Methods in Enzymology

Volume 121

Immunochemical Technique sPart I

Hybridoma Technology and

Monoclonal Antibodie s

John J. Langone Helen Van Vunakis

CONTRIBUTORS TO VOLUME 121 Xiii

PREFACE XXi

VOLUMES IN SERIES xxii i

Section I . Production of Hybridomas

A . Immunization and Cell Fusio n

1 . Factors Affecting Production ofMonoclonal Anti- REGINE J . WESTERWOUDT

3bodie s

2 . In Vitro Immunization for the Production of Anti- CHRISTOPHER L . READING

1 8

gen-Specific Lymphocyte Hybridomas

3 . An Improved in Vitro Immunization Procedure for BARBARA D . Boss

27

the Production of Monoclonal Antibodie s

4 . "Single-Shot" Intrasplenic Immunization for the M . SPITZ

3 3

Production of Monoclonal Antibodies

5 . Method for Generating a High Frequency of Hy- DAWN E . COLWELL ,

bridomas Producing Monoclonal IgA Anti- SUZANNE M . MICHALEK ,

bodies

AND JERRY R . MCGHEE

42

6 . Production of IgA-Secreting Rat x Rat Hybrid- C . J . DEAN, L . A . GYURE ,

omas

J . G . HALL, AN D

J . M . STYLES

5 2

7 . The Use of Conventional Antisera in the Produc- KENDRA B . EAGER AN D

tion of Specific Monoclonal Antibodies

ROGER H . KENNETT

5 9

8 . Preparation of Monoclonal Antibodies to Prese- M . ZOUHAIR ATASSI

6 9

lected Protein Region s

9 . The Auto-Anti-Idiotypic Strategy for Preparing W. Louis CLEVELAND AND

Monoclonal Antibodies to Receptor Combining BERNARD F . ERLANGER

9 5

Site s

10. Optimal Strategies for Developing Human- PAUL G . ABRAMS ,

Human Monoclonal Antibodies

JEFFREY L . Rossio ,

H . C . STEVENSON ,

AND K . A . FooN

107

11. Comparative Phenotypic Analysis of Available DANUTA KOZBOR ,

Human Hybridoma Fusion Partners

JOHN C . RODER,

MICHAEL E . SIERZEGA ,

SUSAN P . C . COLE ,

AND CARLO M . CROCE

120

12 . The EBV-Hybridoma Technique

J . C . RODER ,

S . P . C . COLE ,AND D . KOZBOR

140

13 . Generation of Human Monoclonal Antibodies by STEVEN K . H . FOUNG ,

Fusion of EBV-Activated B Cells to a Human- EDGAR G . ENGLEMAN ,Mouse Hybridoma

AND F . CARL GRUMET

168

14 . Improved Hybridoma Technology : Spleen Cell P . VAN MOURIK AND

Separation and Soluble Growth Factors

W . P . ZELILEMAKER

17 4

15 . High Efficiency Fusion Procedure for Producing RICHARD D . LANE ,

Monoclonal Antibodies against Weak Immu- ROBERT S . CRISSMAN ,

nogens

AND STEPHEN GINN

18 3

16 . Methods for High Frequency Production of Solu- MAURIZIO CIANFRIGLIA ,

ble Antigen-Specific Hybridomas ; Specificities MASSIMO MARIANI ,

and Affinities of the Monoclonal Antibodies Ob- DORETTA ARMELLINI ,

tained

ANNALISA MASSONE ,

MARGHERITA LAFATA ,

RIvo PRESENTINI ,

AND GUIDO ANTONI

19 3

17 . Bispecific Monoclonal Antibodies from Hybrid M . R . SURESH ,

Hybridomas

A. C . CUELLO, AND

C . MILSTEIN

21 0

18. Human x (Mouse x Human) Hybridomas

