THE JOURNAL
`OF
`
`EXPERIMENTAL MEDICINE
`
`EDITED BY
`MACLYN McCARTY
`RENE DUBOS
`HENRY G. KUNKEL
`
`ADVISORY EDITORS
`
`VINCENT G. ALLFREY
`
`JEROME GROSS
`
`JAMES G. HIRSCH
`ALEXANDER G. BEARN
`FRANK L. HOR SF AL L, JR.
`B AR UJ BENACERRAF
`RU PERT E. BILLINGHAM COUNCILMAN MORGAN
`HANS J. M'ULLER - EBERHARD
`ALBERT H. COONS
`C HANDLER A S T ETSON
`JONATHA N W. UHR
`W . BARRY WOOD, JR.
`
`FRANK J. D I XON
`HARRY EAGLE
`HAROLD S. G I NSBERG
`
`VOLUME 132
`1970
`
`PUBLISHED MONTHLY BY
`
`T HE ROCKEFELLER UNIVERSITY PRESS
`
`LIBRARY
`'UNIVERSITY OF CALIFOR.m4
`DAVIS
`
`1 of 22
`
`BI Exhibit 1103
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`COPYRIGHT@ 1970 BY THE ROCKEFELLER U NIVERSITY PRF.SS
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`CONTENTS
`
`l O. 1, JULY 1, 1970
`
`1
`
`31
`
`44
`
`PAGE
`G. L. Asn:ERSON and M. ZEMBAI.A . Contact sensitivity in the mouse. IV.
`The role of lymphocytes and macrophages in passive transfer and the
`mechanism of their interaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`J3ARRY R. BLOOM, Lms JIMENEZ, and PHILIP I. lVIARcus. A plaque assay
`for enumerating antigen-sensitive cells in delayed-type hypersen-
`sitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`·
`. . . . . . . . 16
`ERWIN DIENER and MARC .fELDll'IANN. Antibody-mediated suppression of
`the immune response in vitro. II. A new approach to the phe-
`nomenon of immunological tolerance. . . . . . . . . . .
`RooERT C. SEEGER and JoosT J. OPPENHEIM. Synergistic interaction of
`macrophages and lymphocytes in antigen-induced transformation of
`lymphocytes... .
`. . . . . . . . . . . . . . . . . . . . .
`D. C. BENJAMIN and W. 0. WEIGLE. The termination of immunological
`unresponsiveness to bovine serum albumin in rabbits. I. Quantita-
`Live and qualitative response to cross-reacting albumins. . . . . . .
`Bmc;1~R ANDERSSON. Studies on the regulation of avidity at the level of
`the single antibody-forming cell. The effect of antigen close and time
`after immunization . . . . . . . . . . . . .
`. . . . . . . . .
`G101<CIO TONJETTI, MICHAEL B. A. OLDSTONE, and FRANK J. DIXON. The
`effect of induced chronic viral infections on the imm.unologic diseases
`of New Zealand mice . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . .
`H. WIGZELL and O. MA.KELA. Separation of normal and immune lymphoid
`cells by antigen-coated columns. Antigen-binding characteristics of
`membrane antibodies as analyzed by ha.pten- protein antigens. . .
`s. FAI CHEUK, HELMUT H. HAHN, DOUGLAS M . .MOORE, DONALD N .
`KRAUSE, PETER A. TOMASULO, and W. BARRY WOOD, JR. Studies
`on the pathogenesis of fever. XX. Suppression and regeneration of
`pyrogen-producing capacity of exudate granulocytes. . . . . . . . . . . . . . 127
`D. C. GILMOUR, G. A. THEIS, and G. J. TliORBECKE. Transfer of anti-
`body production with cells from bursa of Fabricius .. . ... . .. . . . . . . . 134
`SUSAN ZOLLA, JOEL BuxBAUM, E. C. fnANKLIN, and :iVI. D. SCHARFF.
`Synthesis and assembly of immunoglobulins by malignant human
`plasmacytes. I. Myelomas producing -y-chains and light chains . . . . 148
`]. WAYNE STREILEIN and R . E. BILLINGHAM. An analysis of graft-versus(cid:173)
`bost disease in Syria.n hamsters. I. The epidermolytic syndrome:
`description and studies on its procurement. . ..... . ..... . ... . .. .. 163
`
`66
`
`77
`
`89
`
`110
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`lV
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`CONTENTS
`
`J. WAYNE STEILEIN and R. E. BILLINGHAJvC. An analysis of graft-versus(cid:173)
`host disease in Syrian hamsters. II. The epiderrnolytic syndrome:
`studies on its pathogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
`
`PAGE
`
`199
`
`No. 2, AUGUST 11 1970
`A. L . L UZZATI, R. j\I[. Tosr, and A. 0. CAR!lONARA. Electrophoretically
`homogeneous ant ibody synthesized by spleen foci of irradiated
`repopulated m ice.... . . . . . .
