186,121-126

ANALYTICALRIOCHEMISTRY

(1990)

Labeled Antigen Capture Assay: A Method for Detecting Monoclonal Antibodies to Cloned Gene Products Robert

G. Urban

Department

Received

and Lawrence

of Microbiology,

A. Dreyfus’

The University

of Texas Medical

Branch,

Galveston,

Texas 77550

July 21, 1989

The relative easeof monoclonal antibody (MAb)’ production has revolutionized immunochemical analysis of

proteins, peptides, and other biologically significant antigens. Often, however, the antigen of interest is the product of a cloned gene and little,‘if any, information on the structure, biological function, or enzymatic activity is available. This limitation does not necessarily preclude the generation of MAbs to the gene product of interest; however, the cryptic nature of the gene product does complicate strategies for screening a hybridoma library for specific MAbs. One method for the production of MAbs to a specific cloned gene product involves an analysis of the deduced amino acid primary sequence to predict surface epitopes on the native molecule. Peptides corresponding to the predicted epitopes are synthesized, coupled to a carrier protein, and used as immunogen (l-3). Although repeatedly successful, this approach relies on prior knowledge of the gene sequence and the costly preparation of synthetic peptides. Another currently used method is to construct a fusion between the cloned gene of interest and a “reporter” gene, such that a hybrid protein, possessing enzymatic activity, is generated (4). The hybrid protein is then purified, relying on reporter activity for assay, and subsequently used as immunogen. Although lesscostly and time consuming than the previously mentioned procedure, this protocol still requires a source of purified hybrid protein when screening hybridoma supernatants for specific monoclonal antibodies. In this report we present a novel use of the previously described T7 bacteriophage expression system (5) to simplify the identification of MAbs specific for the product of a cloned gene of interest. The system, as described by Tabor and Richardson (5), relies on the high activity and rifampicin resistance of the T7 RNA polymerase, thus allowing selective high-level expression and exclusive metabolic radiolabeling of any T7-promoted gene and its encoded product, respectively. The protocol we describe uses the radiolabeled product as a source of an-

’ To whom correspondence should be addressed. * Abbreviations used: MAb, monoclonal antibody; LACA, labeled antigen capture assay; LB, Luria broth; Cb, carbenicillin; Km, kanamycin; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl

sulfate-polyacrylamide gel electrophoresis; lium; ELISA, enzyme-linked immunosorbent 4-chloro-3-indolyl phosphate.

We report here a relatively easy and highly sensitive assay for detecting monoclonal antibodies to the product of virtually any cloned gene. The protocol, termed labeled antigen capture assay (LACA), is a solid-phase type radioimmunoassay which uses a bacteriophage T7 expression system to generate exclusively radiolabeled antigen. Thus, to generate radiolabeled antigen for screening, the gene encoding the protein of interest need only be subcioned downstream of a T7 promoter, and the new construct transformed into an Escherichia coli strain harboring a compatible plasmid which encodes a thermal inducible copy of the T7 RNA polymerase. Expression of the T7-promoted gene in the presence of rifampicin and [35S]cysteine (or methionine) yields labeled antigen, which is then “captured” by specific monoclonal antibody and detected by autoradiography. Our results indicate that as little as 30 ng of specific monoclonal antibody can be detected using the LACA protocol. The protocol is applicable to the product of any cloned gene but is particularly useful in the case where the biochemical properties of the gene product of interest are unavailable. In this report we use the LACA protocol to screen a hybridoma library for monoclonal antibodies to the STb heat-stable enterotoxin (STb) of E. coli. Mice were immunized with a genetically constructed, affinity-purified Protein A-STb hybrid protein, and following spleen cell fusion and HAT selection, hybridomas were screened by the described LACA protocol for production of STb-specific monoclonal antibody. Of over 1500 hybridomas tested 138 were positive, by primary LACA screening, for STbspecific IgG monoclonal antibodies. 0 1990 Academic Press,

Inc.

0003-x97/90 Copyright All rights

$3.00 0 1990 by Academic Press, of reproduction in any form

Inc. reserved.