LARS OSTBERG

22 8

19 . Generation of Rat-Rat Hybridomas with the Use LIEVE DE CLERCQ ,

of the LOU IR983F Nonsecreting Fusion Cell FRANcoISE CORMONT ,

Line

AND HERVE BAZIN

234

20 . Production of Syrian and Armenian Hamster FRANCISCO SANCHEZ-MADRI D

Monoclonal Antibodies of Defined Specificity AND TIMOTHY A . SPRINGER 239

21 . Production and Characterization of Bovine Im- ALBERT J . GUTDRY ,

munoglobulins from Bovine x Murine Hybrid- S . SRIKUMARAN ,

omas

AND RICHARD A . GOLDSBY 244

B . Growth and Cloning of Hybridomas

22 . Serum-Free Medium for the Growth of NS-1 ToMOYUKI KAWAMOTO ,

Mouse Myeloma Cells and the Isolation of NS-1 J . DENRY SATO ,

Hybridomas

DoN B . MCCLURE, AN D

GORDON H . SATO

266

23 . Serum-Free Medium for Hybridoma and Parental JAN KovAii AN D

Myeloma Cell Cultivation

FRANTI g EK FRANEK

277

24 . Endothelial Cell Growth Supplements for Promot- JANET H . RANSOM

29 3ing the Growth of Monoclonal Antibody-Pro-

ducing Hybridoma Cell s

25 . Feeder Effect of Enriched Human Monocytes on THOMAS BRODIN ,

Human Hybridoma, Myeloma, and Lymphoma LENNART OLSSON, AND

Cell Lines

HANS OLOV SJÖGREN

295

26 . Removal of Bovine Immunoglobulin from Serum P . A . UNDERWOOD

30 1in Hybridoma Culture Media Using Protein A

27 . A Single-Step Technique for Selecting and Clon- JOHN M . DAVIS

30 7

ing Hybridomas for Monoclonal Antibody Pro-ductio n

28 . Cloning of Murine Hybridoma Cells in Ultra-Low CURT I . CIVIN ANDGelation Temperature Agarose

M . L . BANQUERIGO

32 2

29 . A Simple Method for Cloning Hybridomas in 20-µ1 MARVIN B . RITTENBERG ,Hanging Drops

ABIGAIL BUENAFE ,

AND MCKAY BROWN

327

30 . Clonal Isolation of Hybridomas by Manual Single- ALBERT BOEY$

33 2Cell Isolatio n

31 . Semiautomated Plaque Method for the Detection BEVERLEY L . PIKE

34 1of Antibody-Forming Cell Clones

32. Production of Monoclonal Antibodies by Agarose- K . NlLssoN ,

Entrapped Hybridoma Cells

W . SCHEIRER,H . W . D . KATINGER,AND K . MOSBACH

35 2

33 . Automated Production of Monoclonal Antibodies S . FAZEKAS DE ST .GROTH

36 0in a Cytostat

34 . The Effect of Pristane on Ascites Tumor Forma- NICHOLAS J . HOOGENRAAD AN Dtion and Monoclonal Antibody Production

CHRISTOPHER J . WRAIGHT

375

35 . Production of Ascites and Rescue of Hybridomas PAMELA L . WITTE ANDby Intrasplenic Inoculation

ROSALIE BER

38 1

36 . Growth of Rat-Mouse Hybridomas in Nude Mice DHIRENDRA N . MISRA ,and Nude Rats

SAAD A . NOEMAN ,

HEINZ W. KUNZ, ANDTHOMAS J . GILL III

38 6

37 . Poisson Statistical Analysis of Repetitive Subclon- HILARY A . COLLER AN D

ing by the Limiting Dilution Technique as a BARRY S . COLLER

41 2Way of Assessing Hybridoma Monoclonalit y

38 . A Method for Freezing Hybridoma Clones in 96- DAVID E . WELLS AN DWell Microculture Plates