`. . . . . . . . . . .
`TAX TE \i\fu and ELVIN A. KABAT. An analysis of the sequences of the
`variable regions of Bence J ones proteins and myeloma light chains
`and their implications for antibody complementarity. . . . . . . . . . . . 211
`J AMES H . GORDON and R ENE Dunos. The anaerobic bacterial flora of the
`mouse cecum .. . . .. ... . . .. . . ........ . . . . . .. . ..... .. . . .. . ... . . . 251
`DAVID H. KATZ, Wu.LIAM E. PAUL, EDMOND A. GomL, and BARUJ
`BENACERRAF. Carrier function in anti-hapten immune responses.
`I. Enhancement of primary and secondary anti-hapten antibody
`responses by carrier preimmunization . . . . . . . . . . . . . . . . . . . . . . . . . . 261
`WrLLIAM E. PAUL, DAVID H. K.ATz, EDMOND A. GomL, and BARUJ
`BENACERRAF. Carrier function in anti-hapten immune responses.
`II. Specific properties of carrier cells capable of enhancing anti-
`hapten antibody responses . .. .. ..... ... .. .. . .. . . ... ... .. . ... . . . 283
`ROBERT C. SKARNES. H ost defense against bacterial endotoxernia:
`m.echanism in normal animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
`HENRY R. HitGARJ). Synergism of thymus and bone marrow in the pro(cid:173)
`duction of g raft-versus-host splenomegaly in X -irradiated hosts. .. . . 317
`PETER C. H ARPEL. Human plasma alpha 2-macroglobulin. An inhibitor of
`plasma k allikrein. . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
`J AllillS C. K ENNEDY, P ERRY E . TREADWELL, and EDWIN S. LENNOX.
`Antigen-specific synergism in the immune r esponse of irradiated mice
`given marrow cells and peritoneal cavity cells or extracts ... .... . 353
`PAvlI> OsonA. Some physical and radiobiological properties of immu-
`nologically r eactive mouse spleen cells . . . .
`. . . . . . . . . 368
`
`No. 3, SEPTEMBER 1, 1970
`L. vV. CLEM and P.A. SMALL, J1~. P hylogeny of immunoglobu lin structure
`and function. V. Valences and association constants of teleost anti-
`bodies to a haptenic determinant . . . . . . . . . . . . . . .
`. .... . . . . . . .. 385
`RICHARD J. M ANGl and MICHAEL R. MARDINEY, JR. The in v itro trans(cid:173)
`formation of frozen-stored lymphocytes in the rn.ixed lymphocyte
`reaction and in cul ture with phytohemagglutinin and specific antigens . 401
`MARTINE Y. K. ARMSTRONG, ERNST GLEICIIMANN, HELGA GLEICHMANN,
`
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`CONTENTS
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`v
`
`448
`
`l'AGE
`Lo1mAINE BELDOTTI, JANINE ANm~E-SCHWARTz, and ROBERT S.
`ScHWAll'fZ. Chronic allogeneic chsease. II. Development of lym-
`phomas. . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
`Nm:r, L. WARNER and LEONORE A. HERZENBERG. Tolerance and im(cid:173)
`munity to maternally derived incompatible IgG2 .. -globulin in mice. . 440
`f.RED G. GuDAT, T. N. HARRIS, SUSANNA HARRIS, and KLAUS HUMMELER.
`Studies on antibody-producing cells. I. Ultrastructure of 19$ and
`7S antibody-producing cells .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`JuDITI-1 C. KNECHT, GERALD SCHIFFMAN, and ROBERT AUSTRIAN. Some
`biological properties of pneumococcus Type 37 and the chemistry of
`its capsular polysaccharide . . . . . .. ...... ...... . ....... . ......... 475
`Dov L. Bonos and KENNETH S. WARREN. Delayed hypersensitivity-type
`grnnuloma formation and dermal reaction induced and elicited by
`a soluble factor isolated from Schistosorn.a mansoni eggs. . . . . . . . .
`H. G. KUNKEL, F. G. JOSLIN, G. M. PENN, and J. B. NATVIG. Genetic
`variants of ')'G4 globulin. A unique relationship to other classes of
`')'G globulin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`R. J. NORTH. The relative importance of blood monocytes and fixed
`macrophages to the expression of cell-mediated immunity to infec-
`tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`R. J. NORTH. Suppression of cell-mediated immunity to infection by an
`antimitotic drug. Further evidence that migrant macrophages ex-
`press immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`:Efox. B. RICHERSON, HAROLD F. DvoRAK, and SIDNEY LESKOWITZ.
`Cutaneous basophil hypersensitivity. I. A new look at the Jones-Mote
`reaction, general characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`HAlWLD F. DVORAK, ANN M. DVORAK, BLANCHE A. SIMPSON, HAL n.
