Anal. Chem. 1992, 64, 1698-1701
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Automated Rate Nephelometric Determination of Apolipoproteins A I and B in Human Serum by Consecutive Addition of Antibodies Fotis K.Fotiou' Sanofi Diagnostics Pasteur, Inc.. Immunology Research and Development, 1000 Lake Hazeltine Drive, Chaska, Minnesota 55318
A preclse, rapld, automated, rate nephelometric Immunoassay for apollpoproteln A I (APA) and apollpoproteln B (APB) Is described. Both analytes are determlned by a "one-pot" procedurewhich uses consecutlveaddltknof the correspondIng antlbodles. Poly(oxyethylene) type nonlonlc surfactants are used to selectlvely lnhlblt the APB reactlon after the maxlmum reactlon rate has been reachedand to enhance the APA lmmunoreactlvlty. The assay range for APA Is 0.3-3.8 g/L and for APB 0.2-3.0 g/L. The new assay was compared to cmmerclal rate nephelometricmethodology. Both methods were shown to be mutually unblased for APA and APB determlnatlons In human serum.
INTRODUCTION Lipids play an important role in all aspects of biological life. They are transported to tissues and organs via micelles known as lipoproteins. The outer layer of lipoproteins contains polar compounds, such as proteins (apolipoproteins), cholesterol, and phospholipids, while the inner structure consists of triglycerides and cholesteryl esters. The lipoproteins are divided into different classes based on their density: chylomicrons, very low density lipoproteins (VLDL), lowdensity lipoproteins (LDL), intermediate-density lipoproteins (IDL) and high-density lipoproteins (HDL). Apolipoprotein AI (APA) is the major protein moiety of HDL, and apolipoprotein B is the major protein moiety of LDL and VLDL. The clinical utility of the measurement of APA and APB in assessing the risk for the development of coronary artery disease has been recently established. In particular, measurement of the APAIAPB ratio has been considered a more effective indicator than conventional lipid markers such as cholesterol and triglyceride measurements.192 Various immunological assays that measure the concentration of these analytes in human serum and plasma have been developed such as radioimmunoassay (RIA),electroimmunodiffusion (EID),radial immunodiffusion (RID),enzymelinked immunosorbent assay (ELISA), and immunonephelometry (INAl.1-5 Fruchard developed an EID for the simultaneous quantification of APA and APB by incorporating both antibodies in the agarose gel matrix.6 EIDs however, are not amenable to automation, and the analysis time is in the order of hours. In rate (kinetic) INA the maximum rate of change of scattered light intensity
* Present address: American Cyanamid, Agricultural Research Division, Analytical Physical and Biochemical Research Section, Princeton, NJ 08543. (1)Bachorik, P.S.;Kwiterovich, Jr. P. 0. Clin.Chim. Acta 1988,178, 1-34. (2)Naito, H. K.Ann. N.Y. Acad. Sci. 1985,454,230-238. (3)Labeur, C.; Shepherd, J.; Rosseneu, M. Clin.Chem. 1991,36,591597. (4)Bojanovski, M.; Gregg,R. E.; Wilson, D. M.; Brewer, Jr. H. B. Clin. Chim. Acta 1988,178, 159-170. (5)Albers, J. J.; Adolphson, J. L. J. Lipid Res. 1988,29,102-108. (6) Fruchard, J. C.; Kora, I.; Cachera, C.; Clavey, V.; Duthilleul, P.; Moschetto, Y. Clin. Chem. 1982,28,59-62.
