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ally present in human sera may lead to false-positive signals when intact IgG is used. ~5Generally, such interference can be blocked by the addition of 1% mouse serum when high-affinity antibodies and, as a consequence, short incubation times are used; with prolonged incubation times, however, the addition of mouse serum may be insufficient. For this reason, F(ab)2 fragments of MAb 2 are used in the MAb 2/MAb 6 immunoassay. The characteristics of the two immunoassays are given in Table I. Considering 4.2-6.5/zg/liter as the upper limit of normal range, the sensitivity of both assays is sufficiently high for the measurement of CEA in serum samples. The assay characteristics of the MAb 2/MAb 4 EIA are quite comparable to those of immunoassays not based on the avidinbiotin system. The assay correlates well with commercial CEA immunoassays. The lower precision of the MAb 2/MAb 6 immunoassay is probably due to the lower affinity of MAb 6. Acknowledgment This research was supported by grants from the Deutsche Forschungsgemeinschaft, Wa-473/4-1, and from the National Large Bowel Program, National Cancer Institute, Grant CA 37808. We wish to thank Birgit Esser, Frances Crawford, and Karen Rickard for expert technical assistance, and Dr. Y. H. Joy Yang for production of monoclonal antibodies. is C. Wagener, Ann. Clin. Biochem. 24, Suppl. 2, 208 (1987).
[60] S e l e c t i v e P r e c i p i t a t i o n o f B i o t i n - L a b e l e d A n t i g e n s or M o n o c l o n a l A n t i b o d i e s b y A v i d i n for D e t e r m i n i n g E p i t o p e S p e c i f i c i t i e s a n d Affinities in S o l u t i o n - P h a s e A s s a y s B y CHRISTOPH WAGENER, ULRICH KRUGER, and JOHN E. SHIVELY
Solid-phase assays in which either antigen or antibody are adsorbed to a solid support and the second reactant is present in solution may impose several problems in the analysis of the antigen-antibody interaction. The degree of adsorption to the solid matrix depends on the properties of the individual proteins. ~In addition to the potential denaturation of proteins,2 J. D. Andrade, in "Surface and Interfacial Aspects of Biomedical Polymers, Protein Adsorption" (J. D. Andrade, ed.), Vol. 2, p. 1. Plenum, New York, 1985. 2 M. E. Soderquist and A. G. Walton, J. Colloid Interface Sci. 75, 386 (1980).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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adsorption to solid supports may modify or obscure epitopes of native antigens 3 or may create neoepitopes not present on the native antigen. 4,5 As the solid-phase support is effectively a ternary component of the reaction, the nonspecific interaction of antigens or antibodies with the solid phase may interfere both in binding and in inhibition experiments, especially when the affinity of the antigen-antibody interaction is moderate or low. 1 The above problems do not apply to homogeneous assays in which both reactants are present in solution phase. However, a prerequisite for binding and inhibition studies in solution phase is the effective separation of bound from free antigen or antibody. Methods used for the precipitation of polyclonal antibodies may not be suitable for monoclonal antibodies. Precipitation by second antibodies is limited by the formation of soluble complexes between the monoclonal IgG and anti-mouse IgG antiserum at high monoclonal antibody excess. The precipitation of antigenantibody complexes by ammonium sulfate or polyethylene glycol is complicated by high levels of nonspecific binding and incomplete precipitation of the complexes .6 In order to perform binding and inhibition experiments in solution phase using a constant amount of antigen and variable amounts of antibody, a general method for the selective precipitation of antigen would be desirable. Here we describe the use of the avidin-biotin system to selectively and quantitatively precipitate either biotin-labeled antigen or antibody from solution by the addition of avidin at optimal ratios of avidin to biotinylated reactant. Using this technique, inhibition studies and saturation experiments can be performed in solution phase at constant concentrations of biotinylated antibody or antigen. General Procedures
Monoclonal antibodies (MAbs) against the carcinoembryonic antigen (CEA) are produced as described. 7 MAbs are elicited by immunizations either with purified CEA (MAbs 1, 3, and 5) or with the transplanted colon carcinoma cell line T84 (MAbs 2 and 4). CEA is purified according to 3 F. J. Stevens, J. Jwo, W. Carperos, H. K6hler, and M. Schiffer, J. lmmunol. 137, 1937 (1986). 4 A. D. Smith and J. E. Wilson, J. lmmunol. Methods 94, 31 (1986). 5 H. C. Vaidya, D. N. Dietzler, and J. H. Ladenson, Hybridoma 4, 271 (1985). 6 C. Wagener, B. R. Clark, K. J. Rickard, and J. E. Shively, J. Immunol. 130, 2302 (1983). 7 C. Wagener, L. Wickert, and J. E. Shively, this volume [59]; C. Wagener, Y. H. J. Yang, F. G. Crawford, and J. E. Shively, J. Immunol. 130, 2308 (1983).
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Coligan et al. ,8 and the T84 cell line is that of Murakami and Masui. 9 IgG fractions of MAbs are purified from ascites fluid by protein A affinity chromatography according to Ey et al. 1° The IgG concentration is calculated from the absorbance of the solutions at 280 nm, assuming an extinction coefficient [1% (w/v); 1 cm] of 14.2.11 CEA concentrations and concentrations of Fab fragments are determined by amino acid analysis.
Binding and Inhibition Studies with Biotin-Labeled Antibodies and Avidin as Precipitating Agent Materials
PBS: 50 m M sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride and 0.1% NaN3 Avidin-PEG: avidin (Sigma Chemical Co., St. Louis, MO) 0.1 mg/ml in PBS containing 5% (w/v) polyethylene glycol (PEG 6000, Sigma) BNHS: N-hydroxysuccinimidobiotin (Sigma) DMF: dimethylformamide (J. T. Baker, Phillipsburg, NJ), distilled from ninhydrin Preparation of lesI-Labeled Antigen. Purified CEA is lzSI-labeled by the chloramine-T procedure of Hunter and Greenwood. 12 Biotinylation Procedures. MAb is biotinylated as described elsewhere in this volume. 7 As a biotinylated carrier protein, either normal rabbit or goat serum can be used. After dialysis against PBS, carrier serum (2 ml) is treated with 2 ml of freshly made aqueous BNHS, prepared by dissolving 20 mg of BNHS in 0.5 ml of DMF and diluting to 2 ml with 1.5 ml of distilled water. The remainder of the procedure is as described for preparing the biotinylated MAbs. Assay Overview. The protocol is similar to that described by Clark and Todd 13 and Wagener et al. 6 Determination of Kaff is performed in two steps. First, binding curves for each MAb are determined. A constant amount of radiolabeled antigen (0.5 ng of [IzsI]CEA) is titered against serial 2-fold dilutions of MAb. The concentration of MAb IgG yielding half-maximal binding is determined. This concentration is used in the second step. The second step comprises a competitive RIA involving the s j. E. Coligan, J. T. Lautenschleger, M. L. Egan, and C. W. Todd, lmmunochemistry 9, 377 (1972). 9 H. Murakami and H. Masui, Proc. Natl. Acad. Sci. U.S.A. 77, 3464 (1980). to p. L. Ey, S. J. Prowse, and C. R. Jenkin, lrnmunochemistry 15, 429 (1978). H H. N. Eisen, E. S. Simms, and M. Potter, Biochemistry 7, 4126 (1968). 12 W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). 13 B. R. Clark and C. W. Todd, Anal. Biochem. 121, 257 (1982).