NBT, nitroblue assay; BCIP,

tetrazo5-bromo-

121

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AND

tigen in a labeled antigen capture assay (LACA) for screening hybridoma supernatants and the ultimate identification of specific MAb. The LACA does not require prior sequencing of the cloned gene or purification of the encoded gene product. These features render the assay particularly useful in the case of gene products of uncharacterized biochemical or biological function. We also report application of the protocol to the production and screening of monodonal antibody to the STb heatstable enterotoxin of Escherichia coli. STb is a perfect test immunogen for the assay because, even though the gene encoding STb has been cloned and sequenced (6,7), the toxin is difficult to purify and little is known of its biochemical and biological properties. Limited information on STb stems largely from the lack of an in vitro assay for the toxin; thus, the availability of STb-specific monoclonal antibody would provide a significant benefit to the field. In addition to our specific use of the LACA protocol, we discuss some modifications and variations on the assay and its use. MATERIALS Bacterial

AND

Strains,

METHODS Media, and Plasmids

Bacteria were grown in Luria broth (LB (8)) or M9 medium (8). When appropriate, carbenicillin (Cb) and kanamycin (Km) were added to the growth medium at 100 and 50 pg/ml, respectively. E. coli HBlOl (F-, hsdS.20, recAl3, supE44, ara-14, galK2, lacYI, proA2, rpsL20, ~~1-15, leu, m&l) was used in all expression experiments. The plasmids pT7-5 and pGPl-2 (5) were supplied by Stan Tabor. The T7-promoted E. coli enterotoxin (STb)-alkaline phosphatase (phoA) gene fusion plasmid, PST-phoA (Fig. l), was constructed in our laboratory. A manuscript describing its construction and the biological properties of the encoded STb-PhoA protein fusion is in preparation. Another gene fusion plasmid, pPD3, which encodes a h PL-promoted Protein A-STb hybrid polypeptide is briefly described below but will be reported in detail elsewhere.

DREYFUS

pulsed for 10 min at 37°C with 10 &i [35S]cysteine (>600 Ci/mmol, Amersham Corp., Arlington Heights, IL). After labeling, the bacterial cells were harvested, washed twice in 5 ml ice-cold PBS, and resuspended in 5 ml of the same buffer. The washed cells were then lysed by three 30-s pulses (at a setting of 75 W) from a Branson Model 185 sonicator. Remaining intact cells and particulate cell debris were removed by centrifugation at 250,OOOgfor 2 h. The supernatant fraction was collected and stored in aliquots at -70°C until used. Analysis of Metabolic Labeling Products of the T7 expression and metabolic labeling experiment were analyzed by SDS-PAGE, Western blot, and autoradiography. Following separation by SDS-PAGE, the 10% acrylamide gel was sliced into identical halves containing lanes loaded with experimental and control lysates. One gel half was stained with Coomassie blue and the other half transferred to nitrocellulose (Schleicher & Schuell Inc., Keene, NH) for Western blot analysis. After blocking with 3% gelatin, TBS (0.5 M NaCl, 0.02 M Tris-HCl [pH 7.5]), the nitrocellulose was reacted with anti-alkaline phosphatase monoclonal antibody (IgG, containing ascites fluid) (CAL-TAG, Inc., San Francisco, CA) for 4 h at room temperature followed by an identical incubation with goat anti-mouse IgG alkaline phosphatase enzyme conjugate (Promega, Inc., Madison, WI). Primary and secondary antibodies were diluted in TBS containing 0.05% Tween 20 (v/v) (TTBS) and 1% (w/v) gelatin. The blot was developed by incubation in buffer (0.1 M NaCl, 0.1 M Tris-HCl ]pH 9.5],0.005 M MgClz) containing BCIP and NBT (Promega). Following development, the blot was exposed to Kodak X-OMAT film for 18 h at -70°C. Radiolabel present in the expressed samples was determined by liquid scintillation counting. The percentage of incorporated label was determined by size-exclusion chromatography on prepacked G-25 disposable columns (Pharmacia, Inc., Piscataway, NJ). Labeled Antigen Capture Assay

T7-Promoted

Expression

of STb-PhoA

Cultures of E. coli HBlOl (pGPl-2, PST-phoA) and a negative control strain HBlOl (pGPl-2, pT7-5) were grown overnight with shaking (200 rpm) in Luria broth containing Cb and Km at 30°C. A 5-ml portion of this culture was pelleted by centrifugation at 3500g for 10 min and washed twice with 25 ml of supplemented M9 medium. The washed cells were resuspended in 25 ml of supplemented M9 medium then incubated for 2 h at 30°C (200 rpm) followed by a shift to 42°C for 30 min to induce expression of the T7 RNA polymerase. Rifampitin (250 pg/ml) was added and the cultures were incubated at 42°C for an additional 15 min. Following this incubation, cultures were placed at 37°C for 20 min to allow degradation of existing E. coli transcripts and then