in an antigen-antibody reaction can be made to occur within minutes after initiation of the reaction and provides a measure of the antigen concentration under antibody excess conditions. Moreover, rate INA assays are easy to automate. The objective of this report was to develop an automated rate INA that allows the detection of both apolipoproteins in "one-pot" with sequential addition of the corresponding antibodies. There are several advantages in quantifying two analytes of interest by a one-pot sequential immunoassay rather than by two separate assays. The analysis time can be significantly reduced, a smaller amount of sample is used, and there is a saving in the usage of buffers and other ancillary reagents. The use of nonionic surfactants to expose APA antigen determinants, masked with lipids, has been described in several reports.' It has also been reported that nonionic surfactants reduce the immunoreactivity in APB INA assays.839 We have used polyoxyethylene type nonionic surfactants (Brij 35,Tween 20, and Triton X100) to alter the APA and APB immunoreactivity in rate INA. Nonionic surfactants with poly(oxyethy1ene) head groups usually do not denature proteinslo and are commercially available in high purity a t low cost. In APA INA immunoreactivity is greatly enhanced while the APB immunoreactivity can be totally inhibited depending on the surfactant concentration. On the basis of this opposite effect, we introduce here a new approach for APA and APB quantification. This procedure allows the measurement of both apolipoproteins in a single rate INA by consecutive addition of the corresponding antibodies. Thus, APB is allowed to react with a monospecific anti-APB antibody and the rate of change of scattered light intensity is monitored. Once the maximum rate is reached, further reaction is inhibited by the addition of a nonionic surfactant. Moreover, the surfactant creates a matrix where APA immunoreactivity is enhanced. Anti-APA antibody addition follows and the maximum rate of change of scattered light intensity caused by the APA reaction is determined. The maximum rates of light scatter increase are correlated to the APA and APB concentrations in human serum.
EXPERIMENTAL SECTION Reagents. Buffer A, pH 7.2, consisted of phosphate salta (0.1 M) and sodium chloride (0.15 M) with sodium azide (0.1 g/L) as preservative. The reaction buffer (buffer B) consisted of phosphate buffer, prepared as above, and 40 g/Lpoly(ethy1ene glycol) (PEG) 6000 (Calbiochem San Diego, CA). Poly(ethy1ene glycol) is used to accelerate the antigen-antibody complex formation." (7) Maciejko, J. J.; Mao, J. S. Clin. Chem. 1982,1 , 199-204. (8)DaCol, P.; Kostner, C. M. Clin. Chem. 1983,29,1045-1050. (9)Hanson, N. Q.;LeGeault, T. S.;Freier, E. F. Clin.Chem. 1987,33, 1882-1884. (10)Helenius, A.; McCaslin, D. R.; Fries, E.; Tanford, C. Methods E n ~ y m o l1979,56, . 734-749. (11)Hellsing, K.Automated Immunoanalysis; Marcel Dekker: New York, 1978;Part 1. 0 1QQ2American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 84, NO. 15, AUGUST 1, 1992
Monospecific goat anti-human APA and goat anti-human APB antisera were obtained from Sanofi Diagnostics Pasteur, Inc. (Chaska, MN). The antisera, diluted in buffer A, were stored at 4 "C. Human serum samples were stored at 4 "Cup to 1month or at -20 O C up to 3 months prior to the analysis time. Commercial rate INA kits for the detection of APA and APB were obtained from Sanofi Diagnostics Pasteur, Inc. The kits consisted of antibody packs, controls, and a calibrator. Nonionic surfactants, Tween 20, Brij 35, and Triton X100, were obtained from Calbiochem (San Diego, CA). Dilutions of surfactants were made in buffer B and stored at 4 "C. Apparatus. A programmable automated rate nephelometer (QM300, Sanofi Diagnostics Pasteur, Inc.) was used to measure the rate nephelometric response and to quantify APA and APB. The reaction cells of the nephelometer are irradiated with a 660nm high-intensityLED, and the scattered light is monitored at a right angle. The analyzer was programmed in C to perform the combined APA and APB assay procedure. Procedures. The APA and APB concentrations of the samples were determined using the commercial APA and APB assays on the QM300nephelometer followingthe manufacturer's instructions. The analyzer was programmed to perform the combined APA and APB assay in six steps: (1)the sample was prediluted 1/36 (v/v)in buffer A by the analyzer dilutor; (2) 35 pL of the diluted sample, 500 pL of buffer B, and 35 pL of the APB antibody solution were delivered into the reaction cell; (3)the solution was mixed continuously,and the rate of scatter change was monitored; (4)as soon as the maximum