`RICHERSON, SIDNEY LESKOWITZ, and MORRIS J. KARNOVSKY. Cu(cid:173)
`taneous basophil hypersensitivity. IL A light and electron microscopic
`description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`OsrAs STU'.1'.MAN, EDMOND J. YUNrs, and ROBERT A. GooD. Studies on
`thymus function. L Cooperative effect of thymic function and
`lymphohemopoietic cells in restoration of neonatally thymectomized
`mice.... . . . .. .. ........ .... . .. . .. ..... . . . .
`OSIAS STUT:MAN, EDMOND J. YUNIS, and ROBERT A. Goon. Studies on
`thymus function. IL Cooperative effect of newborn and embryonic
`hemopoietic liver cells with thymus function .. . . .... .. . . . . . ..... 601
`
`488
`
`508
`
`521
`
`535
`
`546
`
`558
`
`583
`
`No. 4, OCTOBER 1, 1970
`EDNA MOZES, G. M. SHEARER, and MICHAEL SELA. Cellular basis of the
`genetic control of immune responses to synthetic polypeptides. I.
`
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`Vl
`
`CONTENTS
`
`l'AG£
`
`613
`
`623
`
`694
`
`702
`
`721
`
`Differences in frequency of splenic precursor cells specific for a syn(cid:173)
`thetic polypeptide derived from multichain polyproline ([T, Gj-
`Pro- -L) in high and low responder inbred mouse strains.
`G. CunKow1cz, G. M. SHEARER, and T. ho. Cellular differentiation
`of the immune system of mice. VI. Strain differences in class dif-
`ferentiation and other properties of marrow cells. .
`J. I). DunEY, NANCY L. MILLER, and ]. IC FRENKEL. The Towplasnza
`gondii oocysl from cat feces. . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . 636
`STEPHEN I. MORSE and BRUCE A. BARRON. Studies on the leukocytosis
`and lymphocytosis induced by Bordetelta pertitSSt-s. III. The distribu-
`. tion of transfused lymphocytes in pertussis-treated and normal
`mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663
`Fl~ANK R. ScmnD, IVAN M. RoITT, and l\IIA.1uA J. ROCHA. Complement
`fixation by a
`two-component antibody system : immunoglobulin
`G and immunoglobulin lVI anti-globulin (rheumatoid factor). Para-
`doxical effect related to immunoglobulin G concentration . .... . . . .. 673
`GARY D. OVERTURF and EDWARD A. MORTIMER, ]R. Studies on the
`relationship between the production of bacteriocines by Group A
`streptococci and acute glomerulonephritis.... . ...... . .........
`CELSO IlrANCo, RICHARD PATRICK, and VICTOR NussENZWEIC. A popula(cid:173)
`tion of lymphocytes bearing a membrane receptor for antigen(cid:173)
`antibody- complement complexes. I. Separation and characteriza-
`tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`ALAN E . BEER and R. E. BILLINGHAM.
`Implantation, transplantation,
`and epithelial-mesenchymal relationships in the rat uterus. . . . . . .
`MICHAEL PorrnR and RosE LIEBERMAN. Common individual antigenic
`determinants in five of eight BALB/c IgA rnyeloma proteins that
`bind phosphoryl choline . ... . . . . . . . . . . . . . . . . . . . . .
`. . . 737
`Gurno Bioz:a, RICHARD AsOFSKY, RosE LIEBERMAN, CLAUDE STIF.li'EL,
`DENISE Mou'.I'ON, and BARUJ B ENACERRAF. Serum concentrations
`and allotypes of immunoglobulins in two lines of mice genetically
`selected for "high" or "low" antibody synthesis...... ... .......
`B . SHANNON DANES, J. E. SCOTT, and ALEXANDER G. BEARN. Further
`studies on metachromasia in cultured human fibroblasts. Staining
`of glycosaminoglycans (mucopolysaccharides) by Alcian b lue in salt
`solutions . . . .. ...... .. . . . .. .. . ..... . . .... . ...... . . . ...... . .. . . 765
`NEIL R. COOPER and HANS J. MuLLER-ElrERHARD. The reaction
`mechanism of human CS in immune hemolysis . . ..... . ........ . . 775
`RAJ.PH VAN FURTH, ]AMES G. HmscH, and MARTHA E. F.rmoRKO. Mor(cid:173)
`phology and peroxidase cytochemistry of mouse promonocytes,
`rnonocytes, and macrophages.
`. . . . . . . . .
`
`752
`
`794
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`CONTENTS
`
`Vil
`
`l'AGE
`
`RALPH VAN F URTH and MARTINA .M. C. DmssELHOFF-DEN DULK. The
`kinetics of promonocytes and monocytes in the bone marrow. . . . . . 813
`D. SJ·:J.LlN, M . Ki:ivARY, U. ROTHER, and K. ROTHER. Intrarenal com-
`plement fixation by cytotoxic antibodies . . . . . . . . . . . . . . . . . . . .