Nitrocellulose, wetted in PBS, was fitted into a dotblot apparatus (Bio-Rad, Richmond, CA) as described by the manufacturer. Wells were either precoated or not with affinity-purified polyclonal goat anti-mouse Fc gamma IgG (Accurate Chemical and Scientific Corp., New York, NY) at 25 pug/ml in PBS. Diluted precoating antibody (100 ~1) was added to the wells and allowed to incubate for 2 h at room temperature. The wells were then emptied by the application of vacuum. The test MAb used to design the assay was the anti-alkaline phosphatase antibody described above. Serial twofold dilutions of the IgG containing ascites fluid were prepared using spent culture medium from the SP2/0 myeloma cell line (9) as diluent. To duplicate precoated wells 100 ~1of the 1:200 to 1:3200 antibody dilutions was

LABELED

ANTIGEN

added. The 1:3200 antibody dilution contained approximately 150 ng of antigen-specific IgG-l/ml. The level of amplification resulting from precoating with goat antimouse antibody was assessed by the addition of 100 ~1 of the 1:lOO dilution of antigen-specific antibody to duplicate nonprecoated wells. Nonspecific binding of radiolabeled antigen to the nitrocellulose was determined in wells that were precoated but lacked antigen-specific antibody. The wells were allowed to incubate at room temperature for 2 h, after which they were emptied by the application of vacuum. The wells were washed five times with PBS to remove any nonabsorbed antibody. The nitrocellulose was removed from the dot-blot apparatus and blocked by incubation in 3% gelatin, TBS for 30 min at room temperature. The nitrocellulose was then cut into strips containing rows of serially diluted antibody. Each strip was placed into a sealable bag with 5 ml TTBS-gelatin containing various amounts of the labeled antigen preparation. The strips were rotated at room temperature for 2 h and then washed 5X in 50 ml TTBS to remove unbound radioactivity. Bound STbPhoA was detected by direct assay of the alkaline phosphatase activity associated with the fusion protein following the addition of the PhoA-insoluble substrates NBT/BCIP and by autoradiography. The relative quantity of bound radiolabel was determined by scanning the developed autoradiogram with a Model GS300 scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). Source of Test Immunogen, Hybridoma Production

Immunizations,

and

The genetic construction of the test immunogen, an STb heat-stable enterotoxin-Protein A protein fusion, is to be described in a subsequent communication. Briefly, a BgZII-PstI fragment containing the carboxyterminal 41 codons of the STb encoding segment was removed from pCHL6 (6) and ligated into pPD1 (a derivative of the Protein A fusion vector pRIT2T [Pharmacia LKB Biotechnology, Inc., Piscataway, NJ] which lacks the Bgl II site) previously digested with BamHI and P&I. The reading frames of Protein A and STb were realigned with complementary synthetic oligonucleotides which also reformed the 7 additional amino-terminal codons of mature STb (submitted for publication). The final plasmid, pPD3, is a X Pn-promoted Protein A-STb gene fusion vector. When harbored in E. coli N4830-1 (~18857) (Pharmacia LKB Biotechnology) at 30°C little or no Protein A-STb is made; however, at 42°C PR is derepressed and high-level expression of Protein A-STb follows. Protein A-STb was prepared by culturing E. coli N4830-1 (pPD3) to the midlog phase (OD600nm 1.0) in 500 ml of LB containing carbenicillin (100 pg/ml) at 30°C. The culture was then shifted to 42°C for 30 min to inactivate the cI857-encoded repressor and then maintained for 2 h at 37°C to allow maximum expression of

CAPTURE

FIG. (other phoA,

ASSAY

123

1. Schematic map of pSTb-phoA plasmid. Abbreviations than restriction enzyme cleavage sites) are bla, @-lactamase; alkaline phosphatase; ~$10, bacteriophage T7 promoter.

