[59]

EIAs FOR CEA

507

TABLE I REPRODUCIBILITY OF RESULTS WITH PROTOCOL I V TO MEASURE VARIOUS LEVELS OF I g E IN DIFFERENT-DAY RUNS

Test; day run (peak A450,m)a IgE (IU/ml) 200 20 2 0.2 0.02

1 0.75 0.38 0.27 0.10

[0.71-0.77] b [0.35-0.40] [0.24-0.29] [0.09-0.11] n.d. c

2 0.77 0.32 0.19 0.07 0.03

3

[0.73-0.79] [0.28-0.36] [0.16-0.24] [0.05-0.09] [0.02-0.04]

0.68 0.39 0.26 0.08 0.03

[0.64-0.72] [0.36-0.41] [0.24-0.28] [0.06-0.10] [0.02-0.05]

° Tests were run in triplicate in different days. Values shown represent the peak of the reading curve after addition of substrate for peroxidase. b Arithmetic mean [range; n = 3]. c Not done.

(Fig. 2). 13 This was a consistent trend confirmed in different runs performed on different days using either the same or different reagent batches. For example, the mean factor of increase obtained with Protocol IV compared with I was 3.20 (n = 4). Protocol IV also consistently detected low levels of IgE (Table I). 13 IgE levels are expressed in International Units (IU), based on a World Health Organization reference serum. One International Unit is equivalent to 2.4 ng IgE.

[59] Quantification of Carcinoembryonic Antigen in Serum and Analysis of Epitope Specificities of Monoclonal Antibodies By

CHRISTOPH WAGENER, L U C I A WICKERT,

and JOHN E . SHIVELY

The gene coding for the carcinoembryonic antigen (CEA) is a member of the irnmunoglobulin supergene family.l Similar to other members of this family, the CEA gene is a member of a subfamily of genes coding for glycoproteins of close structural relationship. Monoclonal antibodies i R. J. Paxton, G. Mooser, H. Pande, T. D. Lee, and J. E. Shively, Proc. Natl. Acad. Sci. U.S.A. 84, 920 (1987).

METHODS IN ENZYMOLOGY, VOL. 184

Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.

508

APPLICATIONS

[59]

(MAbs) induced against CEA may be directed against CEA-specific epitopes or more or less common epitopes present on CEA as well as on structurally related antigens. 2 In this regard, a logical sequence of experimental steps can be designed in order to use monoclonal antibodies for the quantification of a single antigen or possibly several antigens in body fluids. The following strategy has thus been used for the characterization of monoclonal anti-CEA antibodies: (1) selection of MAbs with different epitope specificities3,4; (2) binding of the selected MAbs to different antigens of the CEA family, e.g., in Western blots or related techniquesS.6; (3) determination of affinity constants of antibodies with interesting antigenbinding patterns7; (4) establishment of monoclonal antibody-based immunoassays. For many techniques involved in the approach outlined above, either antigen or antibody have to be tagged by a label. Radiolabels such as 1z5I have found wide application. However, because of the usually high number of antibodies to be handled and the time course of the experiments, the instability of radiolabels represents a significant drawback. In addition, radioactive hazards have to be taken into account. We have found the biotin label to be adequate or superior to radioactive labels in several aspects. Once antibodies and antigen are labeled by biotin, all of the experiments involved in the above approach can be performed, including initial antibody screening, determination of affinity constants, and double monoclonal, sandwich-type immunoassays. In this context, the use of avidin-enzyme conjugates for solid-phase immunoassays and the complementary application of avidin for the precipitation of either antigen or antibody in solution-phase assay 7 are particularly helpful. Here we describe the use of the avidin-biotin system for the initial screening for epitope specificities of MAbs and for their ultimate use in double-monoclonal immunoassays for the sensitive detection of antigens in biological fluids. The precipitation of biotin-labeled antibodies or antigens from solution for the determination of affinity constants and epitope specificities is described elsewhere in this volume. 7 2 j. E. Shively and J. D. Beatty, CRC Crit. Rev. Oncol./Hematol. 2, 355 (1985). 3 C. Wagener, Y. H. J. Yang, F. G. Crawford, and J. E. Shively, J. Immunol. 130, 2308 (1983). 4 C. Wagener, U. Fenger, B. R. Clark, and J. E. Shively, J. lmmunol. Methods 68, 269 (1984). 5 M. Neumaier, U. Fenger, and C. Wagener, J. Immunol. 135, 3604 (1985). 6 M. Neumaier, U. Fenger, and C. Wagener, Mol. Immunol. 22, 1273 (1985). 7 C. Wagener, B. R. Clark, K. J. Rickard, and J. E. Shively, J. Immunol. 130, 2302 (1983); C. Wagener, U. Kriiger, and J. E. Shively, this volume [60].