`
`829
`
`' 1
`
`858
`
`868
`
`885
`
`898
`
`916
`
`No. 5, NovmmER 1, 1970
`v. s. HYERS and E. E. SERCARZ.
`Induction and reversal of immune
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845
`paralysis in vitro.. . . . . . .
`M. VAN WrNKLl': and L. LEVY. Further studies on the reversibility of
`serum sickness cholesterol-induced atherosclerosis. . . . . . . . . .
`PETER B. LAMBERT and HOWARD A. FRANK. Cellular and vascular com(cid:173)
`ponents of
`the allograft reaction. Evidence from returned skin
`nllografts...
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`NATHAN TRAININ and MYRA SMALL. Studies on some physicochemical
`properties of a thymus humeral factor conferring immunocom-
`petence on lymphoid cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`OTTO GOTZE and HANS J. ::V.l"tiLLER-Enm:rnARD. Lysis of erythrocytes
`by complement in the absence of antibody. . . . . . . . . . .
`PE'f8R J. McCuLLAGH. The abrogation of sheep erythrocyte tolerance
`in rats by means of the transfer of allogeneic lymphocytes.. . . . . .
`WESLEY \V . BULLOCK and MARVIN B. RITTENBERG . In vitro-initiated
`secondary anti-hapten response. II. I ncreasing cell avidity for
`antigen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926
`N. K. B. DAY, H. GEwunz, R . JOHANNSEN, J. FINSTAD, and R. A.
`Goon. Complement and complement-like activity in lower ver(cid:173)
`tebrates and invertebrates.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 941
`BRUCE W. B1UENT and ALFRED NrsoNOFF. Quantitative investigations of
`icliotypic antibodies. IV. Inhibition by specific haptens of the reaction
`of anti-hapten antibody with its anti-idiotypic antibody.. . . ........ 951
`H. F. JEEJEEBHOY. T he effect of heterologous anti-lymphocyte serum
`on lymphocytes of thymus and marrow origin. . . . . . . . . . . . . . . . . . . . 963
`KNUD D. KNUDSEN, LEWIS K . DAHL, I(EITH T HOMPSON, J UNICHI lWAI,
`MARTHA HEINE, and GEO.RGE LEITL. Effects of chronic excess salt
`ingestion. Inheritance of hypertension in the rat. . . . . . . .
`BENVENUTO PERNIS, LUCIANA FORNI, and L UISA AMANTE. Immuno(cid:173)
`globulin spots on the surface of rabbit lymphocytes . . . . . . . . . . . . . . . . 1001
`VOLI<l·:tl SCHIRRMACHER and KLAUS RAJEWSKY. Determination of anti-
`body class in a system of cooperating antigenic determinants. . . . . . 1019
`R. V. 8r,ANDEN. Mechanisms of recovery from a generalized viral infec-
`tion : mousepox. I. The effects of anti-thyrnocyte serum. . . . . . . . .
`
`976
`
`1035
`
`I
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`Vlll
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`CONTENTS
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`PAGE
`
`No. 6, DECEMBER 1, 1970
`IRUN R. COHEN, LARY STAVY, and MICHAEL FELDMAN. Glucocorti(cid:173)
`coids and cellular immunity in vitro. Facilitation of the sensitization
`phase and inhibition of the effector phase of a lymphocyte anti-
`fibroblast reaction ...... .. ... . ... . .. . .. .... ................... 1055
`ELLEN BoRENFREUND, YuJI HoNoA, Mn.DRED STEINGLASS, and AARON
`BENDICH. Studies of DNA-induced heritable alteration of mam-
`malian cells .... .. ... ...... . ....................... .. ..... . .. 1071
`JOSEPH D. SCHULMAN and KATIIl<YN H. BRADr;J·:Y. The metabolism of
`amino acids, peptides, and clisulfides in lysosomes of fibroblasts cul-
`tured from normal individuals and those wit11 cystinosis .. ...... . . .. 1090
`MICHAEL HEIDELBERGER and MOREY E. SLODKC. Predicted and un(cid:173)
`predicted cross-reactions of an acetylphosphogalactan of Sporobolo-
`myces yeast. II . ... .... ................... . ..... ... ......... .. 1105
`D. G. COLLEY, A. Y. SmH Wu, and B. H . WAKSMAN. Cellular differenti-
`ation in the thymus. III. Surface properties of rat thymus and lymph
`node cells separated on density gradients .............. .......... 1107
`HAROLD C. MILLER and GusTAvo Cunxow1cz. Antigen-specific cells in
`mouse bone marrow. I. Lasting effects of priming on inununocyte
`production by transferred marrow .............................. 1122
`JOEL G. BASEMAN, A. M. PAPPEN'HEThfER, Jn., D. M. GILL, and ANNABEL
`A. HARPER. Action of diphtheria toxin in the guinea pig.....