reaction rate was reached, 35 pL of surfactant solutionwas injected to inhibit further APB reaction; (5) 35 p L of the anti-APA antibody was added next, and the rate of scatter change due to the APA reaction was monitored until its maximum value was reached; (6) the solution was allowed to flow to waste. Calibration Curves. To construct calibrationcurves, serial dilutionsof the commercial calibratorserum (Sanofi Diagnostics Pasteur, Inc.) were prepared in buffer A and assayed with the combined assay format. The maximum rates for the APA and APB reactions were determined and the results were fitted to a fourth degree polynomial equation. The calibration range was 0.3-3.8 g/L for APA and 0.2-3.0 g/L for APB. Precision Study. The assay precision was tested using three human serum samples with 0.61, 1.62, and 2.08 g/L APA values and 0.59,0.64,and0.88 g/L APB values. Between assay precision was determined by assaying each sample over a period of 7 days. Within assay precision was determined by performing 10separate assays of each sample in the same day. Recovery Study. A human serum sample was diluted gravimetrically at compositions ranging from 90% down to 10% in buffer A. The APA and APB content of each dilution was quantified. Correlations. Twenty three human serum samples with APA levels ranging from 0.3 to 2.3 g/L and APB levels ranging from 0.3 to 1.3 g/L were assayed using the combined APA and APB procedure. The same samples were also assayed with the commercial kits. In all cases single determinations were performed.
RESULTS AND DISCUSSION The nonionic surfactant level in the reaction matrix is an important variable in the development of the APA and APB immunoassays. Figure 1A shows the maximum value of the APA/anti-APA reaction rate (maximum change of scattered light intensity over time, R,,) as a function of the surfactant (Tween 20, Brij 35, and Triton X100) concentration. The value of R , increases with surfactant level. The highest R,, value was obtained when the surfactant concentration was approximately equal to the critical micelle concentration (cmc) and remained unchanged with an additional amount of surfactant. Surfactants replace lipids in the vicinity of apolipoprotein portion of the lipoprotein particles. APA epitopes masked with lipids become accessible to the antiAPA antibody. Furthermore, the poly(oxyethy1ene) portion of the surfactant creates a hydrophilic network on the apolipoprotein particle which competes with the anti-APA
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Flgure 1. (A) Effect of the concentration of surfactantson the maxlmum reactlon rate (R,) of the APA reaction (APA concentration: 2.08 g/L). (E) Inhibition effect of surfactants on APB reaction (APE concentration: 0.87 g/L).
interaction. R, values are higher when Tween 20 is used instead of Brij 35 and Triton X100. Brij 35 is linear in shape and has a smaller head structure than Triton Xl00 and Tween 20. It is probably more effective in coating the APA particle and affectingadversely the APA immunoreactivity. Similarly, Brij 35 was observed to be a more efficient masking agent of the silanol groups in capillary electrophoresis than the Tween series of surfactants.l2 Triton X100, having a linear poly(oxyethylene) chain, would be more efficient than the branched Tween 20 in coating the APA particle. In contrast to the favorable effect on the APA reaction, the APB reactivity is significantly reduced when the same surfactants are present, as shown in Figure 1B. This seems to corroborate that, under our experimental conditions, changes in the physicochemical properties of APB occur which affect the binding characteristics with the anti-APB antibody. APB is a large protein having a molecular mass of about 400 OOO Da and a marked insolubility in aqueous solutions in the absence of denaturants or amphiphiles. Its physical, biologic, or immunologic properties strongly depend on the composition of the APB solution. For example it has been shown that when the lipids of the spherical LDL are replaced by poly(oxyethylene) surfactants (Triton X100) the Triton-APB product becomes very asymmetric, having hydrodynamic properties different from LDL, and that under certain conditions aggregation of APB can be promoted.13 Tween 20, a t the 0.1% level, was selected for the development of the combined immunoassay. The effect of Tween 20 addition during the course of the APB reaction is illustrated in Figure 2. The scattered light intensity stops increasing (Figure 2A), and the nephelometric reaction rate intensity (12)Towns,J. K.;Regnier, F. E. Anal. Chem. 1991, 63, 1126-1132. (13)Patterson, B.W.;Kilgore, L. L.; Chun,P.W.J . Lipid Res. 1984, 25, 763-769.