[59]

EIAs FOR CEA

509

General Procedures M o n o c l o n a l Antibodies. The production and characterization of MAbs against CEA are described in detail by Wagener et al. 3 In the original publications, the following abbreviations for the monoclonal antibodies were used: MAb 1, CEA.66-E3; MAb 2, T84.1-E3; MAb 3, CEA.41C-12. l-D8; MAb 4, T84.66-A3.1-HI 1; MAb 5, CEA.281-H5; MAb 6, CEA. 1l-H5. The IgG fractions of MAbs are purified from ascitic fluids over protein A-Sepharose. 8 The purified IgG can be dialyzed against water or a low ionic strength buffer (1 mM sodium phosphate, pH 7.0), lyophilized, and stored at - 2 0 °. The A280of a 10 mg/ml solution of mouse IgG is 14. 9 CEA is purified according to Coligan et al. l°

Determination of Epitope Specificities Using Biotinylated Monoclonal Antibodies in Competitive Solid-Phase Enzyme Immunoassay Materials

PBS: 50 mM sodium phosphate (pH 7.2) containing 0.15 M NaC1 and 0.1% NaN3 Na2CO3, 0.2 M (pH 9.3-9.4) BSA-PBS: PBS containing 1% (w/v) bovine serum albumin (Sigma Chemical Co., St. Louis, MO) PBR (phosphate buffer-rabbit serum): 0.2 M potassium phosphate (pH 6.5) containing 20% (v/v) normal rabbit serum APC: avidin-peroxidase conjugate (Sigma) diluted 1 : 100 in PBR 3 M HCI: dilute 260 ml concentrated HC1 in 1 liter of distilled water 6 mM H202: dilute 68 ~1 of 30% H202 (w/v) in 100 ml of distilled water Citrate buffer: 0.1 M sodium citrate (pH 5.0) Substrate 1: 6 mM H202 (freshly prepared) and 40 mM o-phenylenediamine (Sigma) in citrate buffer BNHS: N-hydroxysuccinimidobiotin (Sigma) DMF: dimethylformamide (J. T. Baker, Phillipsburg, NJ), distilled from ninhydrin Preparation o f Biotin-Labeled M A b . A modification of the method of Clark and Todd ~ is employed. An equal volume of MAb (70 mg/ml in s p. L. Ey, S. J. Prowse, and C. R. Jenkin, lmmunochemistry 15, 429 (1978). 9 H. N. Eisen, E. S. Simms, and M. Potter, Biochemistry 7, 4126 (1968). ~0j. E. Coligan, J. T. Lautenschleger, M. L. Egan, and C. W. Todd, lmmunochemistry 9, 377 (1972). ii B. R. Clark and C. W. Todd, Anal. Biochem. 121, 257 (1982).

510

APPLICATIONS

[59]

PBS, 0.1 ml) is added to a freshly prepared solution of BNHS (1 mg/ml, 0.1 ml). 12The BNHS solution is immediately added to the MAb dropwise with constant stirring, allowed to react 2 hr at room temperature, diluted to 1 ml with PBS, and dialyzed versus 3 to 4 changes of PBS. The degree of biotinylation can be changed by increasing or decreasing the amount of BNHS by a factor of 2-3 if desired. Aliquots of the biotinylated MAb are stored at - 2 0 ° until used. EIA Protocol. The protocol is similar to that described by Wagener et al. 4 Wells of a 96-well polyvinyl microtiter plate are coated with 100/xl of CEA (20/zg/ml) in sodium carbonate buffer for 12-18 hr at room temperature. Nonspecific binding is blocked by incubating antigen-coated wells with BSA-PBS for 2 hr at 37° followed by rinsing with PBS 5 times. A constant amount (50/zl; see below) of biotinylated MAb is added to 50/zl of serial 2-fold dilutions of unlabeled test MAb in PBS-BSA. Aliquots (50/zl) are added to the wells, after which the plate is incubated for 2 hr at 37° and washed 3 times with PBS. The wells are washed once with PBR, incubated with APC conjugate (100/zl of conjugate diluted 1 : 100 in PBR) for 2 hr at 37°, washed 5 times with 0.1 M citrate buffer (pH 5.0), and incubated with 100/xl of substrate for 30 min at room temperature in the dark. The reaction is stopped with 100/zl of HC1, and the absorbance is read at 492 nm. The dilution of biotinylated antibody was chosen to give a maximum A492 value of 0.8-1.5 in the absence of unlabeled MAb. Controls include wells not coated with antigen, incubations with no MAb, and incubations with normal mouse serum. Nonspecific binding should not exceed 0.05 absorbance units. EIA Results. Sample results for epitope analysis of four MAbs are shown in Fig. 1. The results are equivalent to those published for a competitive solid-phase radioimmunoassay.3 See next section for further discussion.

