Anal. Chem. 1991, 63,468-474
460
Quantification of Antibodies to Human Growth Hormone by High-Performance Protein G Affinity Chromatography with Fluorescence Detection Alice Riggin and Fred E. Regnier*
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
J. Richard Sportsman Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285
The technique of high-performance affinity chromatography (HPAC) is applied to the quantltative determination of antibodies to human growth hormone (hGH) in serum from patients. An afflntty column consisting of covalently hnmobtlized protein G on a rigid support is used to capture the antibodies. Texas Red labeled hGH (hGH-TR) is used as a fluorescence probe for detectlng the anti-hGH antibodies. Callbration curves are established by uslng a well-characterized monoclonal antibody to hGH (GHC101). The minimum detectable concentration (MDC) of anti-hGH antibody in serum is 250 ng/mL (this represents 10 ng of anti-hGH injected onto the protein G column). Analytical recoveries are 92-110% for seven replicates with 250-4000 ng/mL of GHC101. A precision of 15% relative standard deviation (RSD) can be achieved at the MDC. The preclslon Is better above the detection limit. The linear dynamic range of the method is approximately 2 orders of magnitude. The total fluorescence recovery from the affinity column is 296%. Sample analysis times are on the order of 20 min. The HPAC technlque gives results in absolute units of concentration that correlate well with blnding capaclty values determlned by radioimmunoassay.
INTRODUCTION The quantification of antibodies in serum samples from patients is essential for evaluation of the immune response to therapeutic proteins and for understanding the immunological properties of these proteins. Traditionally, many relative or semiquantitative terms such as “relative percent binding”, “degree of response”, and ”titer” have been used to describe the quantity of antibodies specific for a given antigen or the extent of the immune response induced by such an antigen ( I , 2 ) . While this approach may be sufficient for the purpose of most diagnosis, it is desirable to express the immune response in explicit concentration terms such as nanograms/milliliter or picomoles/milliliter of the antibody or of the specific antigen binding capacity of this antibody. This is particularly important when we study the immune response following administration of pharmaceuticals to compare the immunogenicity of closely related substances. Using clearly defined concentration terms also simplifies interlaboratory comparisons ( 3 ) . Immune response generally has been determined by either enzyme-linked immunosorbent assays (ELISAs) or radioimmunoassays (RIAs). Data generated by competitive RIAs have been used to estimate the absolute concentration (i.e., explicit concentration units such as nanograms/milliliters or picomoles/milliliter) of antibodies ( 4 ) . However, because competitive binding assays are nonlinear, there is a large quality control issue with running and maintaining them. Multiple controls, large numbers of assays, and curve fitting must be
used to obtain quantitative data from the logarithmic dose response curve. Reported coefficients of variation (CVs) for quantification of antibody response to hGH by competitive RIAs are 20-60% ( 4 ) . This method generally requires 2-3 days and is difficult to automate. In addition, the method uses radioiodinated hGH, which, in conjunction with safety issues, is expensive to prepare, handle, and discard. When ELISAs are used to detect specific antibody, usually antigen is immobilized on microtiter plates and used to capture the induced antibody (5,6). Following antibody capture, a variety of “sandwich” techniques may be used to detect antigen-specific antibodies. Despite the sensitivity and convenience of such ELISAs, there are limitations. The first problem is that the signals generated by sandwich detection schemes do not directly represent the quantity of specific immunoglobulin G (IgG). Certain assumptions or arbitrary reference standards are therefore needed to make such conversions possible. ELISAs, in this widely used format, actually measure a combination of affinity and concentration (7). Since the relative contribution of these components varies according to the assay conditions, the concentration of antibodies can only be reported in relative terms, such as “titer”. Although such ELISA data for serum antibody levels may be made internally self-consistent, they are not the basis for determination of antibody content in units of absolute concentration. The second problem with the ELISA methods arises from their use of immobilized antigens. When protein antigens are adsorbed or bonded to a surface, the biologically active regions (epitopes) may be oriented toward the sorbent surface and be unavailable for binding. As a consequence,components of the antibody response may be missed (8). The third disadvantage of these ELISA methods is that the response of the “reporter molecules” (Le., labeled second antibodies, protein A, or protein G) varies with different animal species. Therefore, an interspecies comparison or standardization will not be legitimate. Here we introduce a new approach for specific antibody quantification which should overcome the above problems encountered with current immunoassay methods. This procedure employs the following strategies. First, we allow the complexation of antigen and antibody to occur in solution. This eliminates changes in the antigen or antibody that may occur after immobilization and circumvents avidity effects induced by solid phases (9). Second, we use the fluorescence-labeled antigen in excess to saturate all specific antibody binding sites. This produces linear reponse curves and allows direct quantification of the immune response. Third, we effect the separation of bound and unbound antigen by protein G affinity, which does not involve the specific antigen-binding sites of antibody (10, 11). T o carry out this procedure, we choose to use a chromatographic format rather than ELISA or RIA formats because the chromatographic columns can provide more capacity for IgG binding than in either of the