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addition of increasing amounts of unlabeled antigen (0.5-100 ng of CEA) which competes with a fixed amount of radiolabeled antigen (0.5 ng of [125I]CEA). The present RIA version required the separation of free from bound radiolabeled antigen. This is accomplished by precipitation of the biotinylated MAb with avidin. Since only a minute amount of MAb is present, the precipitation is driven to completion by adding a biotinylated carrier protein mixture. Binding RIA Protocol with Biotinylated Antibodies. The RIA is performed in 400-/xl polypropylene tubes (Bio-Rad, Richmond, CA) which can be tightly capped and centrifuged in a Beckman microcentrifuge. Serial 2-fold dilutions of biotinylated MAb (50 /zl; 0.01-1,000 ng) in biotinylated carrier serum (I : 800 dilution in either underivatized normal rabbit or goat serum diluted 1 : 40 in PBS) are incubated with 200/xl of PBS containing 6.5% PEG and 72 m M EDTA and with 10/xl of [125I]CEA (0.5 ng in PBS; -104 cpm) for 18 hr at 37 °. The [125I]CEA cocktail may include 57Co as a volume marker if desired. Biotinylated MAb and biotinylated MAb-antigen complexes are coprecipitated with biotinylated carrier proteins by addition of 10/zl of avidin (1.0 mg/ml in PBS containing 5% PEG 6000), and the mixture is incubated for 1 hr at room temperature. The tubes are centrifuged, the supernatant fluids are removed, and the precipitates are counted. The binding of constant amounts of [125I]CEA by decreasing amounts of monoclonal antibody IgG is shown in Fig. 1. At high IgG excess, MAb 3 binds over 90% of labeled antigen, thus indicating quantitative precipitation of the antibody. The broad plateau regions of the binding curves for MAbs 2 and 4 show that nonprecipitable soluble complexes between biotin-labeled monoclonal antibody and avidin were not observed when excess IgG concentrations were used. Nonspecific precipitation of labeled CEA as determined by the use of biotin-labeled carrier without specific antibody was in the range of 1-3%. CEA Inhibition Assay Protocol. The assay tubes, centrifugation protocol, and reagents are the same as in the previous section. To each assay tube (in duplicate) are added 10/zl of 125I-labeled CEA (0.5 ng in PBS) and 50/xl of biotinylated MAb at concentrations indicated in the legend to Fig. 2. The dilution of MAbs is performed in a 1 : 800 dilution of biotinylated goat serum in either underivatized normal goat or rabbit serum (1 : 40 in PBS) and 200/zl o f a CEA inhibitor solution (0.5-100 ng in PBS with 6.5% PEG). The mixture is incubated, precipitated with 10/zl of avidin (1.0 mg/ ml in PBS with 5% PEG), and counted as before. For inhibition experiments, IgG concentrations corresponding to 4060% of the maximum binding of [125I]CEA (see plateau regions in Fig. 1)
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100
9O
81) o
i
70
•
•
•
6O
i10
0
10000
I 1,000
I 100
I 10
I
0.1
0.01
igo (rig]
FIG. 1. Binding curves for radiolabeled antigen to MAb in a solution-phase, avidinbiotin-based RIA. Radiolabeled CEA was titered with decreasing dilutions of biotinylated MAbs. The biotinylated MAbs and M A b - C E A complexes were precipitated with avidin in the presence of 5% PEG. ©, MAb 1; 41, MAb 2; [~, MAb 3; I , MAb 4; &, MAb 5. [From C. Wagener, B. R. Clark, K. J. Rickard, and J. E. Shively, J. lmmunol. 130, 2308 (1983), with permission.]
are chosen for MAbs 1, 2, 3, and 5. Because of the low binding plateau of MAb 4, inhibition experiments are performed (in this case) at a higher relative binding of [12sI]CEA. The inhibition curves for MAbs 1-5 are shown in Fig. 2. Calculation of Affinity Constants. Affinity constants are calculated from inhibition experiments by using a general form of the equation derived by Miiller. 14The original equation refers to 50% inhibition of tracer binding, whereas the general form of the equation can be applied to a broader range of inhibitor concentrations. Thus, gaff =
(1 --
b(1 - r ) - r(1 - b) r)(1 - b)[r(Tt + It) -- bTt]
where Tt is the total concentration of tracer, It the total concentration of i4 R. MOiler, J. lmmunol. Methods 34, 345 (1980).
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SOLUTION-PHASE IMMUNOASSAYSFOR CEA
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100
cO .Q J= ¢:
5O
0.
I
1
I
10
100
1,000
CEA [ng] FIG. 2. Inhibition curves for calculating affinity constants of five MAbs. Constant amounts of radiolabeled CEA and biotinylated MAb were incubated with increasing amounts of unlabeled CEA. The biotinylated MAb and MAb-CEA complexes were precipitated with avidin in the presence of 5% PEG. The IgG concentrations of MAbs used were as follows: ©, MAb 1, 26.5 ng IgG per tube; O, MAb 2, 1.4 ng IgG per tube; [], MAb 3, 96.9 ng IgG per tube; II, MAb 4, 0.9 ng IgG per tube; A, MAb 5, 171.6 ng IgG per tube. [From C. Wagener, B. R. Clark, K. J. Rickard, and J. E. Shively, J. ImmunoL 130, 2308 (1983), with permission.]