Protein A-STb. Following this incubation the cells were pelleted, washed, resuspended in 1:20 original volume, and disrupted by passage through a French press. Cell debris and particulate material were removed by centrifugation at 20,OOOg for 2 h. The clarified extract was then passed over a column (5 ml) of IgG-Sepharose (Pharmacia LKB Biotechnology) which was washed with 10 vol of 50 mM Tris, 150 mM NaCl, 0.05% Tween 20 (pH 7.8) followed by 2 vol of 5 mM ammonium acetate, pH 5.0, to remove unbound and loosely bound material, respectively. Protein A-STb was eluted by washing with 5 vol of 0.5 M acetic acetate (pH 3.4 by addition of ammonium acetate). The eluted material was lyophilized and then reconstituted to approximately 2 mg/ml in deionized HzO. SDS-PAGE analysis of the eluted material demonstrated the presence of a single major band of protein approximately 5000 Da larger than purified Protein A. Mice (two BALB/c females) were immunized ip with 50 pg of Protein A-STb in Freund’s complete adjuvent. After 30 days mice received 50 pg Protein A-STb (ip) in Freund’s incomplete adjuvent. Five days following the booster immunization, spleens of immune mice were removed and splenocytes harvested. Polyethylene glycol fusion of isolated splenocytes and Sp2/O myeloma cells was performed exactly as described elsewhere (10). Fused cells in HAT medium were cultured at 37°C in 5% CO2 in 96-well plates. Following 2 weeks of HAT selection, wells containing hybridomas at 50% confluency were tested for STb-specific antibody production by the LACA protocol. For initial screening, 100 11 of culture medium was removed from the wells to be tested and assayed as described above. RESULTS

The product of the T7-promoted E. coli STb-phoA gene fusion construct (Fig. 1) is a hybrid protein of approximately 50 kDa possessing both alkaline phosphatase activity and intestinal secretory activity (manuscript in preparation). Expression and exclusive radiolabeling of the STb-PhoA protein fusion are shown

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lS.O14.8 -

FIG. 2. Analysis of metabolic labeling. Following induction and radiolabeling cells were lysed and fractionated on 10% SDS-PAGE. Lane 1, strain harboring pSTb-phoA and pGPl-2; lane 2, negative control strain harboring pT7-5 (T7 promoter vector) and pGPl-2. (A) Coomassie-stained gel. (B) Western blot of identical gel shown in A probed with monoclonal anti-phoA then second antibody enzyme conjugate. (Cl Autoradiogram of Western blot shown in B.

in Fig. 2. The negative control strain harboring pT7-5 and pGPl-2 is also shown. The stained protein profiles of the pSTb-PhoAand pT7-5containing strains are shown in Fig. 2A. The relative molecular mass of the STb-PhoA protein fusion (marked by the asterisk) is approximately 5000 Da larger than that of the wild-type alkaline phosphatase (not shown). Expression of the STb-PhoA fusion in the presence of rifampicin and [35S]cysteine results in high-level expression (Fig. 2B) and exclusive radiolabeling of the T7-promoted gene fusion as depicted in the autoradiogram shown in Fig. 2C. The absence of any anti-PhoA-reactive or radiolabeled product in the pT7-5-containing strain (Figs. 2B and 2C, lane 2) is due to the lack of a T7-promoted cloned insert. The lysate from which the sample in lanes 1 in Fig. 2 contained about 2500 cpm/pl of incorporated radiolabel and was used as the source of labeled antigen for the LACA assay. The LACA protocol, schematically represented in Fig. 3, was designed to assay supernatants of microtiterplate-grown hybridomas for antigen-specific MAbs. The assay can be quickly set up and several sheets prepared ahead of time with precoating and test supernatants. Once blocked, the sheets can be stored at 4°C for several days prior to testing. Although not intentional, the use of the STb-PhoA protein fusion as antigen allows an examination of both the captured radiolabel and the alkaline phosphatase activity associated with the hybrid protein (Fig. 4). Figure 4A depicts strips that were either precoated or not, with anti-mouse Fc and dilutions of SP2/0 spent medium “doped” with anti-alkaline phosphatase MAb. The well containing the 1:1600 dilution of

DREYFUS

MAb (i.e., the last clearly positive well) contained approximately 30 ng of antigen-specific IgG-1. The results of the LACA are best appreciated by the data obtained by scanning the autoradiogram (Fig. 5). Precoating the wells with anti-mouse Fc greatly increased the sensitivity of the assay. Initial tests determined that Protein A was not as effective as anti-Fc (not shown). The amount of antigen added to the wells also clearly influenced the sensitivity of the assay. At the highest level tested, nonspecific binding of radiolabel was not observed. Presumably, the addition of more label may improve the sensitivity of the assay; however, as described here, LACA is both highly specific and sensitive. A major research interest in our laboratory is the biochemical and biological characterization of the STb heat-stable enterotoxin of E. coli. The toxin is unique among E. coli enterotoxins since it appears to mediate intestinal ion secretion by an uncharacterized cyclicnucleotide-independent mechanism (11). Progress in our understanding of STb, however, suffers from the lack of an in vitro assay and a readily obtainable source of purified toxin. Further, although STb-specific monoclonal antibody would provide a powerful tool for several facets of STb research, the source of STb immunogen and the availability of screening antigen and/or a manageable method to accommodate the screening of hun-