Determination of Epitope Specificities Using Biotinylated Antigen in Competitive Solid-Phase Enzyme Immunoassay Materials

PBS: 10 m M sodium phosphate buffer (pH 7.4) containing 0.15 M NaC1 PBS-Tween: PBS plus 0.05% (v/v) Tween 20 (Sigma) BSA-PBS: PBS containing 1% (w/v) bovine serum albumin (Sigma) ~2The BNHS (2.0 mg) is addedto 0.5 ml of DMF, stirredfor 5 min, and dilutedto 2.0 ml with 1.5 ml of water.

[59] A

EIAs VORCEA

Biotinyloted

0.9 0.8

~

0.6

Mob

1

511

C Biotinyloted

1.or~b2

Mob

5

=

/-~b



3

b2

°'e~-'~

*

-

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O6. ~ 2

*,

0.5

'~ 0.4 0.3 0.2 ~z I I I I 2625 1313 656 328 164 82

Mob

I 41

I 20

I 10

M°bl~

I 5

~,~

"

" / 6

I I I I I 2625 1313 656 328 164 82

2

O

Biotinyloted

1.4

Mob

I 41

I 20

I I0

I 5

I 2

4

1.6

/--Mob 3

1.*k£

~

0.4 0.3 0.2 0.1

"-Mob 1

0.1

B Biotinyloted 1.6[Mob 5

"o

o

.

1.2 I~ 1.0

4 0.4

~[ 0.6 0.4

~-Mob 2

/

0.2

Mob 4

0.2

2625 1313 656 328 164 82

ng ]QG

i 41

i

20

i 10

i 5

i 2

I I I I I I 2625 1313 656 328 164 82 41 ng IQG

I 20

I 10

I 5

FIG. 1. Epitope analysis using a competitive avidin-biotin enzyme immunoassay. The wells of a microtiter plate were coated with C E A and incubated with a biotiny|ated MAb preparation and decreasing dilutions of the given unlabeled MAb. [From C. Wagener, U. Fenger, B. R. Clark, and J. E. Shively, J. Immunol. Methods 68, 269 (1984), with permission.]

Reaction buffer: 20 mM sodium phosphate (pH 6.8) Coating buffer: PBS containing 0.1% NaN3 APC: avidin-peroxidase conjugate (Sigma) diluted 1:100 in PBSTween 1% H202: dilute 30% H202 1 : 30 in distilled water Substrate 2:1 mg/ml of 5-aminosalicylic acid (Sigma) in preheated (56°) reaction buffer; add 5 mg active charcoal per 100 ml and filter; add 100/zl of freshly prepared 1% H202 per 10 ml of above 3 M NaOH: dissolve 12 g of NaOH in 100 ml of distilled water BNHS: N-hydroxysuccinimidobiotin (Sigma) DMF: dimethylformamide, distilled from ninhydrin EIA Protocol. The wells of a 96-well polystyrene microtiter plate (Costar, Cambridge, MA) are coated with MAb (5 /zg MAb/ml in coating

I 2

512

APPLICATIONS

[59]

