Anel. Chem. 1999, 65, 613-616
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Liquid-Phase Binding Assay of Human Chorionic Gonadotropin Using High-Performance Liquid Chromatography Kenji Nakamura, Shinji Satomura,' and Shuji Matsuura Osaka Research Laboratory, Wako Pure Chemical Industries Ltd., 6-1 Takada-cho Amagasaki, Hyogo 661, Japan
A new enzyme immunoassay technlque (LBA Ilquld-phase blndlng assay) to examlne the characteristicsof Ilquld-phase antigen-antibody reactions k described. Antlgen (human chorknk gonadotropln: h a ) and peroxidase(POD)-labeled antl-hCQ monoclonal antlbody (Fab'-POD) oolutlons were mbced, Incubated, and analyzed directly by gel flltratlon high performancellquld chromatography with postcolumn enzyme actlvlty measurement. Using the system, bound (hCG-Fab'POD) and froe (Fab'-POD) forms of enzyme-iabeled antlbody WM separated by thelr molecular m a r dMerence, and the POD actlvlty d the ConJugatewas determined fluorophotomddcaWy. All analytos becameboundupon addl#onof excess Fib'-POD, becauu reactioncondltlonsIn a llquld phase could bo easlly altered. Thus, hCG moleculecould be measuredvla the actlvlty d one POD molecule. And the Ilquhlphase antibody roactlon was very fast and quantitattve. On the bask of thk stdchlometrlc relatlonrhlp, equlllbrlum and rate constantr, optimum pH, and tmperature effects were eadly examined. The method k simple and convenient for examInatlon of the antigen-antlbody reaction and Is appncable for antigen assays requlrlng an accurate deflnltlon of concentratlons.
performed via gel filtration.6 Properties of an anti-human albumin monoclonal antibody were examined spectrophotometrically without the need for both a solid phase and an immobilization technique. However, the sensitivity of the spectrophotometric method was not sufficient to investigate additional properties of the antibody. The present study employed enzyme-labeled antibody and a postcolumn fluorophotometric detection in combination with the gel-filtrationtechnique to get higher sensitivity.Using the sensitive technique, rate and equilibrium constants, optimum pH, effects of temperature, and other characteristics of an anti-hCG monoclonal antibody were investigated.
Specific binding reactions of monoclonal and polyclonal antibodies have been applied frequently for many assays. Characteristics of these binding reactions have been measured by various methods. Antibody specificities have been well studied, but examination of rate and equilibrium constants is rarely done. The most commonly used method in this field is equilibrium dialysis, but it can only be used for hapten antigens.' Immunoaffmity chromatography and immunoprecipitation were used for protein antigens, but these methods were semiquantitative and tedious and could not determine binding rate ~onstants.2~3A more convenient method is needed to accurately measure binding rate constants and other parameters of the antigen-antibody reaction. High-performance liquid chromatography (HPLC) can rapidly and accurately separate protein and thus can be used for protein measurements. A HPLC method was combined with competitive immunometric binding in an insulin assay.4 However the labeled insulin-antibody complex formed in the method did not elute out from the column. Thus, the method could not be used for investigation of antibody binding parameters. In our previous study, separation of bound antibodyprotein antigen from free antibody (B/Fseparation) was
EXPERIMENTAL SECTION Materials. Anti-hCG monoclonal antibodies (Clone No. 2530 and B-101) were selected from our panel of antibodies and purified. These antibodies recognize different hCG epitopes. Immunoglobulin G (Clone No. 25-30) was digested with pepsin followedby reduction of F (ab%to form Fab'. This single binding site fragment (Fab') was conjugated to peroxidase (POD, Grade I-C, Toyobo, Osaka, Japan.) by a sulfo-SMCC method (Pierce, Rockford, IL).6 The Fab'-POD was homogeneous as judged by HPLC using gel filtration. The Fab'-POD concentration was determined by ita POD enzyme activity. A hCG was used having a specific activity of 10 mIU/ng (National Institutes