inhibitor, b the fraction of tracer bound in the absence of inhibitor, and r the fraction of tracer bound in the presence of inhibitor. A model calculation is given for MAb 2. Inhibition experiments were performed with 0.5 ng o f [~25I]CEA per tube (260/xl). In the absence of inhibitor, 31.1% o f labeled CEA was bound (b = 0.311). In the presence of 1.94 ng of unlabeled CEA, 22.9% of labeled CEA was precipitated (r = 0.229). This corresponds to a percent binding inhibition of (1 - r/b) x 100 = 26.4% (see Fig. 2). Assuming an Mr of 180,000 for CEA, the concentration of tracer is 10.7 x 10 -12 M, and the concentration of inhibitor is 41.5 × 10 -12 M. Using the above formula, Kaff = 1.8 x 10 m M -1. In the presence o f 7.7 ng o f unlabeled CEA (164.5 × 10 -~2 M), 12.1% of the tracer was precipitated (r = 0.121), corresponding to a percent inhibition of 61.1% (see Fig. 2). F r o m these values, K, ff = 1.8 x 10 ~0 M -1 is calculated. As shown in Fig. I, the fraction of radiolabeled CEA bound at high IgG excess is different for each of the MAbs. Since over 90% of unlabeled CEA is bound by the different MAbs, 6 the decrease of the immunoreactiv-
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TABLE I AFFINITY CONSTANTS OF MONOCLONAL ANTI-CEA ANTIBODIESa'b
MAb
Affinityconstants (M-~)
1
3.7 ×
2 3 4 5
1.8 × 1.0 × 2.6 × 3.8 ×
109 10I° 108 101° 108
For calculation, see text. b Data from C. Wagener, B. R. Clark, K. J. Rickard, and J. E. Shively, J. lmmunol. 130, 2302 (1983).
ity of radiolabeled CEA is most probably due to partial radioiodination damage of individual epitopes. If only 70% immunoreactivity of labeled CEA is assumed for MAb 2 (see plateau region in Fig. I), the calculated affinity constants increase to 2.2 × 10 l° to 2.5 x 1010M -l, depending on the inhibitor concentration used for calculation. For MAb 4, the affinity constants obtained for 30% immunoreactivity of tracer are in the range of 4.9 x 1010 to 5.7 × 10 l° M -1. The affinity constants calculated for 5 MAbs from inhibition experiments without corrections for immunoreactive tracer are shown in Table I. The K ~ values for MAbs 1, 2, and 4 are sufficiently high to warrant their use in an immunoassay for CEA. The final choice of the optimum MAb is also dependent, however, on the required epitope specificity. A discussion of this topic and the problem of cross-reacting antigens is given by Wagener et al. 6
Determination of Epitope Specificities and Kaff Using Biotinylated CEA and Avidin as Precipitating Agent Reagents PBS: see above Avidin-PEG: avidin (Sigma) 5 mg/ml in PBS containing 6.5% (w/v) PEG 6,000 (Sigma) B N H S and DMF: see above Preparation of 125I-Labeled Fab fragments. Fab fragments are prepared from MAbs 2 and 4 according to the procedure described by
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Parham.~5 Molecular weight determinations and purity control are performed by SDS-PAGE using the Phast System (Pharmacia) with preformed gradient gels of 8-25% acrylamide. Under nonreducing conditions, molecular weights of 48K are obtained for the Fab fragments of both antibodies. The procedure is not generally applicable to all monoclonal antibodies as MAb 1 is completely digested by pepsin treatment. For iodination, the concentration of Fab fragments is determined by amino acid analysis. The Fab fragments are labeled with ~25I by the chloramine-T method according to Bolton and Hunter. 16The protein iodination is stopped by the addition of excess free tyrosine. The specific activity is approximately 2 MBq//zg for both Fab fragments. Preparation of Biotinylated CEA and Carrier Protein. An equal volume of CEA (1 mg/ml in PBS, 0.1 ml) is added to a freshly prepared solution of BNHS (3.5 mg/ml, 0.1 ml). The preparation of the BNHS solution and the biotinylation procedure are performed as described for the monoclonal antibodies.7 Biotin labeling of carrier protein (normal rabbit serum) is performed as described above. RIA Overview. The RIA procedure employed here represents an inverse version of the RIA described above. Prior to epitope analysis and determination of Kaff, titration curves are established. Constant amounts of radiolabeled Fab fragments are titered against serial 2-fold dilutions of biotinylated CEA. From the plateau region, the fraction of immunologically active radiolabeled Fab fragment is determined. For inhibition experiments, the CEA concentration yielding 15-27% binding of labeled Fab is determined. This concentration is used for competitive inhibition studies involving the addition of increasing amounts of unlabeled monoclonal antibody IgG or Fab fragments respectively, which compete for the binding of a fixed amount of labeled Fab fragment at constant concentrations of biotinylated CEA. In case of the present RIA version, separation of bound and free antibody is required. This is accomplished by precipitation of the biotinylated antigen with avidin in the presence of a biotinylated carrier protein. The method allows the determination of epitope specificities and affinity constants in a single experiment. Binding Assay Protocol. To 200/zl of 6.5% PEG (w/v) in PBS containing biotinylated CEA at 2-fold serial dilutions, an aliquot (50/xl) of PBS containing labeled Fab fragments (-l04 cpm, corresponding to -0.1 ng), 15 p. Parham, in "Handbook of Experimental Immunology, Immunochemistry" (D. M. Weir, L. A. Herzenberg, C. Blackwell, and L. A. Herzenberg, eds.), Vol. 1, p. 14.1. Blackwell, Oxford, 1986. t6 A. E. Bolton and W. M. Hunter, in "Handbook of Experimental Immunology, Immunochemistry" (D. M. Weir, L. A. Herzenberg, C. Blackwell, and L. A. Herzenberg, eds.), Vol. 1, p. 26.1. BlackweU, Oxford, 1986.
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7O
t~ LL
~5 t~ ._= t-
32O0
IO0 3.1 Biotin-labeled CEA [ng]
0.1
FIG. 3. Binding curve of radiolabeled Fab fragments of MAbs to antigen in a solutionphase, avidin-biotin-based RIA. Radiolabeled monoclonal Fab fragments were titered with increasing dilutions of biotinylated CEA. The biotinylated CEA and CEA complexes were precipitated with avidin in the presence of 5% PEG. O, MAb 2; II, MAb 4. [From U. Kriiger, L. Wickert, and C. Wagener, J. lmmunol. Methods 117, 25 (1989), with permission.]
