesterol values of these samples were compared to apo B levels (Table I and Fig. 3B). Quantification of apo B (triplicate determinations per point) in a series of normolipidic subjects (age range 24-70 years) gave an average value of 0.92 & 0.22 g/l (n = 41). The intra-assay and interassay coefficients of variation were 5.2% (n = 23) and 7.9% (n = 12) respectively.
321
s
2 5
9
101
1
102
103
104 Plasma, LDL hi)
Plasma. LDL hl) I
I
101
102
I
103
1
104
1
I
105
101
I
1
102
I
103
104
I
105
VLDL (nl)
VLDL (nlf
Fig. 2. Effects of plasma, LDL and VLDL in the apo B ELISA system. Various dilutions (100 ~1) of whole plasma, LDL and VLDL (prepared by sequential ultracentrifugation) were incubated (3 h, room temperature) with microtitre wells precoated with anti apo B IgG. The wells were subsequently processed by the standard procedure. A, absolute absorbance; B, absorbance values normalized to a plateau value of VLDL. 0, plasma; n -A, LDL; * -----a, 1.0. 0 -
Analysis of the ELISA system for its ability to quantify apo B in VLDL subfractions and in intermediate density lipoproteins (IDL) showed that the majority (85%-88%) of the apoprotein in both VLDL subfractions from hyperlipidaemic sera was measured (Table II). Similar observations were made for small VLDL from normolipidaemic sera, although only 73% of apo B in large VLDL from the
TABLE
I
Pearson
correlation
Compared
function
Apo B, EIA Apo B, TMU Cholesterol, Cholesterol,
LDL total
coefficients
for plasma
and LDL concentrations
of apo B and cholesterol
ELISA apo B LDL
Plasma
0.98 0.98
0.76 -
0.99 0.96
0.83 0.81
Apo B levels were measured by ELISA either in LDL (n = 19) obtained by preparative ultracentrifugation or in whole plasma (n = 28). These values were then compared to apo B values measured in the same fractions by EIA or TMU precipitation (LDL only), and LDL and total plasma cholesterol. All Pearson correlation coefficients were significant at p = 0.001. The regression equation for the comparison of serum apo B measured by ELISA and EIA was y = 0.006.x +0.12, with r = 0.76.
32’
TABLE
II
Ape H in VLDL aubfrncttons and IIIlI?~l. I~__ Serum
Suhfracrlcrn
Normal Hyperlipidaemic
VLDL Sf 400-60
VLDL Sf 60-20
IDL Sf 20-12
73.2 x7.7
89.9 x4.7
95.5 95,s
_..
-
VLDL subfractions and IDL were isolated as described in Methods. The apo B content was measured by ELISA and as TMU-precipitable protein and resuk are expressed as percentages: ELISA apo BiTMU ape B x 100.The results are averaged for 4 normal and 7 Ilyp~rlipidaen~ic sera
200 2 d t.2
m
$
100
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Apo B ELISA (g/l)
APO B ELISA (s/l)
Fig. 3. Comparison of apo B values measured in samples by ELISA with (A) apo B values measured by EIA and (B) total cholesterol levels. Twenty-eight samples with a range of apo B and cholesterol values were analysed. Apo B values measured by EIA are expressed in arbitrary units.
normolipidaemic sera was measured. serum was measured.
Over 95% of IDL apo B from both types of
Discussion
The criteria to be applied to an assay being considered for routine clinical analyses are precision, reproducibility, sensitivity and ease of application, the latter encompassing rapidity and the possibility of automation. The ELISA we have developed for apo B satisfies these criteria. The excellent correlations between plasma and LDL levels of apo B as measured by ELISA, EIA and TMU precipitation-the latter was not applied to plasma(Table I), testify to the precision of the ELISA. As corroborative evidence, there are also the excellent correlations between apo B levels measured by ELISA and the
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total and LDL cholesterol concentrations (Table I). Intra- and interassay coefficients of 5.2% and 7.5% respectively indicate that the procedure displays the
of variation
necessary reproducibility. The sensitivity of the assay (working range lo-100 ng) is equivalent to that of radioimmunoassay. Its high sensitivity could lead to imprecision, given that dilutions of serum samples up to 2000-fold may be necessary. However the coefficients of variation affirm the reproducibility of the assay, if the usual laboratory care is taken when diluting the samples. Less sensitive assays such as EIA and radial immunodiffusion necessitate lower plasma dilutions. However their potential for automation is limited, which is not the case with the ELISA system. Immunonephelometry offers a rapid, inexpensive means of measuring apo B, but suffers from the requirement for a similar size of lipoprotein for sample and standard, and dyslipoproteinaemic samples can seriously affect the results [3]. As regards rapidity, the ELISA allows apo B levels to be measured in 6 h, with the potential for extensive automation of the process. We have chosen to use a secondary standard of whole plasma to generate the calibration curve, as suggested by Rosseneu et al 131. The parallelism of the calibration curves for whole plasma and the primary LDL standard (Fig. 2A), and the excellent correlations between apo B values measured by ELISA, EIA and TMU precipitation support this approach. With the ELISA we observed an average concentration of apo B in normolipidic subjects of 0.92 g/l. Values reported in the literature for a variety of methodologies range from 0.8 to 1.59 g/l, whilst radioimmunoassay, which is of equivalent sensitivity, gave values ranging from 0.81 to 0.94 g/l [3], within the range of our assay. Measuring the apo B content of VLDL is an acknowledged problem [3]. When we investigated this point by subfractionating hypertriglyceridaemic sera into large and small VLDL, the majority of apo B (85-S8%) was detected by the ELISA system (Table II). This level of detection is higher than that reported by Holmqvist [7]. Although Fievet et al [8] have reported equivalent reactivity of apo B from VLDL and LDL, the absence of data on this point in their publication means that comparisons cannot be made. TO recapitulate, a non-competitive ELISA has been developed for measuring human plasma apo B. It is based on a solid-phase antibody absorbed to the wells of microtitre plates. The assay is rapid, reproducible and sensitive and can be used both for experimental research and in routine clinical analyses. Acknowledgements We are very grateful to Lan Jornot and Dr. Blaise Martin for their help with the computer-based analyses mentioned in this report. We are also grateful to MarieClaude Brulhardt for the cholesterol analyses, Freda Ruinard for EIA measurements and Brigitte Wojtek for purification of apolipoproteins. The secretarial skills of Florence Kaempfen are also acknowledged. This study was supported by the Fonds National Suisse de la recherche scientifique, 3.808-0.83.
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