`M. A. VEJ\KATACHALAM, M. J. KARNOVSKY, H. D. FAIUMI, and R. S.
`CoTRAN. An ultrastructural study of glomernlar permeability using
`catalase and peroxidase as tracer proteins. . . . . . . . . . . . . . . . . . . . . . . 1153
`M. A. VENKATACHALAM", R. S. Con<AN, and M. J. KARNOVSKY. An
`ultrastructural study of glomerular permeabili ty in aminonucleoside
`nephrosis using catalase as a tracer protein. . . . . . . . . . . . . . . . . . . . . 1168
`T . TAKAHASm, E. A. CARSWELL, and G. J. TIIORDECKE. Surface anti-
`gens of im1mmocompetent cells. I. Effect of 0 and PC.l alloantisera
`on the ability of spleen cells to transfer immune responses. . . . . . . . . . 1181
`N. ABRAMSON, A. F. LoBucuo, J. H. J ANDL, and R. S. CoTRAN. The
`interaction between human monocytes and red cells. Binding char-
`acteristics...................... ...... ................. .
`N. ABRAMSON, E. W. GELFAND, J. H. JANDL, and F. S. RosEN. The in(cid:173)
`teraction between human monocytes and red cells. Specificity for JgG
`subclasses and IgG fragments .................................. 1207
`J. J. T. OWEN and M. C. RAFF. Studies on the differentiation of thymus-
`derived lymphocytes ..... . .. . .......................... ....... 1216
`CURLA S. WALTERS and HA:-.1S WIGZELL. Demonstration of heavy and
`light chain antigenic determinants on the cell-bound receptor for
`
`1138
`
`1191
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`CONTENTS
`
`IX
`
`l'AGE
`
`anligen. Similarities between membrane-attached and humoral
`antibodies produced by the same cell. . . . . . . . . . . . . . . . . . . . . . . . . 1233
`CHAJU,ES HUGGINS, SOL WISEMAN, and A. H. REDDI.
`'11-ansformation
`of fibroblasts by allogeneic and xenogeneic transplants of deminera-
`lizecl tooth and bone. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1250
`vVII.LlAM JOHN MARTIN", LEONARD ELLMAN, IRA GREEN, and BARUJ
`:BENACERRAF. Histocompatibility type and immune responsive-
`ness in random bred Hartley strain guinea pigs. . . . . . . . . . . . . . . . . . 1259
`KLAUS-ULRICH HARTMANN.
`Induction of a hemolysin response in vitro.
`Interaction of cells of bone marrow origin and thymic origin.. ... . . 1267
`JEJ\N-CLAUDE BYSTRYN, MARTIN W. GRAF, and JONATHAN W. UHR.
`Regulation of antibody formation by serum antibody. II. Removal
`of specific antibody by means of exchange transfusion . . . . . . .
`
`1279
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`THE JOURNAL OF EXPERIMENTAL MEDICINE
`JULY 1, 1970
`VOLUME 132, NUMBER 1
`
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`(cid:52)(cid:89)(cid:70)(cid:80)(cid:77)(cid:87)(cid:76)(cid:73)(cid:72)(cid:4)(cid:50)(cid:83)(cid:90)(cid:73)(cid:81)(cid:70)(cid:73)(cid:86)(cid:4)(cid:21)(cid:16)(cid:4)(cid:21)(cid:29)(cid:27)(cid:20)
`
`QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES
`
`IV. INHIBITION BY SPECIFIC HAPTENS OF THE REACTION OF ANTI-HAPTEN
`ANTIBODY WITH ITS ANTI-IDIOTYPIC ANTIBODY*
`
`BY BRUCE W. BRIENT,t M.D., AND ALFRED NISONOFF, PH.D.
`
`(From the Departments of Surgery and Biological Chemistry, University of Illinois at
`the Medical Center, Chicago, Illinois 60680)
`
`(Received for publication 6 July 1970)
`
`Myeloma proteins (1) and antibodies from individual animals (2, 3) have
`been shown to possess individually specific or "idiotypic" (4) antigenic de(cid:173)
`terminants. For example, anti-salmonella antibodies from an individual rabbit
`have determinants that are not detectable in other immunoglobulins of that
`rabbit nor, ordinarily, in antibodies of the same specificity from other rabbits
`(3). Quantitative studies have shown that various fractions (up to 803) of
`purified anti-benzoate antibodies, designated D (5), from a given donor rabbit
`are reactive with anti-idiotypic (anti-D) antisera (6, 7). Precipitin lines in the
`Ouchterlony test were obtained when as little as 2 3 of the donor (D) popula(cid:173)
`tion was precipitable.