Cut Blat Apparatus with N~tracellulose

FItted

N~tracellulose coated with the Immunoadsorbanl. ank-mouse Fc

Addlbon of hybrldoma culture supernalants contzvn~ng anlibodles 01 unknown specllicry

Ankbody coated nltrocellulose removed, blocked, and ncubaled Wh labeled anbgen

FIG.

3.

Hybndomas producl Idenbked

Schematic

of LACA

prodwng cloned gene specific anhbodies by auloradiogtapby

protocol.

LABELED

YN

Y

Y

Y

Y

ANTIGEN

Capture antigen

dreds of potential anti-STb-producing hybridomas have made monoclonal antibody production difficult at best. Using a genetically constructed and affinity-purified Protein A-STb immunogen and the T7-promoted STbPhoA fusion as screening antigen, we identified several hybridomas producing STb-specific monoclonal antibody. Primary screening of hybridoma-containing wells is shown in Fig. 6. In the results of a single plate (Fig. 5), 12 of 96 wells contained anti-STb secreting hybridomas. From a single fusion, in which over 1500 wells were screened by the LACA protocol, 138 hybridomas were found to produce anti-STb IgG antibodies. Antibodies were subsequently determined to be STb-specific by a combination of assays including synthetic STb-peptide ELISA and Western blot against a variety of antigens including STb-PhoA and Protein A-STb, where alka-

of Antigen

125

ASSAY

YNYYY

Y

FIG. 4. LACA detection of anti-alkaline phosphatase MAb. phosphatase substrates (A). Radiolabel associated with captured

Amount

CAPTURE

Y

of STb-PhoA was detected

Y

Precoated

was visualized by incubating nitrocellulose strips following autoradiography of strips shown in A (B).

in

line phosphatase and Protein A served as the negative controls, respectively, and against highly purified STb (kindly provided by Dr. Shannon C. Whipp, USDA/ ARS National Animal Disease Laboratory, Ames, IA) (not shown). Currently, two of the hybridomas giving interestingpatterns of reaction with various antigens have been cloned and determination of their isotype specificity is underway. DISCUSSION The importance of monoclonal antibodies as powerful reagents for the analysis of biologically significant mole-

1

2

3

4

5

6

7

8

9

10

11

12

Adde

0 1:lOO

1:200

I:400

1~300

1 :I ,600

C-1

(+I

(+I

(+I

(+)

Antibody Precoated

dilution wells

FIG. 5. Densitometric quantification of captured label. The autoradiogram shown in Fig. 4B was scanned as described under Materials and Methods. The relative amounts of captured antigen were determined by integration and plotted.

FIG. 6. LACA screening of putative anti-STb hybridomas. Culture medium from a 96well plate containing hybridomas generated following Protein A-STb immunization of mice was tested by LACA protocol as described in text. Letters (vertical) and numbers (horizontal) refer to the 96-well plate (and dot blot apparatus) position of the particular hybridoma tested. The amount of radiolabeled antigen added to the precoated blot was 150,000 cpm. Exposure time was 2 days at -70°C.