WILLIAM F . BIBB

417

Section II . Monoclonal Antibodies

A . Screening Assays for Monoclonal Antibodie s

39. Multivalent

Antibody

Radioimmunoassay J . GHEUENS AN D

(MARIA) for Screening Specific Antibody Se- D . E . MCFARLIN

42 5

cretion by Lymphocyte Hybridoma Culture s

40 . A Rapid Technique Using Radiolabeled Soluble K . F . MILLER,

Antigen to Screen Hybridoma Culture Superna- D . J . BoLT, AN D

tants

R . A . GOLDSBY

43 3

41 . Culture-Well Solid-Phase 'H-RIA for the Isotype- PATRICIA K . A . MONGINI

Specific Measurement of Antibody from Mouse AND CLARE BLESSINGER

43 8

and Human Antibody-Secreting Cell s

42. The Spaghetti Overlay Procedure

W . STEVEN ADAIR

45 3

43. Solution-Phase RIA and Solid-Phase EIA Using JOHN E . SInvELY ,

Avidin-Biotin Systems for Analysis of Mono- CHRISTOPH WAGENER ,

clonal Antibody Epitopes and Affinity Con- AND BRIAN R . CLARK

459

stant s

44 . A Simple Method for Ranking the Affinities of VERONICA VAN HEYNINGEN 47 2

Monoclonal Antibodies

45 . The Hazards of Mycoplasma Contamination in the JAMES P . JAKWAY

48 1

Screening of Hybridoma Supernatants for Ef-

fects on ['H]Thymidine Incorporatio n

46. The Dot Immunobinding Assay

RICHARD HAWKES

484

47 . Detection of Antibody-Secreting Hybridomas GENEVIEVE MOTrA AN D

with Diazobenzyloxymethyl Paper

DANIEL LOCKER

49 1

48 . The Use of Alkaline Phosphatase-Conjugated PETER L . EY AN D

Anti-Immunoglobulin with Immunoblots for LEONIE K . ASHMAN

497

Determining the Specificity of Monoclonal Anti -

bodies to Protein Mixture s

49 . Slide lmmunoenzymatic Assay (SIA) in Hybrid- EVERLY CONWAY DE MACARIO ,

oma Technology

ALBERTO J . L . MACARIO ,

AND ROBERT J . JOVELL

509

50 . Use of Mouse and Human Monoclonal Antibodies MARK C GLASSY AN D

in Enzyme Immunofiltration

PATRICK H . CLEVELAND

525

51 . Measurement of Monoclonal Immunoglobulin KENNETH W . HUNTER, JR . AN D

Concentrations in Hybridoma Cultures by Corn- JOHN M . BOSWORTH, JR .

54 1

petitive Inhibition Enzyme Immunoassa y

52 . The Detection and Characterization of Antigen- M . R . CLARK

548

Specific Monoclonal Antibodies Using Anti-Im-

munoglobulin Isotype Antibodies Coupled to

Red Blood Cells

53 . Agglutination Assay Using Protein A-Sensitized DAVID MALE AN D

Erythrocytes for Detection of Immunoglobulin GARETH PRYCE

55 6

in Tissue Culture Supernatants

54, Immunohistochemical Techniques Using Mono- DANIEL R . CIOCCA AND

clonal Antibodies

ROBERT J . BJERCKE

562

55 . Method for Rapid Detection of Membrane Anti- CLAUDE BOUCHEIx ,

gens by Immunofluorescence and Its Applica- PATRICIA KRIEF ,

tion to Screening Monoclonal Antibodies

JEAN-YVES PERROT ,

MASSOUD MIRSHAHI ,

AND CLAUDE ROSENFELD

58 0

B . Purification of Monoclonal Antibodies and Preparation ofAntibody Fragment s

56. Purification of Mouse Monoclonal Antibodies CLAUDINE BRUCK,

from Ascitic Fluid by DEAE Affi-Gel Blue JEFFREY A . DREBIN ,Chromatography

CORINNE GLINEUR, AN D

DANIEL PORTETELLE

587

57 . Purification and Analysis of Monoclonal Antibod- SCOTT W . BURCHIEL

59 6ies by High-Performance Liquid Chromatog-

raph y

58 . Separation of IgG Idiotypes by High-Performance HECTOR JUAREZ-SALINAS ,Hydroxylapatite Chromatography

GARY S . OTT ,JENG-CHYH CHEN ,

TIMOTHY L . BROOKS ,

AND LARRY H . STANKER

61 5

59 . The Use ofRat Monoclonal Antibodies to Charac- FRANQOISE CORMONT ,

terize, Quantify, and Purify Polyclonal or PATRICK MANOUVRIEZ ,Monoclonal Mouse IgM

LIEVE DE CLERCQ ,

AND HERVE BAZIN

622

60 . Purification of IgG Monoclonal Antibodies from FRANTISEK FRANEK

63 1

Ascitic Fluid Based on Rivanol Precipitatio n

61 . Purification of Rat Monoclonal Antibodies

HERVE BAZIN ,

FRANQOISE CORMONT, AN D

LIEVE DE CLERCQ

63 8

62 . Preparation ofF(ab')2 Fragments from Mouse IgG EDMUNDO LAMOYI

652

of Various Subclasse s

63 . Optimal Conditions for the Preparation of Pro- J . ROUSSEAUX ,teolytic Fragments from Monoclonal IgG of Dif- R . ROUSSEAUX-PREVOST ,ferent Rat IgG Subclasses