0003-2700/91/0363-0468$02.50/00 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 5, MARCH 1, 1991
other systems and the radiaton safety concern encountered in RIA can be avoided. Although protein A, protein G, and immunoaffinity chromatography have already been used as analytical techniques (12-1.5), the potential of these techniques in analytical chemistry is not yet widely exploited. One reason for this may be that absorbance detection is not sufficiently specific to distinguish the signal of interest from background interference, particularly in more complicated sample matrices such as serum or other biological fluids. Fluorescence detection can provide sufficient specificity and sensitivity for such quantification. However, the fluorescence of most commonly used fluorescence tags, such as fluorescein, is quenched a t the low pH conditions often used as the desorbing step in affinity chromatography. In the studies reported here, Texas Red dye (16) has been identified as a suitable fluorescence tag for detection under acidic conditions. Unlike most other fluorophors, Texas Red's fluorescence is not quenched a t pH 2. It has an excitation maximum a t 596 nm and an emission maximum a t 615 nm (16-28), a region that is well removed from most spectral interferences in serum. The affinity column used here is immobilized protein G, which is of a recombinant DNA origin with the sequence coding for the albumin binding site cloned out (19). Protein G was chosen over protein A because it binds to a wider range of IgG species and subclasses than does protein A. Protein G also does not bind any IgM, IgA, or IgE antibodies (29,20). In this paper, we demonstrate that protein G affinity chromatography and fluorescence labeling of target antigen can be combined into a rapid and sensitive method for quantifying specific antibodies to hGH. Results can be obtained in absolute units of concentration.
EXPERIMENTAL SECTION Materials. Texas Red (TR) was purchased from Molecular Probes, Inc. (Eugene,OR). BSA (bovine albumin, fraction V, 98% pure by electrophoresis)was purchased from Cal Biochem-Behring Corp. (La Jolla, CA). Human y-globulin (purified from Cohn fractions I1 and 111, electrophoretic purity approximately 99%) was purchased from Sigma Chemical Company (St. Louis, MO). Tris [tris(hydroxymethyl)aminomethane]was crystallized ultrapure grade supplied by Boehringer Mannheim Biochemicals (Indianapolis, IN). Reagent water was obtained from a Millipore Milli-Q water purification system. All other chemicals were analytical reagent grade unless otherwise indicated. Human growth hormone (hGH, molecular mass is 22.1 kDa) was natural sequence protein (somatropin) of recombinant DNA origin, produced by Eli Lilly and Co. (Indianapolis, IN), and had a purity greater than 99% monomeric hCH by size exclusion high-performance liquid chromatography (HPLC) (21). The purity determined by reversed-phase HPLC was greater than 95%. Texas Red Labeled hCH (hCH-TR). The method described by Titus et al. (16)was followed for the preparation of hGH-TR. A dye-to-protein ratio (wt/wt) of 0.1 was added in carbonate buffer (0.1 M, pH 9.1). The reaction mixture was gently stirred in an ice bath in dark for 2 h. It was then desalted into Tris buffer (25 mM, pH 7.5) by using NAP-25 columns (Sephadex G-25, DNA grade, Pharmacia), to remove excess dye. The desalted reaction mixture was then injected onto an anion-exchange column (Mono Q, HR10/10, Pharmacia) and eluted at 2 mL/min using 30% acetonitrile/70% Tris buffer (50 mM, pH 7.5) as the mobile phase. The elution program consisted of three parts, one isocratic and two linear gradient segments, an initial isocraticsegment at 0.035 M NaCl for 10 min, a 0.035-0.14 M NaCl gradient for 60 min, and a 0.14-0.3S M NaCl gradient for 10 min. Multiple peaks were detected by monitoring the absorbance at 280 and 590 nm (Figure 1). Peak A was collected, lyophilized, and reconstituted. The protein concentration was 2.0 mg/mL as determined by amino acid analysis, which agreed with the value of 2.1 mg/mL determined by UV absorption at 280 nm after correction for the contribution of Texas Red. This value was used to calculate concentrations of hGH-TR in the subsequent studies reported below. The average conjugation ratio of this peak was determined
1
A
469
I! 0:315 0.280 0.245
3 0
0.210
E
L
0.175 0.140
5
0.105
$
0.070
z
0.035
1 . . . . ..,,
O.Oo0
B
,.,, . . . . I . " . ~ . . . . I ' . . ' 1 . ' . . ' . . . . I . . I . !o 480.0 960.0 1440. 1920. 2400. 2880. 3360. 3840. 4320. 4800. lime (secands)
Figure 1. Anion-exchange separation of Texas Red labeled hGH (solid line) and hGH (broken line). Full scale is 0.05 absorbance unit.