normal rabbit serum (1:40 dilution in PBS), and biotin-labeled normal goat serum (1 : 100 dilution in PBS) is added. The carrier serum is prepared as described in the previous section. The assay mixture is incubated for 18 hr at 37°. Subsequently, 10/zl of 6.5% PEG (w/v) in PBS containing 50/zg egg-white avidin (Sigma) is added. After 1 hr at room temperature, the mixture is centrifuged (10 min, 15,000 g). A sample (200/zl) of the supernatant is removed. The remaining volume is counted in a 3' scintillation counter. The percentage of radioactivity precipitated is calculated from the total counts and the volume removed, thus obviating the addition of a volume marker. Two-fold serial dilutions of biotinylated CEA are performed in the range of 6400-0.012 ng per tube (Fig. 3). For the MAb 2 Fab fragment a plateau was reached with 400-1600 ng of biotinylated CEA and with 3200-6400 ng CEA for the MAb 4 Fab fragment. Depending on individual Fab preparations and radioiodination, 40-60% of the radiolabeled Fab fragment is bound in the plateau region. The finding that 100% binding is
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not reached at high antigen excess may be due to the following: (1) the presence of nonimmune IgG in the ascitic fluid, (2) partial degradation during the preparation of Fab fragments, and (3) damage by the radioiodination procedure. The possibility that soluble complexes between biotinylated CEA and avidin may be responsible for the binding of less than 100% of the labeled Fab fragments has been excluded for the concentration range of biotinylated CEA used for the binding studies. Less than 10% of total biotinylated CEA is present in the supernatant when levels of 5,000 ng per tube are used. At higher amounts of antigen, soluble complexes between avidin and CEA-biotin may develop. The nonspecific binding is less than 3%. Inhibition Assay Protocol. For inhibition experiments, concentrations of CEA-biotin of 5-25 ng per tube are chosen. Under these conditions, 15-27% of the total radioactivity is bound. To 200/zl of 6.5% PEG (w/v) in PBS containing the appropriate amount of biotinylated CEA and 2-fold serial dilutions of unlabeled monoclonal antibody IgG or Fab fragments, 50 ~1 of PBS containing labeled Fab fragments (-104 cpm), normal rabbit serum (1:40 dilution), and biotin-labeled normal goat serum (1 : 100 dilution) is added. The rest of the procedure is identical to that described above for the binding assay protocol. A representative inhibition curve is shown in Fig. 4. Using labeled Fab fragments of MAb 2, increasing concentrations of unlabeled MAb 2 Fab fragments result in complete binding inhibition. The remaining MAbs do not inhibit binding of the labeled Fab fragments. These results are comparable with those obtained by competitive solid-phase a s s a y s . 7A7 Calculation of Affinity Constants. In addition to epitope specificities, K~f can be calculated by using the general form of the equation derived by Mialler 14 given above. In the case of inhibition experiments performed with constant amounts of biotin-labeled antigen and uSI-labeled Fab fragments in the presence of increasing amounts of unlabeled Fab fragments, It is the concentration of unlabeled Fab fragments, Tt the concentration of labeled Fab fragments, b the fraction of tracer bound in the absence of inhibitor, and r the fraction of tracer bound in the presence of inhibitor. In the inhibition experiment presented in Fig. 4, Tt = 0.17 ng per tube. In the absence of inhibitor, 26.1% of the radioactivity is precipitated. In the presence of 8.85 ng per tube of unlabeled Fab fragments, 14.8% of the tracer is precipitated, corresponding to a percent binding of 56.7% (see Fig. 4). Given a reaction volume of 260/zl and an Mr for Fab fragments of 4 8 , 0 0 0 , T t -- 13.8 x 10-12 M, It = 709 x 10-J2M, b = 0.261, and r = 0.148. From these values, Kaff = 1.7 x 109 M -I. 17 C. Wagener, U. Fenger, B. R. Clark, and J. E. Shively, J. ImmunoL Methods 68~ 269 (1984).
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90
-~ 8O ,% 70
'~ 50
~ ~o ~ 3o 20 10
1
2
k
8
16
32
6/,
128
256
~ga[ng] Fab [rig]
FIG. 4. Inhibition curves for determination of epitope specificities and affinity constants of monoclonal antibodies. Constant amounts of radiolabeled Fab fragments of MAb 2 and biotinylated CEA were incubated with increasing amounts of unlabeled intact MAbs or Fab fragments, respectively. Biotinylated CEA and CEA-antibody complexes were precipitated with avidin in the presence of 5% PEG. The total amount of radiolabeled Fab fragments was 0.1 ng per tube, and the concentration of biotinylated CEA was 6.25 ng per tube. S, Fab fragment of MAb 2; the rest of the symbols are as in Fig. 1. [From U. Kri~ger, L. Wickert, and C. Wagener, J. lmmunol. Methods 117, 25 (1989), with permission.]
As shown in Fig. 3, only 58% of labeled Fab fragments from MAb 2 is precipitated at high excess of biotin-labeled CEA. It therefore follows that only part of the labeled Fab is immunoreactive. As discussed above, the reason for partial immunoreactivity may be the presence of nonimmune IgG in the IgG preparation from ascitic fluid, partial damage during the preparation of Fab fragments, or damage during radioiodination. Since the immunoreactive fraction of the unlabeled Fab fragments is unknown, K ~ can be estimated assuming partial immunoreactivity either of labeled Fab fragments alone or of both labeled and unlabeled Fab fragments. Assuming 58% immunoreactivity of the labeled fragment and 100% immunoreactivity of the unlabeled fragment, Tt = 8.02 × 10-~2 M, It = 709 × 10-12 M, b = 0.261/0.58 = 0.45, and r = 0.148/0.58 = 0.255. From these
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values, Kaff = 2.7 × l09 M-L If 58% immunoreactivity of both tracer and inhibitor is assumed, then It = 411 x 10-12 M, and Kale = 4.6 × l09 M -1. The calculated affinity constants are lower than that shown for the intact antibody in Table I. This finding may be due to a variety of reasons, including the use of Fab fragments instead of intact antibodies, the presence of nonimmune IgG in the ascitic fluid, and/or the corrections made for the immunoreactive fraction of the radiolabeled Fab fragments or antigen. Acknowledgments This research was supported by grants from the Deutsche Forschungsgemeinschaft, Wa473/4-1, and from the National Large Bowel Program, National Cancer Institute, Grant CA 37808. We wish to thank Karen Rickard for expert technical assistance, and Dr. Y. H. Joy Yang for production of monoclonal antibodies.
[61] I m m u n o a s s a y s for D i a g n o s i s o f I n f e c t i o u s D i s e a s e s
By ROBERT H. YOLKEN Introduction Traditionally, the diagnoses of infectious diseases have been based on the isolation of the infecting microorganisms in pure culture. Recently, however, infectious disease diagnoses have relied more heavily on the direct identification of infecting organisms in blood and other body fluids of the ill individual. The diagnosis of infectious diseases by the direct detection of microorganisms has been particularly important for the identification of viral agents since these agents are particularly difficult to detect in short periods of time by means of standard cultivation methods. 1 Direct detection methods are also highly useful in the detection of bacterial, parasitic, and fungal pathogens that are fastidious or difficult to cultivate under standard laboratory conditions. 2 Most of these assays have relied on the measurement of the binding of antigens to defined antimicrobial antibodies. Immunoassays have a number of advantages for infectious disease diagnosis. These advantages are based on the sensitivi D. A. Fuccillo, I. C. Shekarchi, and J. L. Sever, in "Manual of Clinical Laboratory Immunology" (N. R. Rose, H. Friedman, and J. L. Fahey, eds.), 3rd ed., p. 489. American Society for Microbiology, Washington, D. C., 1986. 2 N. J. Schmidt, Med. Clin. North Am. 67, 953 (1983).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ally present in human sera may lead to false-positive signals when intact IgG is used. ~5Generally, such interference can be blocked by the addition of 1% mouse serum when high-affinity antibodies and, as a consequence, short incubation times are used; with prolonged incubation times, however, the addition of mouse serum may be insufficient. For this reason, F(ab)2 fragments of MAb 2 are used in the MAb 2/MAb 6 immunoassay. The characteristics of the two immunoassays are given in Table I. Considering 4.2-6.5/zg/liter as the upper limit of normal range, the sensitivity of both assays is sufficiently high for the measurement of CEA in serum samples. The assay characteristics of the MAb 2/MAb 4 EIA are quite comparable to those of immunoassays not based on the avidinbiotin system. The assay correlates well with commercial CEA immunoassays. The lower precision of the MAb 2/MAb 6 immunoassay is probably due to the lower affinity of MAb 6. Acknowledgment This research was supported by grants from the Deutsche Forschungsgemeinschaft, Wa-473/4-1, and from the National Large Bowel Program, National Cancer Institute, Grant CA 37808. We wish to thank Birgit Esser, Frances Crawford, and Karen Rickard for expert technical assistance, and Dr. Y. H. Joy Yang for production of monoclonal antibodies. is C. Wagener, Ann. Clin. Biochem. 24, Suppl. 2, 208 (1987).