`Our present investigations indicate that the reactions of anti-p-azobenzoate
`antibodies (D) with their anti-idiotypic antisera are strongly inhibited by
`benzoate derivatives, i.e. by specific haptens, whereas the reactions of D with
`anti-Fab antibodies are affected to a much smaller extent. The possible relation(cid:173)
`ship of idiotypic determinants to the antigen-combining site of a donor antibody
`is discussed.
`
`Materials and M etlwds
`
`The following methods and materials have been described previously (6, 7): preparation
`and specific purification of anti-p-azobenzoate antibodies of the IgG class; polymerization
`of these antibodies with glutaraldehyde for the purpose of immunization; preparation and
`labeling with 125IC1 of F(ab'h fragments of purified anti-benzoate antibodies (each such
`preparation containing a 20-fold excess of nonspecific F[ab'h fragments); preparation of goat
`antiserum specific for rabbit fragment Fe; and determination of allotypes of rabbit sera.
`Anti-allotype antisera were the generous gift of Dr. Sheldon Dray.
`
`*This work was supported by grants from the National Science Foundation (GB-5424)
`and the National Institutes of Health (Al-06281). It was presented in part at the annual
`meeting of the Federated Societies for Experimental Biology and Medicine, April, 1970
`(Fed. Proc. 29:773).
`t Supported by a National Institute of Medical Sciences Surgery Academic Training Grant,
`U. S. Public Health Service grant No. GM 1930-0lAl.
`
`951
`
`11 of 22
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`

`

`(cid:52)(cid:89)(cid:70)(cid:80)(cid:77)(cid:87)(cid:76)(cid:73)(cid:72)(cid:4)(cid:50)(cid:83)(cid:90)(cid:73)(cid:81)(cid:70)(cid:73)(cid:86)(cid:4)(cid:21)(cid:16)(cid:4)(cid:21)(cid:29)(cid:27)(cid:20)
`
`952
`
`QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES. IV
`
`Percentages of 1251-F(ab'h fragments of D antibodies precipitable were determined by
`an indirect method utilizing excess goat antibody directed to the Fe fragment of rabbit IgG
`to precipitate complexes of 1251-F(ab'h fragments with anti-D antibodies. The method used
`corresponds to that reported previously (7) with two exceptions. First, ovalbumin (50 µg
`per test) was used in place of bovine serum albumin to minimize adherence of the labeled
`protein to glass. Serum albumin was not used because it is known to bind various small
`molecules which were tested as inhibitors. Second, controls were run by utilizing anti-dinitro(cid:173)
`phenyl (anti-DNP) antiserum in place of anti-D. (Formerly anti-oval bu min was used as a con(cid:173)
`trol) . Percentages of radioactivity precipitated in control experiments did not exceed 2.83 .
`The control value was subtracted in each series from percentage values obtained with anti-D
`antiserum.
`When small molecules such as haptens were tested as competitors of the reaction of F(ab')2
`fragments of D with anti-D, the competitor was first incubated with the labeled F(ab')2
`fragments and SO µg of ovalbumin for 2 hr at 37°C, pH 8. Anti-D antiserum was added, in(cid:173)
`cubation was continued for 1 hr, and goat anti-rabbit-Fe was then added to the mixture.
`After standing for 3 days in the refrigerator, the percentage of radioactivity precipitated was
`determined with a gamma scintillation counter (7). A minimum of 3000 counts was recorded
`for each precipitate-supernatant pair. Experiments were carried out in triplicate. All haptens
`and other small molecules tested as competitors were crystallized from water or from an
`ethanol-water mixture, with the exception of p-nitrobenzene sulfonic acid, and used as the
`sodium salts. Solutions were adjusted to pH 8.
`Preparation of Anti-Idiotypic (Anti-D) Antibodies.-Specifically purified anti-benzoate
`antibodies (D) from individual rabbits were injected into recipients of allotype matched to
`the donor with respect to the following specificities: al, a2, a3, b4, bS, b6, b9, c7, c21.
`Rabbit 9Q was immunized with purified D antibody from rabbit AZS, according to the
`second protocol given in reference 6; 3-S mg of monomeric D were used for the initial inocu(cid:173)
`lations with Freund's adjuvant, and polymerized D was used for subsequent intravenous
`inoculations. A pool of antiserum from rabbit 9Q was made from several bleedings which
`showed strong antibody activity by the method of indirect precipitation.