AND H . BAZIN

663

64 . Tritium Radiolabeling of Antibodies to High Spe- UDO KUMMER

67 0cific Activity with N-Succinimidyl [2,3-3 H]Pro-pionate : Use in Detecting and Analyzing Mono-clonal Antibodies

65 . Determination of the True Immunoreactive Frac- TORE LINDMO AN Dtion of Monoclonal Antibodies after Radiola- PAUL A . BUNN, JR .

67 8beling

Section III . Selected Applications of Monoclonal Antibodie s

66 . Ultrasensitive Cooperative Immunoassays with PAUL H . EHRLICH AND

Mixed Monoclonal Antibodies

WILLIAM R . MOYLE

69 5

67 . Use of Monoclonal Antibody to Increase the Sen- ROBERT R . MONTGOMERY ,

sitivity and Specificity of Precipitating Immu- THOMAS J . KUNICKI, AN D

noassays and Cell Surface Binding Immuno- L . MICHAEL GLODE

702

assay s

68 . Immunoaffinity Isolation of Membrane Antigens TIMOTHY V . UPDYKE AN D

with Biotinylated Monoclonal Antibodies and GARTH L . NICOLSON

71 7

Streptavidi n-Agarose

69 . An Indirect Rosette Technique for the Identifica- KINGSTON MILLS

72 6

tion and Separation of Lymphocyte Population s

by Monoclonal Antibodie s

70 . Separation of T Cell Subpopulations by Mono- RÜDIGER W . BRAU N

clonal Antibodies and Affinity Chromatography AND GUNTHER KÜMEL

73 7

71 . A Rapid Micromethod for the Enumeration of T IAN H . FRAZER ,

Cell Subsets in Blood by Epifluorescence Mi- IAN R . MACKAY ,

croscopy

AND FRANK BATTYE

74 8

72 . Cytotoxicity Assays Based on the Use of Anti- J . MARBROOK

75 9

body Secretion as a Measure of Hybridoma Via-

bilit y

73 . Use of Iron- or Selenium-Coupled Monoclonal MARK BOTHWELL AN D

Antibodies to Cell Surface Antigens as a Posi- TIMOTHY BLOCK

77 1

tive Selection System for Cell s

74 . Screening for Monoclonal Antibodies to Human DANIEL P . ESKINAZI ,

Cellular and Soluble Antigens

WILLIAM C . EBY, AN D

GIUSEPPE A . MOLINARO

78 3

75 . Epitope Ratio Analysis for the Simultaneous BENNF,TT KAUFMAN AN D

Study of Several Cell Surface Antigens

RICHARD GOLDSBY

79 7

76 . Use of Monoclonal Antibodies as Radiopharma- DAVID COLCHER ,

ceuticals for the Localization of Human Carci- JOSE ESTEBAN, AN D

noma Xenografts in Athymic Mice

FRANSIOISE MORNEX

80 2

77 . Coating of Liposomes with Subunits of Mono- YOSHIYUKI HASHIMOTO ,

clonal IgM Antibody and Targeting of the Lipo- MINORU SUGAWARA ,

somes

TOSHIO KAMIYA ,

AND SHINYA SUZUKI

817

78 . Immunohistological Studies with Monoclonal An- WAYNE W . HANCOCK ANDtibodies

ROBERT C . ATKINS

82 8

79 . Use of Alkaline Phosphatase-Conjugated Antibod- BRYAN M . TURNER

84 8ies for Detection of Protein Antigens on Nitro -cellulose Filters

80 . Stepwise Amplified Immunoenzyme Staining P . M . LANSDORP ,Techniques for the Detection of Monoclonal A . W . WOGNUM, AN DAntibodies and Antigens

W . P . ZEIJLEMAKER

85 5

Section IV . Summary

81 . Other Articles from Methods in Enzymology Re- HELEN VAN VUNAKIS AN D

lated to Immunochemical Techniques

JOHN J . LANGONE

87 1

AUTHOR INDEX 877

SUBJECT INDEX 911