buffer) for 2 hr at 37° and overnight (8-12 hr) at 4 °. Biotinylated CEA (50/xl of a 1.0/zg/ml solution) is added to separate test tubes containing 50 /zl of the test MAb dilution (0.5-1000 ng of IgG is a good range), and the tubes containing the biotinylated C E A - M A b mixture are incubated overnight at 37°. The plates are washed 3 times with PBS, incubated for 1 hr at room temperature with 150/zl of B S A - P B S to block nonspecific binding, and washed 3 times with PBS. The biotinylated C E A - M A b mixture (100 /~1) is added to the wells and incubated for 90 min at room temperature. The plates are washed 5 times with PBS-Tween, treated with 100/xl of APC, incubated for 2 hr at room temperature, and washed as follows: (1) rinse once with PBS; (2) fill wells to top with PBS, wait 5 min, discard, and repeat 2 times; (3) rinse wells with distilled water 3 times. The wells are then incubated with 200/xl of substrate 2 for 15 min, and the reaction is stopped with 100/zl of NaOH. Absorbance is read at 450 nm. The usual controls are run (see previous section). Nonspecific binding should be less than 0.01 absorbance units. Comments. The inhibition analysis of five MAbs versus two MAbs coated on microtiter wells is shown in Fig. 2. The binding of biotinylated CEA to immobilized MAb 4 is inhibited by low amounts of the identical unlabeled antibody (Fig. 2A). At higher amounts of IgG, MAb 1 also quantitatively inhibits the binding of biotinylated CEA to MAb 4. MAb 2 shows partial binding inhibition at high IgG excess. Except for the latter inhibition by MAb 2, the results are comparable with those of the inhibition assay using biotinylated MAb 4 (Fig. ID). The binding of biotinylated CEA to wells coated with MAb 2 is not inhibited by MAbs 1, 3, 4, or 5 (Fig. 2B). This result is equivalent with the result shown in Fig. 1B for the competition assay with biotinylated MAb 2. Discussion of Inhibition EIA Results for Epitope Analysis of Monoclonal Antibodies. The results of the above inhibition assays are influenced by several factors, such as the affinity of different MAbs for the respective epitopes; the relative position of the respective epitopes; the number of epitopes recognized by the different MAbs; the effect of immobilization on the antigen-antibody interaction; and the effect of labeling on the antigen-antibody interaction. Negative results in the inhibition assays do not necessarily mean that the antibodies in question are directed against unrelated epitopes. If the affinity of the labeled antibody (first assay version) or of the immobilized antibody (second assay version) is much higher than the affinity of the competing antibody, negative or inconclusive results may be obtained even at high excess of the low-affinity antibody. For this reason, labeled and competing antibodies should be exchanged against each other in the first assay version. The exchange of competing and immobilized antibod-

[59]

EIAs FOR CEA

513

A Mob 4 on Plote

0.50 1

/-Mob

0.40

-

°"°2Y / ~

1000

~

250

,. ~. . .w . I . 62

I

t5

I

4

I~

I

I

1

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0.40

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~Mab4

o 0

0.50 0.20 0.10

~W.

-

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~ ~-I I I l I I I I

250

62

15

4

1

ng IgG FIG. 2. Epitope analysis using biotinylated antigen. Microtiter wells coated with monoclonal antibody IgG were incubated with constant amounts of biotinylated CEA and decreasing dilutions of MAbs. The bound biotinylated CEA was detected with the avidinperoxidase conjugate. (From J. E. Shively, C. Wagener, and B. R. Clark, this series, Vol. 121, p. 459.) ies in assay version 2 does not demand additional labeling procedures. Occasionally, MAbs do not bind efficiently to the solid phase. In these cases, assay version 1 is more appropriate. The relatively complex inhibition patterns obtained with labeled MAb 1 (Fig. 1A) and M A b 4 (Fig. 1D) or with immobilized MAb 4 (Fig. 2A), can be explained as follows. MAbs 1 and 2 are directed against repetitive epitopes on C E A , whereas MAbs 4, 5, and 6 are directed against singular

514

APPLICATIONS

[59]

epitopes. 13(The epitope specificies of MAbs 5 and 6 are identical, see Fig. 1C.) The presence of more than one epitope on the protein moiety of CEA is probably due to the fact that CEA is built up of three highly homologous repeat units.14 Though MAbs l, 4, and 5 (6) exhibit totally different antigen specificities, one of the MAb 1 epitopes overlaps with the epitopes of MAbs 4 and 5 (6). For this reason, the binding of labeled MAb 1 to immobilized CEA is partially inhibited by MAbs 4 and 6 (Fig. 1A). In contrast, MAb 1 approaches 100% binding inhibition at high antibody excess when MAb 4 is used either as biotinylated MAb (Fig. 1D) or as immobilized MAb (Fig. 2A). A possible objection to an immunoassay requiring the labeling of an antigen or antibody is that the labeling process may affect binding. In solid-phase assays, a further objection is that either an antigen or an antibody may undergo a conformational change affecting binding. By reversing the roles of antigen and antibody in the two tests described above, it is possible to probe the effect of both parameters on the formation of the antigen-antibody complex. Determination of CEA and Related Antigens in Serum Using Biotinylated Monoclonal Antibodies in Additive Solid-Phase Enzyme Immunoassays Materials