[60] S e l e c t i v e P r e c i p i t a t i o n o f B i o t i n - L a b e l e d A n t i g e n s or M o n o c l o n a l A n t i b o d i e s b y A v i d i n for D e t e r m i n i n g E p i t o p e S p e c i f i c i t i e s a n d Affinities in S o l u t i o n - P h a s e A s s a y s B y CHRISTOPH WAGENER, ULRICH KRUGER, and JOHN E. SHIVELY
Solid-phase assays in which either antigen or antibody are adsorbed to a solid support and the second reactant is present in solution may impose several problems in the analysis of the antigen-antibody interaction. The degree of adsorption to the solid matrix depends on the properties of the individual proteins. ~In addition to the potential denaturation of proteins,2 J. D. Andrade, in "Surface and Interfacial Aspects of Biomedical Polymers, Protein Adsorption" (J. D. Andrade, ed.), Vol. 2, p. 1. Plenum, New York, 1985. 2 M. E. Soderquist and A. G. Walton, J. Colloid Interface Sci. 75, 386 (1980).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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adsorption to solid supports may modify or obscure epitopes of native antigens 3 or may create neoepitopes not present on the native antigen. 4,5 As the solid-phase support is effectively a ternary component of the reaction, the nonspecific interaction of antigens or antibodies with the solid phase may interfere both in binding and in inhibition experiments, especially when the affinity of the antigen-antibody interaction is moderate or low. 1 The above problems do not apply to homogeneous assays in which both reactants are present in solution phase. However, a prerequisite for binding and inhibition studies in solution phase is the effective separation of bound from free antigen or antibody. Methods used for the precipitation of polyclonal antibodies may not be suitable for monoclonal antibodies. Precipitation by second antibodies is limited by the formation of soluble complexes between the monoclonal IgG and anti-mouse IgG antiserum at high monoclonal antibody excess. The precipitation of antigenantibody complexes by ammonium sulfate or polyethylene glycol is complicated by high levels of nonspecific binding and incomplete precipitation of the complexes .6 In order to perform binding and inhibition experiments in solution phase using a constant amount of antigen and variable amounts of antibody, a general method for the selective precipitation of antigen would be desirable. Here we describe the use of the avidin-biotin system to selectively and quantitatively precipitate either biotin-labeled antigen or antibody from solution by the addition of avidin at optimal ratios of avidin to biotinylated reactant. Using this technique, inhibition studies and saturation experiments can be performed in solution phase at constant concentrations of biotinylated antibody or antigen. General Procedures
Monoclonal antibodies (MAbs) against the carcinoembryonic antigen (CEA) are produced as described. 7 MAbs are elicited by immunizations either with purified CEA (MAbs 1, 3, and 5) or with the transplanted colon carcinoma cell line T84 (MAbs 2 and 4). CEA is purified according to 3 F. J. Stevens, J. Jwo, W. Carperos, H. K6hler, and M. Schiffer, J. lmmunol. 137, 1937 (1986). 4 A. D. Smith and J. E. Wilson, J. lmmunol. Methods 94, 31 (1986). 5 H. C. Vaidya, D. N. Dietzler, and J. H. Ladenson, Hybridoma 4, 271 (1985). 6 C. Wagener, B. R. Clark, K. J. Rickard, and J. E. Shively, J. Immunol. 130, 2302 (1983). 7 C. Wagener, L. Wickert, and J. E. Shively, this volume [59]; C. Wagener, Y. H. J. Yang, F. G. Crawford, and J. E. Shively, J. Immunol. 130, 2308 (1983).
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Coligan et al. ,8 and the T84 cell line is that of Murakami and Masui. 9 IgG fractions of MAbs are purified from ascites fluid by protein A affinity chromatography according to Ey et al. 1° The IgG concentration is calculated from the absorbance of the solutions at 280 nm, assuming an extinction coefficient [1% (w/v); 1 cm] of 14.2.11 CEA concentrations and concentrations of Fab fragments are determined by amino acid analysis.
Binding and Inhibition Studies with Biotin-Labeled Antibodies and Avidin as Precipitating Agent Materials
PBS: 50 m M sodium phosphate buffer (pH 7.0) containing 0.15 M sodium chloride and 0.1% NaN3 Avidin-PEG: avidin (Sigma Chemical Co., St. Louis, MO) 0.1 mg/ml in PBS containing 5% (w/v) polyethylene glycol (PEG 6000, Sigma) BNHS: N-hydroxysuccinimidobiotin (Sigma) DMF: dimethylformamide (J. T. Baker, Phillipsburg, NJ), distilled from ninhydrin Preparation of lesI-Labeled Antigen. Purified CEA is lzSI-labeled by the chloramine-T procedure of Hunter and Greenwood. 12 Biotinylation Procedures. MAb is biotinylated as described elsewhere in this volume. 7 As a biotinylated carrier protein, either normal rabbit or goat serum can be used. After dialysis against PBS, carrier serum (2 ml) is treated with 2 ml of freshly made aqueous BNHS, prepared by dissolving 20 mg of BNHS in 0.5 ml of DMF and diluting to 2 ml with 1.5 ml of distilled water. The remainder of the procedure is as described for preparing the biotinylated MAbs. Assay Overview. The protocol is similar to that described by Clark and Todd 13 and Wagener et al. 6 Determination of Kaff is performed in two steps. First, binding curves for each MAb are determined. A constant amount of radiolabeled antigen (0.5 ng of [IzsI]CEA) is titered against serial 2-fold dilutions of MAb. The concentration of MAb IgG yielding half-maximal binding is determined. This concentration is used in the second step. The second step comprises a competitive RIA involving the s j. E. Coligan, J. T. Lautenschleger, M. L. Egan, and C. W. Todd, lmmunochemistry 9, 377 (1972). 9 H. Murakami and H. Masui, Proc. Natl. Acad. Sci. U.S.A. 77, 3464 (1980). to p. L. Ey, S. J. Prowse, and C. R. Jenkin, lrnmunochemistry 15, 429 (1978). H H. N. Eisen, E. S. Simms, and M. Potter, Biochemistry 7, 4126 (1968). 12 W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). 13 B. R. Clark and C. W. Todd, Anal. Biochem. 121, 257 (1982).