`Purified D antibodies from rabbits A4 and I-14 were injected exclusively in the poly(cid:173)
`merized form into recipients 14E and lOX, respectively, and D antibody from rabbit AS was
`injected into 3 recipients, 7A, 7C, and 7D. Two subcutaneous inoculations of 3 mg each in
`complete Freund's adjuvant were given 3 wk apart. These were followed by intravenous in(cid:173)
`oculations at intervals of 2-4 wk. Bleedings were taken S-8 days after an injection. Antisera
`from individual recipients which gave strong reactions by the method of indirect precipitation
`were pooled. Evidence that antibodies produced were directed to idiotypic determinants, and
`not to allotypic or hidden determinants, has been described in detail for D antibodies of rab(cid:173)
`bits AS and AZS (7, 8). This evidence includes the failure of sera taken from donor rabbits
`prior to immunization to inhibit reactions of anti-D with 1251-F(ab'h fragments of D, the loss
`of inhibitory capacity of whole D serum after precipitation of antibenzoate antibodies, and
`the specificity of the reactions when various anti-D sera and D antibodies were utilized.
`Similar results were obtained in a large number of preliminary control experiments with
`the D antibodies of rabbits I-14 and A4.
`The percentages of F(ab'h fragments of specifically purified anti-p-azobenzoate anti(cid:173)
`bodies that were precipitable by anti-D antisera, using the indirect method, were : rabbit AZS,
`S7 ±3% ; rabbit AS, 37 ±2% ; rabbit 1-14, 31 ±1 % ; rabbit A4, 23 ±1%.
`
`RESULTS
`The effects of specific haptens and other small molecules on the reactions of
`125I-F(ab1)2 fragments of D antibodies with anti-D antisera are shown in Tables
`
`12 of 22
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`

`

`(cid:52)(cid:89)(cid:70)(cid:80)(cid:77)(cid:87)(cid:76)(cid:73)(cid:72)(cid:4)(cid:50)(cid:83)(cid:90)(cid:73)(cid:81)(cid:70)(cid:73)(cid:86)(cid:4)(cid:21)(cid:16)(cid:4)(cid:21)(cid:29)(cid:27)(cid:20)
`
`BRUCE W. BRIENT AND ALFRED NISONOFF
`
`953
`
`TABLE I
`Effect of Haptens and Other Small Molecules on the Reaction of 125!-F(ab'h Derived from D
`Antibodies of Rabbit AZ5 with Anti-D Serum*
`
`Competitor
`
`Final molar concentration of competitort
`1.6 x 10-•
`5 x 10-•
`5 x 10-•
`
`Rel. K(9)
`
`p-(p' -hydroxy )-phenylazoben-
`zoate
`benzoate
`p-nitrobenzoate
`m-nitrobenzoate
`o-nitrobenzoate
`p-aminobenzoate
`m-aminobenzoate
`o-aminobenzoate
`p-bromobenzoate
`m-bromobenzoate
`o-bromobenzoate
`p-chlorobenzoate
`m-chlorobenzoate
`o-chlorobenzoate
`p-iodobenzoate
`o-iodobenzoate
`p-methylbenzoate
`m-methylbenzoate
`o-methylbenzoate
`
`"'l-F(ab'h precipitated,% of control§
`57 (1)
`61 (5)
`74 (4)
`
`73 (4)
`69 (2)
`74 (3)
`90 (1)
`88 (1)
`93 (3)
`76 (1)
`75 (1)
`81 (2)
`95 (1)
`64 (2)
`78 (7)
`81 (3)
`64 (2)
`87 (1)
`71 (2)
`87 (2)
`92 (2)
`
`90 (4)
`70 (3)
`86 (3)
`98 (1)
`96 (1)
`91 (1)
`85 (1)
`86 (1)
`98 (1)
`103 (1)
`77 (2)
`83 (3)
`97 (1)
`75 (2)
`97 (1)
`81 (1)
`97 (3)
`100 (1)
`
`95 (1)
`82 (2)
`85 (4)
`89 (2)
`97 (2)
`96 (3)
`93 (2)
`78 (2)
`93 (4)
`96 (1)
`91 (2)
`89 (2)
`87 (5)
`90 (1)
`
`91 (1)
`102 (2)
`102 (1)
`
`22
`
`1.0
`11.5
`0.4
`<0.1
`0.9
`0.3
`1.5
`5.0
`1.3
`0.1
`3.7
`0.8
`0.2
`
`2.6
`0. 7
`0.1
`
`94 (1)
`98 (1)
`97 (1)
`112 (1)
`
`99 (1)
`102 (2)
`102 (1)
`104 (1)
`
`99 (1)
`101 (3)
`95 (1)
`94 (2)
`
`Sodium acetate
`Potassium iodide
`Potassium bromide
`p-(p' -dimethylamino)-phenyl-
`azobenzene sulfonate
`* Anti-D serum was from rabbit 9Q. The indirect method of precipitation was used.
`Each test contained 0.5 µg 1251-F(ab'h of the D antibody, 9.5 µg nonspecific F(ab'h, and 50
`µg ovalbumin in a final volume of 0.31 ml. 10 µl of anti-D serum and 0.4 ml goat anti-rabbit
`Fe were then added. In the absence of competitors 57 ± 3% of the radioactivity was pre(cid:173)
`cipitated with anti-D serum and 1.2% in the control utilizing 10 µl anti-DNP antiserum.