Coating buffer: 20 m M sodium acetate buffer (pH 5.5) PBS: 50 m M sodium phosphate buffer (pH 7.2) containing 0.15 M NaC1 and 0.01% (w/v) NaN3 BSA-PBS: PBS containing 1% (w/v) BSA Incubation buffer: 0.1 M potassium phosphate (pH 7.3) containing 20% (v/v) fetal calf serum (FCS), 0.02% (v/v) Tween, and 4% (v/v) normal mouse serum APC: avidin-peroxidase conjugate (Sigma) APC buffer: 0.2 M potassium phosphate buffer (pH 6.5) containing 20% biotin-free fetal calf serum Washing buffers: 0.1 M potassium phosphate buffer (pH 6.5) and 0. I M sodium citrate buffer (pH 5.0) Substrate solution: 6 m M H202 (freshly prepared) and 40 mM o-phenylenediamine (Sigma) in 0.1 M citrate buffer (pH 5.0) DMF and BNHS: see above 13 B. M. Giannetti, M. Neumaier, and C. Wagener, Fresenius Z. Anal. Chem. 324, 253 (1986). 14 S. Oikawa, H. Nakazato, and G. Kosaki, Biochern. Biophys. Res. Commun. 142, 511 (1987).

[59]

EIAs FOR CEA

515

Preparation of Biotin-Labeled MAbs. Monoclonal antibody IgG is biotin-labeled as described above. 11 As antibodies, MAbs 4 and 6 are used. Molar ratios of BNHS to IgG in the range of 10 to 600 were tested in order to determine which ratio yields the highest sensitivity in the solid-phase EIA. Using MAb 4, a ratio of 60-70 was found to be optimal. Preparation of F(ab)e Fragments. The IgG1 fraction of MAb 2 is purified from ascitic fluid over protein A-Sepharose. 8 IgG1 (6-12 mg/ml) is digested with pepsin (3%, w/v) in 0.1 M sodium acetate buffer (pH 4.1) for 18 hr at room temperature. The solution is then brought to pH 8.1 by the addition of 0.1 M Tris-HC1 (pH 9.0). The reaction mixture is passed over a protein A-Sepharose column with a 0.1 M sodium phosphate solution (pH 8.1) as column and elution buffer. The nonbound fraction is characterized by gel-permeation HPLC (TSK 3000, LKB, Gr/ifelfing, FRG) and SDS-PAGE under reducing and nonreducing conditions. Protocol of Additive Solid-Phase EIAs. The quantification of CEA in serum samples is performed by double monoclonal, sandwich-type immunoassays. MAb 2, which is a broadly cross-reactive, high-affinity antibody, 5-7 is immobilized on a solid support. As second antibodies, either biotinylated MAb 4 or MAb 6 is used. Each of the three antibodies binds to distinct epitopes on CEA. MAb 4 binds CEA with high affinity and specificity. 3-7 MAb 6, which has a moderate affinity for CEA, binds a 128kDa tumor-associated CEA variant in addition to CEA. 5 Neither MAb 4 nor MAb 6 binds to nonspecific cross-reacting antigens present in normal tissues and body fluids. Solid-phase antibody and solution-phase antibodies are incubated in a single incubation step. The incubation time depends on the affinity of the respective solution-phase antibody. In a second incubation step, the biotin-labeled antibodies bound to the antigen(s) are reacted with avidin-peroxidase conjugate, followed by the addition of a suitable substrate. Polystyrene beads with a diameter of 1/4 inch (Spherotech, Fulda, FRG) are washed several times in absolute ethanol and then sonicated for 60 min in PBS. The PBS is exchanged after 30 min. Subsequently, the beads are washed with PBS. The beads are coated with the IgG1 fraction or F(ab)2 fragments of MAb 2. Per 100 beads, 30 ml of coating buffer containing 10/zg/ml of either antibody (IgG) or F(ab)z fragments, respectively, are added. Coating is performed for 30 min at room temperature with shaking. Blocking of nonspecific binding sites and storage of the beads are performed in a solution of 2% BSA in PBS. The CEA standard is dissolved in BSA-PBS. The biotinylated antibodies are diluted with incubation buffer to 2.66/xg/ml in the case of MAb 4 and 5.7/zg/ml in the case of MAb 6. The upper concentration limit of biotinylated antibody depends on the nonspecific binding to the beads which, in the present assays, does not exceed A492values of 0.05.