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addition of increasing amounts of unlabeled antigen (0.5-100 ng of CEA) which competes with a fixed amount of radiolabeled antigen (0.5 ng of [125I]CEA). The present RIA version required the separation of free from bound radiolabeled antigen. This is accomplished by precipitation of the biotinylated MAb with avidin. Since only a minute amount of MAb is present, the precipitation is driven to completion by adding a biotinylated carrier protein mixture. Binding RIA Protocol with Biotinylated Antibodies. The RIA is performed in 400-/xl polypropylene tubes (Bio-Rad, Richmond, CA) which can be tightly capped and centrifuged in a Beckman microcentrifuge. Serial 2-fold dilutions of biotinylated MAb (50 /zl; 0.01-1,000 ng) in biotinylated carrier serum (I : 800 dilution in either underivatized normal rabbit or goat serum diluted 1 : 40 in PBS) are incubated with 200/xl of PBS containing 6.5% PEG and 72 m M EDTA and with 10/xl of [125I]CEA (0.5 ng in PBS; -104 cpm) for 18 hr at 37 °. The [125I]CEA cocktail may include 57Co as a volume marker if desired. Biotinylated MAb and biotinylated MAb-antigen complexes are coprecipitated with biotinylated carrier proteins by addition of 10/zl of avidin (1.0 mg/ml in PBS containing 5% PEG 6000), and the mixture is incubated for 1 hr at room temperature. The tubes are centrifuged, the supernatant fluids are removed, and the precipitates are counted. The binding of constant amounts of [125I]CEA by decreasing amounts of monoclonal antibody IgG is shown in Fig. 1. At high IgG excess, MAb 3 binds over 90% of labeled antigen, thus indicating quantitative precipitation of the antibody. The broad plateau regions of the binding curves for MAbs 2 and 4 show that nonprecipitable soluble complexes between biotin-labeled monoclonal antibody and avidin were not observed when excess IgG concentrations were used. Nonspecific precipitation of labeled CEA as determined by the use of biotin-labeled carrier without specific antibody was in the range of 1-3%. CEA Inhibition Assay Protocol. The assay tubes, centrifugation protocol, and reagents are the same as in the previous section. To each assay tube (in duplicate) are added 10/zl of 125I-labeled CEA (0.5 ng in PBS) and 50/xl of biotinylated MAb at concentrations indicated in the legend to Fig. 2. The dilution of MAbs is performed in a 1 : 800 dilution of biotinylated goat serum in either underivatized normal goat or rabbit serum (1 : 40 in PBS) and 200/zl o f a CEA inhibitor solution (0.5-100 ng in PBS with 6.5% PEG). The mixture is incubated, precipitated with 10/zl of avidin (1.0 mg/ ml in PBS with 5% PEG), and counted as before. For inhibition experiments, IgG concentrations corresponding to 4060% of the maximum binding of [125I]CEA (see plateau regions in Fig. 1)
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100
9O
81) o
i
70
•
•
•
6O
i10
0
10000
I 1,000
I 100
I 10
I
0.1
0.01
igo (rig]
FIG. 1. Binding curves for radiolabeled antigen to MAb in a solution-phase, avidinbiotin-based RIA. Radiolabeled CEA was titered with decreasing dilutions of biotinylated MAbs. The biotinylated MAbs and M A b - C E A complexes were precipitated with avidin in the presence of 5% PEG. ©, MAb 1; 41, MAb 2; [~, MAb 3; I , MAb 4; &, MAb 5. [From C. Wagener, B. R. Clark, K. J. Rickard, and J. E. Shively, J. lmmunol. 130, 2308 (1983), with permission.]
are chosen for MAbs 1, 2, 3, and 5. Because of the low binding plateau of MAb 4, inhibition experiments are performed (in this case) at a higher relative binding of [12sI]CEA. The inhibition curves for MAbs 1-5 are shown in Fig. 2. Calculation of Affinity Constants. Affinity constants are calculated from inhibition experiments by using a general form of the equation derived by Miiller. 14The original equation refers to 50% inhibition of tracer binding, whereas the general form of the equation can be applied to a broader range of inhibitor concentrations. Thus, gaff =
(1 --
b(1 - r ) - r(1 - b) r)(1 - b)[r(Tt + It) -- bTt]
where Tt is the total concentration of tracer, It the total concentration of i4 R. MOiler, J. lmmunol. Methods 34, 345 (1980).
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SOLUTION-PHASE IMMUNOASSAYSFOR CEA
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100
cO .Q J= ¢:
5O
0.
I
1
I
10
100
1,000
CEA [ng] FIG. 2. Inhibition curves for calculating affinity constants of five MAbs. Constant amounts of radiolabeled CEA and biotinylated MAb were incubated with increasing amounts of unlabeled CEA. The biotinylated MAb and MAb-CEA complexes were precipitated with avidin in the presence of 5% PEG. The IgG concentrations of MAbs used were as follows: ©, MAb 1, 26.5 ng IgG per tube; O, MAb 2, 1.4 ng IgG per tube; [], MAb 3, 96.9 ng IgG per tube; II, MAb 4, 0.9 ng IgG per tube; A, MAb 5, 171.6 ng IgG per tube. [From C. Wagener, B. R. Clark, K. J. Rickard, and J. E. Shively, J. ImmunoL 130, 2308 (1983), with permission.]