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ANALYTICAL CHEMISTRY, VOL. 84, NO. 15, AUGUST 1, 1992 Surfactant
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Figure 3. Calibration curve for APA in human serum in the presence of 0.1 % Tween 20.
Table I. Precision Data concn
I
APA
I
APB
I
(dL)
between rune RSD (%)
within run RSD (%)
0.62 1.62 2.08 0.59 0.64 0.88
4.3 3.2 3.1 4.9 4.0 3.7
3.7 2.2 2.6 3.8 2.8 2.4
Table 11. Human Serum Dilution Recovery Study I '.;
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150
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% recovery
100 90
208 188 160 142 127 101 86.1 60.3 39.4 19.5
100 96 98 102 97 103 97 95 94
80
1
50
dilution factor (%)
200
TIME (SI Figure2. (A) Evolution of the scattered llgM intensityduring the reaction of APB (0.87 g/L) In human serum wlth the antCAPB antibody. (B) Evolution of nephelometrlc rate durlng APB reaction.
becomes zero (Figure 2B). In the example shown,surfactant had been reached. The sharp was added after the R, decrease of the scattered light intensity during the addition of surfactant, shown in Figure 2A, is due to the volume increase and the dilution of the scattering particles and complexes. In most cases, the scattered light intensity remained either unchanged or slightly decreased. This indicates that only a small fraction of the alreadyformed APB/anti-APBcomplexes may dissociate when surfactant is added. The small decrease of scatter appears to be linear, and it is not expected to interfere with the subsequent APA R,, determination because it produces a zero rate. In the combined assay, surfactant is added within 3-4 s after the APB R, is reached, followed by immediate addition of the anti-APA antibody. The analysis time for the quantification of both antigens, by determining the R,, values, was typically less than 3 min. The relation between the APA concentration in the sample, from 0.3 to 3.8 g/L, and the R, is sigmoidalin shape (Figure 3). R,, increased in proportion to the increase in APA concentration. It gradually became saturated above 4.5 g/L, reaching equivalence,14 and fully saturated above 6.0 g/L. The APB calibration curve was also sigmoidal, as that of APA, showing gradual saturation above 3.0 g/L. The calibration curves can be shifted toward higher or lower APA and APB values by increasing or decreasingthe concentration of the corresponding antibody. (14) Whicher, J. T.; Price, C. P.; Spencer, K. CRC Crit.Rev. Clin.Lab. S C ~1983, . 18,213-260.
70 60 50 40 30 20 10
(g/L)
% recovery
87.5 77.3 71.2 59.2 50.2 41.9 34.0 27.6 19.1 9.8
98 102 97 95 96 97 105 109 112
100[APBl
Concentrations outside the calibration curve range are easily determined by adjusting appropriately the sample predilution in buffer A, which in our protocol was 1/36-fold.Thus, the detection limit can be further reduced if needed, by using less dilute serum. The limiting factors are the light intensity scattered by the sample (blank), which may saturate the detector, and any inherent agglutination rate of the sample in the poly(ethy1ene glycol) matrix of the reaction buffer B,14 which may interfere with the R, determination. The assay precision was assessed in the range of human serum concentration, which is approximately 1.0-1.5 g/L for APAand O.&l.Og/L for APB.15 Three human serum samples with high, normal, and low APA levels and high and normal APB levels were assayed. The APA between and within assay precisions for these samplesranged from 2.2 % to 4.3 % (Table I). Similarly, the APB assay showed good precision, which ranged from 2.4 % to 4.9% (Table I). Dilution recovery results are depicted in Table 11. The diluted sample results could be recovered after analysis with sufficient accuracy. An essential hypothesis of the combined assay format is that the R,, value of the APA reaction is not affected by the previously measured APB reaction, since the latter is inhibited. Moreover, the APA reaction is measured in a turbid matrix due to the already formed APB/anti-APB complexes. Scattered light intensity increased as much as %fold after APB was quantified. This increase represented only approximately 6% of the overall linear range of the photodiode (15)Naito,
H.K.J. Clin. Immun. 1986, 9,11-20.