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cules is due primarily to their single antigenic specificity. Just as important to their overall utility, however, is the lack of a requirement for purified immunogen. This feature has allowed the use of complex protein mixtures, even whole cells, as immunogen for the generation and subsequent cloning of a single antibody-producing cell line. An obvious limitation to this strategy, however, involves the subsequent screening of hybridomas for antigen-specific Ig production. Screening hundreds of Igpositive hybridomas by differential binding assays or Western blot analysis is laborious, at best, and may preclude successful identification of the desired antibodyproducing cell line. The prototype LACA system was based on the STbPhoA protein fusion and a commercially available monoclonal antibody to E. coli alkaline phosphatase. As demonstrated by the nanogram-level detection of Igcontaining supernatant, LACA is a simple and sensitive assay for antigen-specific antibody detection. Although the prototype system was modeled for our specific use, the application is for virtually any cloned gene. The assay is particularly useful when the product of a cloned gene of interest is poorly characterized. The ability to generate and screen MAbs to the gene product would therefore be a major advance in characterization of the protein product. In this report we used the LACA protocol to screen a hybridoma library generated to the product of a Protein A-STb gene fusion. The STb heat-stable enterotoxin of E. coli is a perfect example of the utility of LACA. Although described for over a decade (12), STb remains poorly characterized largely due to the lack of an in vitro toxin assay and the complexity of the animal gut models used for study. Monoclonal antibody specific to STb would be a major contribution to the area of research not only for its potential as a diagnostic reagent but also as an affinity absorbant for toxin purification and a probe for studies on intestinal secretory action. Our results indicate that the LACA protocol is an excellent primary assay when screening hundreds of hybridomas. Although the use of nearly purified immunogen increased our chances at obtaining the desired antibody-producing hybridomas, the ability to conveniently screen hundreds of samples makes the use of very crude immunogens also possible. Because our assay system utilized polyclonal goat anti-mouse Fc gamma as the antibody capture agent we selected only IgG-producing clones. Similarly, selection for IgM-producing clones could have been accomplished by use of the appropriate reagent, or a mixture of re-

agents used to detect all desired antibody isotypes. Likewise, the assay could be utilized to select a single isotype in the primary screen. Several variations are possible and the assay can be thus tailored to specific needs. An additional advantageous feature of our prototype LACA procedure was the use of an alkaline phosphatase protein fusion as the capture antigen. Direct detection of captured alkaline phosphatase activity was nearly as sensitive as radiolabel detection. In addition, detection of alkaline phosphatase activity would allow LACA to be performed in a single day, instead of the required overnight incubation for autoradiography. The LACAprotoco1would therefore be an excellent nonradioactive assay for MAbs generated against any alkaline phosphatase protein fusion. Depending on the strength of promotion, it may also be possible to perform an AP-based LACA without subcloning to a T7 vector. ACKNOWLEDGMENTS R.G.U. is a James W. McLaughlin Fund Predoctoral Fellow. We thank Stan Tabor for providing us with the T7 expression vectors. Also we thank Shannon C. Whipp for supplying us with purified STb for analysis of our monoclonal antibodies.

REFERENCES 1. Hirayama,

A., Takagaki,

Y., and

Karush,

F. (1985)

J. Zmmunol.

134,3241-3247. 2. Niman, H. L., Thompson, A. M. H., Yu, A., Markman, M., Willems, J. J., Herwig, K. R., Habib, N. A., Wood, C. B., Houghten, R. A., and Lerner, R. A. (1985) Proc. Natl. Acad. Sci. USA 82, 7924-7928. 3. Ghose A., and Karush, F. (1988) Mol. Zmmunol. 25,223-230. 4. Weinstock, G. M., Berman, M. L., and Silhavy, T. J. (1983) in Gene Amplification and Analysis (Papas, T. S., Rosenberg, M., and Chirikjian, J. G., Eds.), Vol. 3, pp. 27-67, Elsevier, New York. 5. Tabor, S., and Richardson, 82,1074-1078.

C. C. (1985)

Proc. Natl.

Acad. Sci. USA

6. Lee, C. H., Moseley, S. L., Moon, H. W., Whipp, S. C., Gyles, C. L., and So, M. (1983) Infect. Zmmun. 42,264-268. 7. Picken, R. N., Mazaitis, A. J., Maas, W. K., Rey, M., and Heyneker, H. (1983) Infect. Zmmun. 42,269-275. 8. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, p. 440, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 9. Gefter, M. L., Margulies, D. H., and Scarff, M. D. (1977) Somatic Cell &net. 3,231-236. 10. Ausubel, F. M., Brent, F., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (Eds.) (1988) Current Protocols in Molecular Biology, Units 11.6-11.8, Wiley, New York. 11. Kennedy, D. J., Greenberg, Dunn, J. A., Abernathy, R., Ryerse, J. S., and Guerrant, R. L. (1984) Infect. Zmmun. 46,639-643. 12. Burgess, M. N., Bywater, R. J., Cowley, C. M., Mullan, N. A., and Newsome, P. M. (1978) Znfect. Zmmun. 21,526-531.

Labeled antigen capture assay: a method for detecting monoclonal antibodies to cloned gene products.

We report here a relatively easy and highly sensitive assay for detecting monoclonal antibodies to the product of virtually any cloned gene. The proto...
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