516

APPLICATIONS MAb2/MAb4

[59] MAb2/MAb6

100 ~1 sample/normal serum 50 ul BSA-PSS/standard in BSA-PBS 50 ul of biotinylated MAb 6 solution [5.7 mg/I] +

100 pl sample/normal serum 50 ul BSA-PBS/standard in BSA-PBS 50 ,uf of biotinylated MAb 4 solution [2.66 mg/l] +

immobilizedMAb 2 F(ab)2 fragments

immobilized MAb 2 (IgG1)

/ / ~ r o

7hr,

om temperature

200 ~1 avidin-peroxidase solution 30 rain, room temperature 300 jdl ortho-phenylenediamine solution 30 rain, ] room temperature 2 roll M HCl A]92

FIG. 3. Protocols

of two additive, solid-phase enzyme immunoassays for CEA based on the avidin-biotin system.

The assay protocols are given in Fig. 3. The assays are performed in special reaction trays (Abbott, Wiesboden, Federal Republic of Germany). For the analysis of serum samples, 100/~l of serum is mixed with 50/zl of the solution of biotinylated MAb and 50/zl of BSA-PBS. For the determination of CEA standards, 100/zl of normal serum (checked for low CEA content) is mixed with CEA standard solution in B S A - P B S and 50 pJ of antibody solution. After addition of the antibody-coated beads, the MAb 2/MAb 4 immunoassay is incubated for 1 hr at 37 °, and the MAb 2/MAb 6 immunoassay for 17 hr at room temperature, respectively. Subsequently, the beads are washed 4 times with 0. I M potassium phosphate buffer (pH 6.5). The concentration of avidin-peroxidase conjugate is chosen as the maximum concentration which yields negligible nonspecific binding. Compared with 1-5% BSA or 1% gelatin, 20% FCS most efficiently reduces background staining. However, FCS may contain free biotin. Therefore, new batches of FCS are checked for biotin by adsorption to avidin-Sepharose. The serum is tested in the CEA immunoassay

[59]

EIAs FOR CEA

517

prior to and after absorption. If interfering amounts of biotin are present, the FCS has to be adsorbed with avidin-Sepharose prior to its use in the immunoassay. Maximum binding of the avidin-peroxidase conjugate is reached after an incubation time of 20 min at room temperature. An incubation time of up to 80 min does not increase the sensitivity of the assay. In the present assay, an incubation time of 30 min is chosen. The beads are then washed 3 times in 0.1 M citrate buffer (pH 5.0). Subsequently, the substrate solution is incubated with the beads in the dark for 30 min at room temperature. The reaction is stopped with 2 ml of 1 M HC1, and the OD is read at 492 nm within 30 min. Discussion of Assay Results. The assay protocols of the two immunoassays differ in two major aspects (Fig. 3). Owing to the lower affinity of MAb 6, the incubation time of the MAb 2/MAb 6 assay had to be longer than that of the MAb 2/MAb 4 assay in order to reach comparable sensitivities. The increase of incubation time, however, has an undesirable side effect: nonspecific anti-mouse IgG-binding substances that are occasionTABLE I ASSAY CHARACTERISTICS OF TWO DOUBLE-MONOCLONAL, AVIDIN-BIOTIN-BASED ENZYME IMMUNOASSAYSFOR CEA

Immunoassays Assay parameter Sensitivity a (/zg CEA/liter) Measuring range (/zg CEA/liter) Recovery CEA added (/zg/liter) CEA found (/zg/liter) Recovery (%) CEA added (/~g/liter) CEA found (/zg/liter) Recovery (%) Between-run precision Mean CEA (/zg/liter) SD CV (%) n Mean CEA (/zg/liter) SD CV (%) n Upper limit, normal range 95% percentile (~g CEA/liter)

MAb 2/MAb 4 0.55 0.55-64.0 4.8 5.7 119 19.6 19.3 99 4.8 0.55 11.5 8 17.2 1.50 8.7 8 4.2

CEA concentration corresponding to the zero standard +3 SD.

MAb 2/MAb 6 0.63 0.63-64.0 4.8 4.5 94 19.6 16.6 84 5.2 0.80 15.3 15 18.5 2.50 13.4 15 6.5

518

APPLICATIONS

[60]

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.

Quantification of carcinoembryonic antigen in serum and analysis of epitope specificities of monoclonal antibodies.

[59] EIAs FOR CEA 507 TABLE I REPRODUCIBILITY OF RESULTS WITH PROTOCOL I V TO MEASURE VARIOUS LEVELS OF I g E IN DIFFERENT-DAY RUNS Test; day run...
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