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detector in the system used. Typical APA reactions contributed to further increases of the scattered light up to 20% of the linear range, and therefore signal saturation was not observed. Another possible interference which would result in incorrect APA determination is the possibility of the two antibodies to cross-react. In this case the APA rate nephelometric response would include a contribution from the crossreacting anti-APA/anti-APB antibodies and a contribution from the APAIanti-APA reaction. In order to demonstrate that neither the APB reaction nor the APB antibody itself interfere with the APA result, the following sequence of experiments was performed. First, 23 human serum samples were analyzed using the combined assay format. Next, the APB antibody vial was replaced with a vial containing buffer A and the APA assay was repeated without the presence of the APB complexes and of unreacted APB antibody. Both data sets were in excellent agreement, and it is demonstrated that the APA result was determined without interference from either the APB antibody or the turbid baseline. Linear regression analysis between the data produced the following equation: Y = 0.980X 0.4 X (r2= 0.980). The data differences (bias) are plotted in Figure 4A. A two-tailed statisticaltest (t-test)at the 0.05 level of significance indicated that the null hypothesis of the bias being equal to zero cannot be rejected. It was therefore concluded that both methods are mutually unbiased. The same samples were also analyzed with well-characterized commerical kits, and the results were in agreement. Linear regression analysis of the APA data obtained by the described combined assay and by the commercial kit gave the following equation: Y = 0.943X + 5X (r2= 0.951). The linear regression equation for the APB assay was Y = 1.038X - 3 X (r2 = 0.989). Bias plots of the data for both assays are shown in Figure 4B,C. Again, a two-tailed t-test at the 0.05 level of significance indicated that the sequential assays and the commercial kits are also mutually unbiased. Furthremore, a 40% reduction in the analysis time was observed with the combined assay format over separate APA and APB determinations. It should be noted that correlations were performed with commercial kits operating under the same principles (rate nephelometry) and using the sabe reagents to limit the number of unrelated variables and to clearly demonstrate that the combined assay format provides the same results as assays determining APA and APB separately. The principle of the combined assay, the enhancement of the APA reaction and inhibition of the APB reaction with nonionic surfactants,has been observed by other researchers7-9 under experimental conditions and with antisera different from those used in our study. Therefore, it is expected that this assay could be easily adapted to other automated programmable commercial INA immunoanalyzers using different reagents and experimental protocols from those described here. In summary, an automated, simple, and precise rate nephelometric assay was developed that allows the detection of both APA and APB by consecutive addition of monospecific antibodies. The assay has been shown to yield equivalent results with separate determinations of APA and APB. The principle of selectively quenching the first immunochemical reaction after the first antigen has geen quantified and of subsequently measuring a second antigen in the same matrix could be applied to other analytes of interest in biological samples. The described assay can significantly shorten the analysis time and is also useful when only a limited amount
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Flgure 4. (A) Combined assay format: APA result differences (bias) wlth and without anti-APB present. (B)APA bias of results obtalned with the comblned assay and results obtalned using a commercial klt. (C) APB bias of results obtalned with the combined assay and results obtained with a commercial klt.
of sample is available (for example pediatric blood samples).
ACKNOWLEDGMENT We express our appreciation to Margaret Piper and Kathy Horsfall for the support during the development of this work, to Benton McMullen for his assistance in the development of software, and to Richard Creager for the critical reading of the manuscript and his helpful suggestions and comments.
RECEIVED for review February
10, 1992. Accepted April
23, 1992. Registry No. PEG 6000,25322-68-3;TWEEN 20,9005-64-5; BRIJ 35, 9002-92-0; TRITON X100, 9002-93-1.