to be 1.2. This ratio was calculated based on ultraviolet (UV) absorbance at 590 and 280 nm, assuming an extinction coefficient of hGH at 280 nm to be 0.74 and extinction coefficientsof Texas Red at 590 and 280 nm to be 136 and 45, respectively (17,18). Antibodies. Two monoclonal antibodies to hGH, designated GHClOl and GHC072, were obtained from Hybritech, Inc. (La Jolla, CA), and were shown by competitive RIAs to have affinities of 1.4 X 1O'O and 2.1 X lo9 L/mol, respectively. The GHClOl antibody was more than 95% pure IgG as evaluated by electrophoretic analysis. The antibody concentration determined by measuring UV absorbance a t 280 nm (assuming the extinction coefficientof IgG to be 1.4) was 21 mg/mL (corresponding to 5.8 X lo6 ng/mL hGH binding capacity (BC)). This value was used to calculate concentrations of GHClOl reported later. The active antibody concentration determined by competitive RIAs was 20 mg/mL, which was consistent with the above UV data. GHC072 was purified from mouse ascites by a single sodium sulfate precipitation. Samples. Serum samples were obtained from patients enrolled in clinical evaluation of recombinant human growth hormones (somatrem and somatropin). Some patients had previously undergone therapy with pituitary-derived growth hormone while others were naive to treatment (22-24). Normal human serum (NHS) was a pool of sera from 30 normal adult volunteers. All serum samples were handled in accordance with standard NCCLS recommended safety measures (25). Apparatus. Affinity HPLC determinations were conducted on an HPLC system consisting of a Spectra Physics ternary pump system (SP8700XR, extended range LC pump) equipped with a protein G affinity column (500 A, 30 pm, 5 X 0.46 cm i.d., acquired from Chromatochem, Inc., Missoula, MT). The immobilized protein G, GammaBind G (Genex Corp.), is a recombinant product devoid of albumin binding capacity. Sample solutions were introduced by a Rheodyne injection valve (Model 7125) equipped with a 200-pL sample loop (all analyses were carried out with a fixed sample size of 200 pL). The column eluent was monitored by both a UV (Spectra-Physics SP8440 XR UV/visible (vis) detector) and a fluorescence detector (SLMAminco, Model SPF-500C spectrofluorometer, equipped with a flow cell attachment) in series. UV absorbance was monitored at 280 nm. The fluorescence was monitored with an excitation wavelength of 598 nm from a xenon arc lamp and an emission wavelength of 612 nm. The output of both detectors was interfaced with a HPlOOO minicomputer where all data were collected, stored, and processed by using an in-house (Lilly) chromatographic data system. The peak areas were integrated by the data system with the base line drawn from peak start to peak end (valley to valley). The peak areas that represent fluorescence responses were used for sample quantification. Chromatographic Procedure. Tris/acetate buffer (50 mM, pH 7.4 f 0.1) was used as the sample loading buffer. It was prepared by dissolving Tris base in water and adjusting the pH with glacial acetic acid. Bound substances were eluted by using 20% acetic acid (pH 2.1 i 0.1). The protein G column was equilibrated with the sample loading buffer at a flow rate of 2 mL/min followed by cycles of injections of blank Tris/acetate buffer to ensure that there were no interferences from previous analyses. The chromatographic procedure involved pumping the
470
ANALYTICAL CHEMISTRY, VOL. 63,NO. 5, MARCH 1, 1991 (A.) Load a .