inhibitor, b the fraction of tracer bound in the absence of inhibitor, and r the fraction of tracer bound in the presence of inhibitor. A model calculation is given for MAb 2. Inhibition experiments were performed with 0.5 ng o f [~25I]CEA per tube (260/xl). In the absence of inhibitor, 31.1% o f labeled CEA was bound (b = 0.311). In the presence of 1.94 ng of unlabeled CEA, 22.9% of labeled CEA was precipitated (r = 0.229). This corresponds to a percent binding inhibition of (1 - r/b) x 100 = 26.4% (see Fig. 2). Assuming an Mr of 180,000 for CEA, the concentration of tracer is 10.7 x 10 -12 M, and the concentration of inhibitor is 41.5 × 10 -12 M. Using the above formula, Kaff = 1.8 x 10 m M -1. In the presence o f 7.7 ng o f unlabeled CEA (164.5 × 10 -~2 M), 12.1% of the tracer was precipitated (r = 0.121), corresponding to a percent inhibition of 61.1% (see Fig. 2). F r o m these values, K, ff = 1.8 x 10 ~0 M -1 is calculated. As shown in Fig. I, the fraction of radiolabeled CEA bound at high IgG excess is different for each of the MAbs. Since over 90% of unlabeled CEA is bound by the different MAbs, 6 the decrease of the immunoreactiv-
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TABLE I AFFINITY CONSTANTS OF MONOCLONAL ANTI-CEA ANTIBODIESa'b
MAb
Affinityconstants (M-~)
1
3.7 ×
2 3 4 5
1.8 × 1.0 × 2.6 × 3.8 ×
109 10I° 108 101° 108
For calculation, see text. b Data from C. Wagener, B. R. Clark, K. J. Rickard, and J. E. Shively, J. lmmunol. 130, 2302 (1983).
ity of radiolabeled CEA is most probably due to partial radioiodination damage of individual epitopes. If only 70% immunoreactivity of labeled CEA is assumed for MAb 2 (see plateau region in Fig. I), the calculated affinity constants increase to 2.2 × 10 l° to 2.5 x 1010M -l, depending on the inhibitor concentration used for calculation. For MAb 4, the affinity constants obtained for 30% immunoreactivity of tracer are in the range of 4.9 x 1010 to 5.7 × 10 l° M -1. The affinity constants calculated for 5 MAbs from inhibition experiments without corrections for immunoreactive tracer are shown in Table I. The K ~ values for MAbs 1, 2, and 4 are sufficiently high to warrant their use in an immunoassay for CEA. The final choice of the optimum MAb is also dependent, however, on the required epitope specificity. A discussion of this topic and the problem of cross-reacting antigens is given by Wagener et al. 6
Determination of Epitope Specificities and Kaff Using Biotinylated CEA and Avidin as Precipitating Agent Reagents PBS: see above Avidin-PEG: avidin (Sigma) 5 mg/ml in PBS containing 6.5% (w/v) PEG 6,000 (Sigma) B N H S and DMF: see above Preparation of 125I-Labeled Fab fragments. Fab fragments are prepared from MAbs 2 and 4 according to the procedure described by
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Parham.~5 Molecular weight determinations and purity control are performed by SDS-PAGE using the Phast System (Pharmacia) with preformed gradient gels of 8-25% acrylamide. Under nonreducing conditions, molecular weights of 48K are obtained for the Fab fragments of both antibodies. The procedure is not generally applicable to all monoclonal antibodies as MAb 1 is completely digested by pepsin treatment. For iodination, the concentration of Fab fragments is determined by amino acid analysis. The Fab fragments are labeled with ~25I by the chloramine-T method according to Bolton and Hunter. 16The protein iodination is stopped by the addition of excess free tyrosine. The specific activity is approximately 2 MBq//zg for both Fab fragments. Preparation of Biotinylated CEA and Carrier Protein. An equal volume of CEA (1 mg/ml in PBS, 0.1 ml) is added to a freshly prepared solution of BNHS (3.5 mg/ml, 0.1 ml). The preparation of the BNHS solution and the biotinylation procedure are performed as described for the monoclonal antibodies.7 Biotin labeling of carrier protein (normal rabbit serum) is performed as described above. RIA Overview. The RIA procedure employed here represents an inverse version of the RIA described above. Prior to epitope analysis and determination of Kaff, titration curves are established. Constant amounts of radiolabeled Fab fragments are titered against serial 2-fold dilutions of biotinylated CEA. From the plateau region, the fraction of immunologically active radiolabeled Fab fragment is determined. For inhibition experiments, the CEA concentration yielding 15-27% binding of labeled Fab is determined. This concentration is used for competitive inhibition studies involving the addition of increasing amounts of unlabeled monoclonal antibody IgG or Fab fragments respectively, which compete for the binding of a fixed amount of labeled Fab fragment at constant concentrations of biotinylated CEA. In case of the present RIA version, separation of bound and free antibody is required. This is accomplished by precipitation of the biotinylated antigen with avidin in the presence of a biotinylated carrier protein. The method allows the determination of epitope specificities and affinity constants in a single experiment. Binding Assay Protocol. To 200/zl of 6.5% PEG (w/v) in PBS containing biotinylated CEA at 2-fold serial dilutions, an aliquot (50/xl) of PBS containing labeled Fab fragments (-l04 cpm, corresponding to -0.1 ng), 15 p. Parham, in "Handbook of Experimental Immunology, Immunochemistry" (D. M. Weir, L. A. Herzenberg, C. Blackwell, and L. A. Herzenberg, eds.), Vol. 1, p. 14.1. Blackwell, Oxford, 1986. t6 A. E. Bolton and W. M. Hunter, in "Handbook of Experimental Immunology, Immunochemistry" (D. M. Weir, L. A. Herzenberg, C. Blackwell, and L. A. Herzenberg, eds.), Vol. 1, p. 26.1. BlackweU, Oxford, 1986.
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7O
t~ LL
~5 t~ ._= t-
32O0
IO0 3.1 Biotin-labeled CEA [ng]
0.1
FIG. 3. Binding curve of radiolabeled Fab fragments of MAbs to antigen in a solutionphase, avidin-biotin-based RIA. Radiolabeled monoclonal Fab fragments were titered with increasing dilutions of biotinylated CEA. The biotinylated CEA and CEA complexes were precipitated with avidin in the presence of 5% PEG. O, MAb 2; II, MAb 4. [From U. Kriiger, L. Wickert, and C. Wagener, J. lmmunol. Methods 117, 25 (1989), with permission.]