of Health, CR-123). Diol-200 gel filtration columns (8-mm i.d. X 300 mm and 4.6-mm i.d. X 600 mm) were obtained from YMC Co. (Kyoto, Japan). An ELISA kit for hCG assay was obtained from Mochida Co. (Tokyo, Japan). Other reagents used were reagent grade and were manufactured by Wako Pure Chemical Industries,Ltd., Osaka, Japan. Apparatus. A Shimadzu Model LC-6A HPLC system (Shimadzu Co., Kyoto, Japan) fitted with a postcolumn flow-through coil (0.25-mm i.d. X 9 m) was used. A short column (8-mm i.d. X 300 mm) was used with an elution buffer of 50 mM phosphate buffer, pH 7.6, containing 150 mM sodium chloride and 10 mM 3-@-hydroxyphenyl)propionic acid (HPPA,fluorogenicsubstrate for POD) at the flow rate of 1.0 mL/min.' The reaction reagent consisted of 150 mM sodium chloride and 20 mM HzOz in 50 mM phosphate buffer, pH 7.6. After column separation, reaction reagent was added to the effluent at a flow rate of 0.1 mL/min and mixed well. The mixture (the total flow rate. 1.1mL/min) was then incubated in the long coil at 55 O C for 25 s to allow POD enzymatic reaction. When a longer column (4.6-mm i.d. X 600 mm) was used, the effluent flow rate, the reaction reagent flow rate, and the total flow rate and the reaction time were 0.5,0.05, and 0.55 mL/min and 50 8, respectively. For the molecular mass calibration of the system, standard proteins (glutamate dehydrogenase, 290 000 g/mol; lactate dehydrogenase, 142 OOO g/mol; enolase, 67 000 g/mol; adenylate kinase, 32 OOO g/mol;cytochrome c, 12 400 g/mol) were measured spectrophotometrically at 280 nm using the same conditions free from both HPPA and HzOz. A linear relationship exists between the elution volume and the log molecular mass from 12 400 to 290 OOO g/mol. Figure 1shows
(1)Fujio, H.; Karush, F. Biochemistry 1966,5, 1856-1863. (2) Sportsman, J. R.; Liddil, J. D.; Wilson, G. S. Anal. Chem. 1983,55, 771-775. (3) Yagisawa, S.; Tanimori, H.; Kitagawa, T. J. Biochem. 1986, 99, 793-802. ~. (4) Lidofsky, S. D.; Imasaka, T.; Zare, R. N. Anal. Chem. 1979, 51, 1602-1605.
(5) Nakamura, K.; Satomura, S.; Tanaka, T.; Matauura, S. Anal. Sci. 1992,8,157-160. (6) Imagawa, M.; Yoshitake, S.; Hamaguti, Y.; Ishikawa, E.; Niitau, Y.; Urushizaki, I.; Kanazawa, R.; Tachibana, S.; Nakazawa, N.; Ogawa, H. J. Appl. Biochem. 1982,4,41-57. (7) Zaitsu, K.; Ohkura, Y. Anal. Biochem. 1980,109, 109-113. (8)Smith, T. W.; Skubitz, K. M. Biochemistry 1975, 14, 1496-1502.
INTRODUCTION
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Flow through Coil
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Figw 1. Schematic diagram of the HPLC system.
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Course of the immunoreaction. The course of the formation of the immune complex was examined using various concentrations of hCG: (0)11 pM; (0)28 pM; )(. 55 pM; ( 0 )110 pM; (A)220 pM; Flgwe3.
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Time ( min ) Flguro 2. Chromatographic pattern of the reaction product: (A) Fab’POD; (6)reaction mlxtwe of 138 pM Fab‘900 and 120 pM hCG. Chromatographic conditions: column, YMC diol-200 (&mm 1.d. X 300
mm); effluent flow rate, 1 mL/min; reaction reagent flow rate, 0.1 mL/min.
a scheme of the experimental apparatus. The activity of POD in the effluent was determined fluorophotometrically using an excitation wavelength of 320 nm and an emission wavelength of 404 nm. Characterization of Fab’-POD. For immunoreaction analysis, 40 pL of Fab’-POD solution and 20 pL of hCG solution in buffer A (50 mM phosphate buffer, pH 7.6, containing 150 mM sodium chloride and 0.2% bovine serum albumin) were mixed well and reacted at 30 OC for 60 min. Then, 50 p L of the reaction mixture was subjected to HPLC using an 8-mm4.d. X 300-mmlong column. The immune complex was calculated from ita peak height on the chromatogram. Assay of Serum hCG. For the assay of serum hCG, 40 p L of Fab’-POD (Clone No. 25-30) and 40 pL of immunoglobulin G (Clone No. B-101) solution were mixed with 20 pL of serum and incubated at 30 “C for 60 min. Then, 70 p L of the reaction mixture was injected into a YMC-diol column (4.6-mm4.d. X 600 mm) with a flow rate of 0.5 mL/min.