(B.)
Wash
(C.) Desorb Elution buffer
i
Figure 2. Diagram of protein G affinity chromatographic steps for the separation of free (peak A) and bound (peak B) hGH.
loading buffer through the column at 1 mL/min for 5 min following sample injection. After 5 min, the flow rate was increased to 2 mL/min to elute unbound proteins. When the UV absorbance had returned to base line (about 10 min), the column was eluted with 20% acetic acid at a flow rate of 3 mL/min to desorb the bound IgG (about 5 min). Following acidic desorption, the column was reequilibrated with the neutral loading buffer for 5 min at a flow rate of 2 mL/min. To minimize nonspecific binding of labeled hGH to the protein G column, 200 pL of 5% BSA solution was injected onto the column prior to each sample injection. Determination of Total Fluorescence Recovery. To estimate the total recovery of the HPAC system, samples were prepared by mixing hGH-TR and GHClOl at various concentrations. After an overnight incubation at 5 "C, the samples were analyzed by HPAC. Fractions were collected across the entire chromatographic run of each sample, and volumes were reduced with a SpeedVac concentrator (Savant Instruments Inc., Farmingdale, NY) and then adjusted to 2 mL with reagent water. Blank elutions (when Tris buffer was injected) were collected and prepared in the same manner as the enriched samples. These procedure blanks were used as the reference for the blank and for the preparation of the 100% total recovery reference solutions. For the total recovery references, the amount of hGH-TR and GHClOl equivalent to that in each sample was added directly into 2 mL of the procedure blank. The percent recovery was determined by comparing the recovered fluorescence of the collected eluent samples to the fluorescence of the same standard without column elution. RIA for hGH Binding Capacity. Serum samples were diluted 50-fold in assay buffer (10 mM Tris, pH 8.8 containing 140 mM NaCl, 25 mM EDTA, and 1.0% BSA) and analyzed for hGH binding capacity by competition radioimmunoassay (RIA) as reported before (4). Briefly, to duplicate 1oO-pL aliquots of this diluted serum were added a total volume of 400 pL of assay buffer containing 150 pg of *251-labeledhGH and 0-100 ng of unlabeled hGH. After overnight incubation at room temperature, the bound lZ5I-labeledhGH was separated by poly(ethy1ene glycol) precipitation and data were analyzed by nonlinear least-squares curve fitting with use of the SCAFITLIGAND-PC program (26). RESULTS AND DISCUSSION The affinity chromatographic procedure used in this work for quantification of antibodies to hGH involves (i) incubation of the serum samples with an excess of Texas Red labeled hGH (hGH-TR); (ii) injection of this incubation mixture onto the affinity column under neutral pH conditions whereby all IgG, including any complexed with hGH-TR, is retained; (iii) elution of unbound materials; (iv) desorption and dissociation of the antigen-antibody complexes from the affinity column by changing to a denaturing eluent; (v) quantification of the bound-labeled hGH by fluorescence detection (excitation 598 nm/emission 612 nm). A diagram of these affinity chromatographic steps for the separation of free and bound hGH is shown in Figure 2. Since only the hGH-TR retained on the affinity column is specifically bound to the anti-hGH antibodies, the fluorescence response during acidic elution will be proportional to the antibody content of the sample. The
calibration curve of the hGH binding capacity is established by using a well-characterized monoclonal antibody as a calibrator. The antibody response of serum samples, analyzed with the identical procedure, is then interpolated from this calibration curve and reported in units of the calibrator (a reference standard). These results, in equivalent concentration units of GHC101, can also be converted to other concentration units, such as antigen binding capacity, by applying simple stoichiometry. Optimization. The capacity of the protein G column for binding IgG was determined to be 111mg of human IgG per column. A 200-pL aliquot of 55 mg/mL human y-globulin in Tris buffer (50 mM, pH 7.5) was injected onto the column and analyzed by the HPAC procedure. Eluent was collected, and UV absorbance a t 280 nm was measured. Of the total absorbance, 95% was recovered upon the acidic elution. Only 5% of the total absorbance of UV 280 nm eluted with no retention. When this unretained peak was reinjected, all the substance was again eluted with no retention, suggesting this fraction may be impurities other than IgG. In our experiments, the