normal rabbit serum (1:40 dilution in PBS), and biotin-labeled normal goat serum (1 : 100 dilution in PBS) is added. The carrier serum is prepared as described in the previous section. The assay mixture is incubated for 18 hr at 37°. Subsequently, 10/zl of 6.5% PEG (w/v) in PBS containing 50/zg egg-white avidin (Sigma) is added. After 1 hr at room temperature, the mixture is centrifuged (10 min, 15,000 g). A sample (200/zl) of the supernatant is removed. The remaining volume is counted in a 3' scintillation counter. The percentage of radioactivity precipitated is calculated from the total counts and the volume removed, thus obviating the addition of a volume marker. Two-fold serial dilutions of biotinylated CEA are performed in the range of 6400-0.012 ng per tube (Fig. 3). For the MAb 2 Fab fragment a plateau was reached with 400-1600 ng of biotinylated CEA and with 3200-6400 ng CEA for the MAb 4 Fab fragment. Depending on individual Fab preparations and radioiodination, 40-60% of the radiolabeled Fab fragment is bound in the plateau region. The finding that 100% binding is
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not reached at high antigen excess may be due to the following: (1) the presence of nonimmune IgG in the ascitic fluid, (2) partial degradation during the preparation of Fab fragments, and (3) damage by the radioiodination procedure. The possibility that soluble complexes between biotinylated CEA and avidin may be responsible for the binding of less than 100% of the labeled Fab fragments has been excluded for the concentration range of biotinylated CEA used for the binding studies. Less than 10% of total biotinylated CEA is present in the supernatant when levels of 5,000 ng per tube are used. At higher amounts of antigen, soluble complexes between avidin and CEA-biotin may develop. The nonspecific binding is less than 3%. Inhibition Assay Protocol. For inhibition experiments, concentrations of CEA-biotin of 5-25 ng per tube are chosen. Under these conditions, 15-27% of the total radioactivity is bound. To 200/zl of 6.5% PEG (w/v) in PBS containing the appropriate amount of biotinylated CEA and 2-fold serial dilutions of unlabeled monoclonal antibody IgG or Fab fragments, 50 ~1 of PBS containing labeled Fab fragments (-104 cpm), normal rabbit serum (1:40 dilution), and biotin-labeled normal goat serum (1 : 100 dilution) is added. The rest of the procedure is identical to that described above for the binding assay protocol. A representative inhibition curve is shown in Fig. 4. Using labeled Fab fragments of MAb 2, increasing concentrations of unlabeled MAb 2 Fab fragments result in complete binding inhibition. The remaining MAbs do not inhibit binding of the labeled Fab fragments. These results are comparable with those obtained by competitive solid-phase a s s a y s . 7A7 Calculation of Affinity Constants. In addition to epitope specificities, K~f can be calculated by using the general form of the equation derived by Mialler 14 given above. In the case of inhibition experiments performed with constant amounts of biotin-labeled antigen and uSI-labeled Fab fragments in the presence of increasing amounts of unlabeled Fab fragments, It is the concentration of unlabeled Fab fragments, Tt the concentration of labeled Fab fragments, b the fraction of tracer bound in the absence of inhibitor, and r the fraction of tracer bound in the presence of inhibitor. In the inhibition experiment presented in Fig. 4, Tt = 0.17 ng per tube. In the absence of inhibitor, 26.1% of the radioactivity is precipitated. In the presence of 8.85 ng per tube of unlabeled Fab fragments, 14.8% of the tracer is precipitated, corresponding to a percent binding of 56.7% (see Fig. 4). Given a reaction volume of 260/zl and an Mr for Fab fragments of 4 8 , 0 0 0 , T t -- 13.8 x 10-12 M, It = 709 x 10-J2M, b = 0.261, and r = 0.148. From these values, Kaff = 1.7 x 109 M -I. 17 C. Wagener, U. Fenger, B. R. Clark, and J. E. Shively, J. ImmunoL Methods 68~ 269 (1984).
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APPLICATIONS
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90
-~ 8O ,% 70
'~ 50
~ ~o ~ 3o 20 10
1
2
k
8
16
32
6/,
128
256
~ga[ng] Fab [rig]
FIG. 4. Inhibition curves for determination of epitope specificities and affinity constants of monoclonal antibodies. Constant amounts of radiolabeled Fab fragments of MAb 2 and biotinylated CEA were incubated with increasing amounts of unlabeled intact MAbs or Fab fragments, respectively. Biotinylated CEA and CEA-antibody complexes were precipitated with avidin in the presence of 5% PEG. The total amount of radiolabeled Fab fragments was 0.1 ng per tube, and the concentration of biotinylated CEA was 6.25 ng per tube. S, Fab fragment of MAb 2; the rest of the symbols are as in Fig. 1. [From U. Kri~ger, L. Wickert, and C. Wagener, J. lmmunol. Methods 117, 25 (1989), with permission.]
As shown in Fig. 3, only 58% of labeled Fab fragments from MAb 2 is precipitated at high excess of biotin-labeled CEA. It therefore follows that only part of the labeled Fab is immunoreactive. As discussed above, the reason for partial immunoreactivity may be the presence of nonimmune IgG in the IgG preparation from ascitic fluid, partial damage during the preparation of Fab fragments, or damage during radioiodination. Since the immunoreactive fraction of the unlabeled Fab fragments is unknown, K ~ can be estimated assuming partial immunoreactivity either of labeled Fab fragments alone or of both labeled and unlabeled Fab fragments. Assuming 58% immunoreactivity of the labeled fragment and 100% immunoreactivity of the unlabeled fragment, Tt = 8.02 × 10-~2 M, It = 709 × 10-12 M, b = 0.261/0.58 = 0.45, and r = 0.148/0.58 = 0.255. From these
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values, Kaff = 2.7 × l09 M-L If 58% immunoreactivity of both tracer and inhibitor is assumed, then It = 411 x 10-12 M, and Kale = 4.6 × l09 M -1. The calculated affinity constants are lower than that shown for the intact antibody in Table I. This finding may be due to a variety of reasons, including the use of Fab fragments instead of intact antibodies, the presence of nonimmune IgG in the ascitic fluid, and/or the corrections made for the immunoreactive fraction of the radiolabeled Fab fragments or antigen. Acknowledgments This research was supported by grants from the Deutsche Forschungsgemeinschaft, Wa473/4-1, and from the National Large Bowel Program, National Cancer Institute, Grant CA 37808. We wish to thank Karen Rickard for expert technical assistance, and Dr. Y. H. Joy Yang for production of monoclonal antibodies.
[61] I m m u n o a s s a y s for D i a g n o s i s o f I n f e c t i o u s D i s e a s e s
By ROBERT H. YOLKEN Introduction Traditionally, the diagnoses of infectious diseases have been based on the isolation of the infecting microorganisms in pure culture. Recently, however, infectious disease diagnoses have relied more heavily on the direct identification of infecting organisms in blood and other body fluids of the ill individual. The diagnosis of infectious diseases by the direct detection of microorganisms has been particularly important for the identification of viral agents since these agents are particularly difficult to detect in short periods of time by means of standard cultivation methods. 1 Direct detection methods are also highly useful in the detection of bacterial, parasitic, and fungal pathogens that are fastidious or difficult to cultivate under standard laboratory conditions. 2 Most of these assays have relied on the measurement of the binding of antigens to defined antimicrobial antibodies. Immunoassays have a number of advantages for infectious disease diagnosis. These advantages are based on the sensitivi D. A. Fuccillo, I. C. Shekarchi, and J. L. Sever, in "Manual of Clinical Laboratory Immunology" (N. R. Rose, H. Friedman, and J. L. Fahey, eds.), 3rd ed., p. 489. American Society for Microbiology, Washington, D. C., 1986. 2 N. J. Schmidt, Med. Clin. North Am. 67, 953 (1983).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