RESULTS Chromatograms of Immunoreaction Products. A mixture of 136 pM Fab’-POD solution and 120 pM hCG solution was incubated at 30 OC for 60 min. A 50-pL aliquot of the mixture was then resolved by HPLC (Figure 2). When only Fab’-POD solution was injected into the column, only one peak (at 12.3 min) was found (Figure 2A). When the reaction mixture was applied, a new peak appeared (at 10.9 min) having a larger molecular mass than Fab’-POD (Figure 2B). And the new peak was the hCG-Fab’-POD complex
(immune complex). The molecular mass of the immune complex was determined to be 130 OOO g/mol by a calibration curve derived from standard proteins with the same system. hCG and Fab’-POD were found to have molecular masses of 40 OOO and 90 OOO g/mol, respectively, using the same method. The purity of Fab’-POD measured as antibody activity was calculated to be 93 7%. Thus, 7 5% of eluted Fab’-POD did not react with addition of a large excess of hCG. The concentration of Fab’-POD solution used in subsequent experiments was modified to correct for the apparent 7% inactive antibody. The total time needed to separate bound and free forms of antibody-protein in the mixture by gel filtration was less than 20 min. Immunoreaction Time Course and Equilibrium Constant. The time course for formation of immune complex was examined using various concentrations of hCG (11-548 pM) and 136 pM Fab’-POD in the reaction mixture. All immunoreactions reached equilibrium within 60 min. The quantity of each formed immune complex was determined from the equilibrium state, as shown in Figure 3. The equilibrium constant for the reaction was calculated from the data shown in this figure. A Scatchard plot of bound/ free VB bound fitted a straight line (data not shown). Because the x intercept of the Scatchard plot was 131pM, the number of Fab’-POD molecules bound per hCG molecule was 0.96 (131 pM/136 pM). This result shows that only one epitope of hCG was recognized by this antibody. The equilibrium constant was calculated to be 3.7 X 10’0 M-1 from the slope of the Scatchard plot. Determination of Rate Constant of Immunoreaction. In Figure 3, the initial immunoreaction rate was increased with hCG concentration in the mixture. The rate constant for formation of the antibody-antigen complex in buffer solution can be calculated easily by the present method. Let [Agl be the molar concentration of unbound (free) hCG. [Ab] is the molar concentration of unoccupied Fab’-POD, and [AgAb] is the molar concentration of bound hCG that is equal to the occupied Fab’-POD. The formulas for this reaction are
-d[Abl/dt = k[Ab][Ag]
(2)
-d [Ab] /dt = MAgl [Ab]
(3)
where k is the rate constant. The concentration of [Agl, [Ab],
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hCG ( PM ) Fbure 4. Determlnatlon of the immunoreactlon rate constant. The rate constant (k) Is calculated from the slope of the line.
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Time ( min ) Figure 6. Course of the immunoreactlon at various temperatures: (0) 4 O C ; (0)30 OC; )(. 37 O C ; (0) 50 OC. Immunoreactionswere carrkd out using 136 pM Fab'-POD and 110 pM hCG.
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and [AgAbl in the reaction mixture were the values at 4 min, as shown in Figure 3. Figure 4 shows the plot of eq 3. The rate constant k was calculated to be 9.3 X 108 M-1 min-1 by the slope of eq 3 in Figure 4. Effect of pH on Immunoreaction. The optimum pH for binding of the anti-hCG Fab'-POD was found to be pH 9.0 by measuring the amount of immune complex formed in 60 rnin from 102 pM Fab'-POD and 322 pM hCG in the reaction mixture (Figure 5). Decreases of POD activities at several pHs were corrected using known residual activities of intact POD at the same pH. Amounts of immune complex did not increase at extreme pH even when the reaction time was prolonged up to 90 min in spite of the existence of excess hCG. We chose pH 7.6 for the assay, because POD enzymatic activity decreased above pH 8.0. Effect of Temperature. Figure 6 depicta the immunoreaction time course for several temperatures using 136 pM Fab'-POD and 110 pM hCG. The initial immunoreaction rate did not differ between 30 and 50 OC but was lower at 4 "C. The reaction temperature influenced the amount of immune complex at equilibrium, with an increase in the immune complex with a decrease in temperature. This relationship between the amount of immune complex formed and reaction temperature was linear (Figure 7). The effect of temperature on POD activity was taken into account by correction using the free POD activity obtained at each temperature. In order to elucidate this phenomenon, the reaction temperature was cycled alternately between 30 and 50 OC. The amount of immune complex changed reversibly by this treatment (Figure 8). The experiment was repeated using various column temperatures between 20 and 30 "C, but the same results were obtained.
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Temperature ( "C) Figure 7. Effect of temperature on the amount of immune complex. Fab'-POD (136 pM) and hCG (100 pM) were allow to react for 120 mln at 4 and 10 O C or for 60 min at 20, 30, 40, and 50 O C .
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Time ( rnin ) Flgure 8. Change in the amount of immune complex by cycling of the temperature: (0)immune complex; (0)free form of Fab'90D. Fab'POD (136 pM) and hCG (100 pM) were allowed to react at 30 and 50 O C , repeatedly.