total IgG injected on the column never exceeded 2 mg. Thus, the IgG binding capacity of the protein G column is a t least 8-fold in excess of the amount injected in any of the experiments described here. The influence of incubation time on the amount of hGHT R complexed to GHClOl was studied. The fluorescence response of the antibody-complexed hGH-TR, as estimated from the area of the peak retained by the protein G column, reached a constant value and was not influenced by incubation time after 30 min. We routinely incubated our samples at 5 "C overnight for convenience. Conditions for loading and desorbing hGH-TR and antibody complexes are described below. When 50 mM Tris/ acetate buffer (pH 7.4) was used as the loading buffer a t a flow rate of 1 mL/min, the antibody-antigen complex was totally captured by the protein G column. Acetic acid (20% v/v) quantitatively desorbed antibody-bound hGH-TR from the protein G column. Total fluorescence recovery was determined to be 1 9 6 % when excess hGH-TR reacted with GHClOl at 0,8, and 16 pg/mL. It was found that a flow rate of 3 mL/min could be used for rapid analysis. Slower desorption flow rates resulted in reduced peak height and made it more difficult to detect bound hGH-TR at low concentration. Higher flow rates did not appreciably improve the peak height. The affinity column was cleaned with an additional 10 mL of 20% acetic acid a t the end of each assay and stored at 5 "C in the loading buffer with 0.02% sodium azide added. The column maintained its performance after more than 600 cycles over 6 months. Evaluation of hGH-TR as the Fluorescence Probe for Antibody Quantitation. Tris-buffered BSA solutions (TBSA, 0.1% BSA, 0.15 M NaC1, and 0.02% sodium azide in 10 mM Tris, pH 7 . 5 ) containing various concentrations of hGH-TR were incubated with excess monoclonal antibody GHClOl a t 5 "C overnight. The reaction mixtures were analyzed by using the protein G affinity chromatographic method described above. A typical elution profile is shown in Figure 3A. The chromatograms demonstrate that, in the presence of excess antibody, all the hGH-TR was complexed to antibody, retained by the protein G column, and eluted only when the mobile phase was switched to the denaturing condition (20% acetic acid, pH 2.1). As little as 2 ng of hGH-TR, complexed with GHClOl and injected onto the column, can be detected. Instrument response to the antibody-complexed hGH-TR was linear, with a range greater than 3 orders of the magnitude (2 ng to 3 pg). When the concentration of GHClOl was reduced to 6 pg/mL and a 200-pL aliquot of the reaction mixture was injected onto the column, the fluorescence re-
ANALYTICAL CHEMISTRY, VOL. 63,NO. 5, MARCH 1, 1991
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Figure 3. Protein G affinity separation of free and IgG complexed antigen in TBSA: (A) hGH-TR in the presence of excess GHC101, (6) GHClOl in the presence of excess hGH-TR. Chromatographic conditions as described in the Experimental Section.
471
proportional to the size of the fluorescence peak. Similar results were seen when another anti-hGH monoclonal antibody (GHC072) was used in the HPAC technique. The affinities of GHClOl and GHC072 to hGH-TR were determined by competition RIA to be 3.9 X lo9 and 1.3 X lo9, respectively, comparable to their affinities to hGH, 1.4 X 1O'O and 2.1 X lo9, respectively. These data suggest that the fluorescent-labeled hGH binds well with GHC072 also, even though GHC072 and GHClOl bind to different epitopes on the hGH molecule (27-30). The hGH binding capacity value for the GHC072 preparation was 860 Fg/mL by RIA, comparable to the value of 900 Kg/mL determined by HPAC. It was observed that the binding of hGH-TR to GHClOl and ~ ~ ~ ~ ~ ~ ~ " ~ -072 could be completely displaced by a 20-fold excess of unlabeled hGH over an antibody concentration range of 1-16 pg/mL. Similar results were also observed when the source of hGH antibody was a rabbit antiserum or serum from a patient known to be positive for hGH antibody. These observations demonstrate that hGH-TR is immunochemically indistinguishable from hGH in this assay. Control of Nonspecific Binding. The amount of hGHT R nonspecifically bound to the protein G column (i.e., via nonimmune mechanisms) depends upon the characteristics of the column, the concentration of free hGH-TR in the sample, and the efficiency of the washing procedure after sample loading. The level of nonspecific binding increases as the concentration of free antigen increases. In order to optimize the complexation of antibodies to hGH-TR, as discussed later, an