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