Assay of hCG in Human Serum. The m a y method (LBA)was applied to the measurement of human serum hCG. Separation of bound from free forms of Fab'-POD was more complete because of the addition of a second anti-hCG monoclonal antibody to the reaction mixture (Figure 9). The amount of the bound form was calculated from ita peak height on the chromatogram. Figure 10showsa dose-response curve for the hCG assay with 250 pM Fab'-POD and 600 nM second antibody concentrations. This curve indicates a linear relationship between peak height and hCG concentrations below 200 mIU/mL (500 pM) in the sample and is similar to the ideal curve obtained by calculation from the equilibrium
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so far reported were determined to be from lo6to 10s M-’ by
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Time ( min ) Fbur 0. Chromatographicpattern of the reaction product: (A) without hCQ (B) reactionmlxture. Chromatographkconditions: column, YMC dioC2OO (&mm i.d. X 300 mm); effluent flow rate, 1 mL/mln; reaction reagent flow rate, 0.1 mL/mln. Fab’-POD (136 pM); IgG (600 nM), and hCG (100 pM) were allowed to react at 30 ‘C. I
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Flgurr 10. Dose response of hCG. hCG concentration: (A) 0-200 mIU/mL; (B) 0-1000 mIU/mL.
constant. The hCG detection limit was 1mIU/mL (2.5 pM) in the sample with a signal/noise ratio of 5 (S/N ratio: signal peak height divided by base line noise height). The withinassay reproducibilities for six different hCG concentrations (5,9,21,42,77,102mIU/mL)in humanserumwereexamined. The within-assay coefficients of variation (CVs) ranged from 1.596 to 3.2 96 We measured various concentrations of serum hCG using both LBA and a commercially available ELISA kit. The data obtained using both LBA and the ELISA kit showed good correlation (r = 0.984, 24 samples).
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DISCUSSION Gel filtration by HPLC separated formed immune complex from free forms of Fab’-POD. The amount of immune complexwas measured by a postcolumn technique using POD activity with high sensitivity. Although the immunoreaction equilibrium constant has been measured for various antigens using several methods, no similar studies have been undertaken for protein antigens in buffer solution. The equilibrium constants of antibodies (9) Jackson, D. C.; Howlett, G. J.; Neetorowicz, A.; Webster, R. G. J . Immunol. 1983,130,1313-1316.
equilibrium dialysis, immunoaffinity chromatography, enzyme immunoassay, and other methods.1-3*8.9 The equilibrium value of the monoclonal antibody was obtained by LBA as 3.7 X 1Olo M-l without the use of a solid phase. The rate constants for hapten and antibody binding have been reported by many workers, but it has been difficult to measure the rate constant for protein antigen and antibody binding. The rate constant was reported as 5.6 X 104 M-l min-1 for anti-insulin polyclonal antibody (capybara) binding to insulin, obtained by immunoaffiiity chromatography.2 Immunoreaction of the anti-hCG antibody used in the present study on the other hand was faster than this (9.3 X lo8 M-l min-I). Effects of pH on the immunoreaction have not heretofore been investigated in detail. Anti-albumin monoclonal antibody binding to albumin was found by LBA to have a pH optimum between pH 6 and 9, but the antibody used in the present study had a sharp optimum pHS5 Changes in the amounts of immune complexes at several pHs were not due to a change in reaction rate, but rather due to increases in unreactive Fab’-POD. Because of the importance of finding optimum binding reaction conditions, it may be important to routinely determine antibody binding pH optima since the data show such a large effect. The effects of temperature on the immunoreaction have been investigated for hapten antigen using polyclonal antib0dy.l For protein antigen binding, an increase in temperature caused a linear decrease in the immune complex. This change was reversible. These results can be interpreted to mean that temperature causes a change in the threedimensional structure of Fab’-POD or/and hCG and this interferes with the immuno binding reaction. When a large excess of hCG was added, the amount of immune complex was not affected by temperature. This additional fact in view of the above would indicate that the three-dimensional structure of hCG may be more affected by temperature than that of Fab’-POD. Use of LBA allows more detailed analyses of these reactions than previous techniques by virtue of rapid analyses of the equilibrium state. When applied to the assay of human serum hCG, the LBA procedure was modified by addition of a second antibody specific for hCG. This allowed a lower detection limit of hCG because the separation between the free form (Fab’POD) and the bound form (immunoglobulin G-hCG-Fab’POD) was made clear (Figure 9). Normally, it is difficult to measure the concentration of specific monoclonal antibody species in a Fab’-POD solution or other mixtures, but by this method it can be easily quantified. Additionally, the LBA technique allows accurate determination of binding parameters and concentrations of analyte in homogeneous conditions. This obviates complications of nonspecific binding to the solid phase and inactivation of antibody activity by immobilization. The present results show applicability of the LBA method for determination of hCG in human fluids, and it should prove useful in many areas of research that concern binding reactions and measurement of analytes.
ACKNOWLEDGMENT We are grateful to the Center for Population Research of the National Institute of Child Health and Human Development of the National Institutes of Health for providing us with hCG, CR-123. RECEIVEDfor review September 8, 1992. December 1, 1992.
Accepted