excess of hGH-TR (110 Kg/mL) was added to the sample matrices. This excess of hGH-TR results in significant amounts of nonspecific binding. One method for minimizing the nonspecific binding is to wash the protein G column extensively after sample loading. The efficiency of sample washing is affected by the composition and pH of the washing buffer, flow rate, and length of time for washing. Washing the sample with neutral loading buffer a t an increased flow rate of 2 mL/min for 10 min effectively brought the peak of unbound material back to the base line but did not totally remove a small residue of nonspecifically bound hGH-TR. Increasing the flow rate of washing buffer to 3,4, or 5 mL/min also did not reduce this level. It was found that addition of modifiers such as poly(ethy1ene glycol) or TWEEN 20 to the washing buffer decreased the level of nonspecific binding. However, such additions still need to be carefully validated to ensure that these additives, while removing the nonspecifically bound antigen, do not affect the peak of interest. An approach found to be successful in controlling the nonspecifically bound antigen was to load the column with 200 FL of 5% BSA solution to saturate nonspecific binding sites on the column prior to the sample injection. This approach minimized the nonspecific binding level to less than 1% of the initial antigen concentration. This BSA "frontblocking" technique also improved the shape of both the free and bound hGH-TR peaks. MDC, Analytical Recoveries, and Quantification of Antibodies to hGH in Serum Samples. Typical chromatograms for serum samples and controls are shown in Figure 5 . NHS is normal human serum with no hGH-TR added. The negative control was prepared by addition of the usual amount of hGH-TR reagent in NHS. In the elution profile of NHS, most of the fluorescent background is eluted unretained, but there is a small background peak that elutes with the change to acid conditions. The excitation and emission maxima of this peak were determined to be 582 and 655 nm, respectively, by stop flow scanning. The HPAC method, with an excitation and emission wavelength set at 598 and 612 nm, has been optimized to discriminate against this background for the detection of hGH-TR. For the quantification discussed
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200
400
600
800
1000
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Flgure 4. Fluorescence response curve of the fixed amount (1.2 Fg) of GHClOl treated with varying amounts of hGH-TR. sponse reached a plateau at about 400 ng of hGH-TR, as would be expected if all the available binding sites on the antibody were saturated (Figure 4). In a complementary experiment, a limited fixed amount (approximately 700 ng/mL) of hGH-TR was incubated with varying amounts of GHClOl antibody in TBSA. The fluorescence responses of the bound hGH-TR were directly proportional to GHC101. Typical elution profiles for the free and bound hGH-TR are shown in Figure 3B. It can also be seen in the figure that the amount of unbound hGH-TFt varies inversely with the concentration of GHC101. Less than 8 ng (corresponding to approximately 2 ng of hGH binding capacity) of GHClOl antibody injected on the column could be detected. The results (Figure 3) from both of the above experiments suggest that the fluorescenceresponse to hGH-TR upon acidic elution of the affinity column reflects the amount of hGH-TR complexed to IgG. Thus, when excess hGH-TR is used, the amount of IgG injected onto the column will be directly
ANALYTICAL CHEMISTRY, VOL. 63, NO. 5, MARCH 1, 1991
472
Table I. Precision of Antibody Quantification by HPAC and Recoveries from GHClOl Enriched NHS concn of
concn of
GHClOl added, fig/mL
hGHTR, pg/mL
0.25 0.60 0.90 1.20
2 2 2 2
0.25
2.00
10 10
5 3 3
4.00
10
3
Sample 0 /hGH EC < MDLI
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960.0
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1200.
Time (seconds) Figure 5 . Typical protein G affinity chromatograms of serum samples
and controls: blank NHS (no hGH-TR added); negative control (NHS added 10 pg/mL hGH-TR); samples C, E, and G (see Table 11). Chromatographic conditions as described in the Experimental Section.
300 200
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/
8
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5
10
15
20
B
0.0
99 110 92 98 108 100 101
% RSD
15 10 9 4 24 1 0.8
Table 11. hGH Binding Capacity (BC) of Human Serum Samples Assayed by Protein G Affinity Chromatography (AC) and RIA
hGH BC, u.cg/mLn
sample
RIA
AC
A B
3.6 1.5 1.6 2.9 0.5
3.6
C D
2.9 1.8 2.5 0.9 0.9