`Experiments were in triplicate with average deviations given in parentheses.
`t Refers to concentration prior to the addition of goat anti-rabbit Fe antiserum.
`§Expressed as percentage of the quantity precipitated in the absence of competitor.
`
`I-IV. Each table reports data obtained with a different donor-recipient pair of
`rabbits. The last column in each table presents relative binding affinities of
`haptens, obtained by Pressman et al. (9), who measured inhibition of specific
`precipitation by haptens.
`It is evident that specific haptens inhibit the reactions of anti-D sera. By far
`the best inhibitor in each system was p-(p'-hydroxy)-phenylazobenzoate. This
`
`13 of 22
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`

`

`(cid:52)(cid:89)(cid:70)(cid:80)(cid:77)(cid:87)(cid:76)(cid:73)(cid:72)(cid:4)(cid:50)(cid:83)(cid:90)(cid:73)(cid:81)(cid:70)(cid:73)(cid:86)(cid:4)(cid:21)(cid:16)(cid:4)(cid:21)(cid:29)(cid:27)(cid:20)
`
`954
`
`QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES. IV
`
`compound also combines with greater affinity with anti-p-azobenzoate anti(cid:173)
`bodies than any of the other compounds tested. The percentage inhibition ob(cid:173)
`served in the four systems ranged from 43 to 693 when p-(p'-hydroxy)-phenyl(cid:173)
`azobenzoate was present at a final concentration of 1.6 X 10-3 M.
`In three of the four systems (Tables I-III), all para-substituted benzoate
`derivatives tested were quite effective as inhibitors, and were more effective
`than unsubstituted benzoate. This is consistent with the greater binding affini(cid:173)
`ties of p-substituted benzoate derivatives. At a final concentration of 1.6 X
`
`TABLE II
`Effect of Haptens and Other Small Molecules on the Reaction of 1251-F(ab'h Derived from D
`Antibodies of Rabbit A4 with Anti-D Serum*
`
`Competitor
`
`Final molar concentration of competitort
`5 x 10-4
`5 x 10-5
`
`1.6 X 10-c
`
`Rel. K(9)
`
`p-(p' -hydroxy)-phenylazobenzoate
`Benzoate
`p-nitrobenzoate
`m-nitrobenzoate
`o-nitrobenzoate
`
`p-aminobenzene arsonic acid
`p-nitrobenzene sulfonic acid
`Sodium acetate
`Potassium iodide
`Potassium bromide
`p ( (p' -dimethylamino)-phenyl-
`azobenzene sulfonate
`
`"'f-F(ab') precipitated , % of control§
`86 (1)
`42 (1)
`43 (1)
`106 (1)
`95 ( < 1)
`92 ( <l)
`90 (2)
`71 (<l)
`92 (<l)
`102 (1)
`101 (2)
`87 (1)
`101 (1)
`101 (<l)
`103 (1)
`
`22
`
`11.5
`0.4
`<0.1
`
`105 (1)
`100 (<l)
`91 (1)
`96 (1)
`84 ( < 1)
`98 (1)
`
`103 (<1)
`102 (1)
`95 (<1)
`100 (1)
`97 (1)
`99 ( < 1)
`
`100 (1)
`102 (2)
`98 (1)
`94 (2)
`95 (<1)
`77 (<1)
`
`* Anti-D serum was from rabbit 14E. The indirect method of precipitation was used (see
`first footnote of Table I).
`t Refers to concentration prior to the addition of goat anti-rabbit Fe antiserum.
`§ Expressed as percentage of the quantity precipitated in the absence of competitor.
`
`10-3 M, p-nitrobenzoate decreased the percentage of 125I-F(ab'h fragments
`bound to 68-903 of the control value.
`For benzoate derivatives with the same substituent group at various positions
`on the benzene ring, the order of inhibitory capacity in nearly all instances is
`p > m > o, which is the same as the decreasing order of combining affinities
`(9). The only exceptions are the amino derivatives (Table I); however a similar
`anomaly obtains with respect to their combining affinities. This was attributed
`(9) to the capacity of an a-amino substituent to form a hydrogen bond with the
`carboxylate group of benzoate ion.
`A few tests were carried out in two additional systems in which the donor
`antibody again was from rabbit AS and the recipients were rabbits 7A and 7C.
`
`14 of 22
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`BI Exhibit 1103
`
`

`

`(cid:52)(cid:89)(cid:70)(cid:80)(cid:77)(cid:87)(cid:76)(cid:73)(cid:72)(cid:4)(cid:50)(cid:83)(cid:90)(cid:73)(cid:81)(cid:70)(cid:73)(cid:86)(cid:4)(cid:21)(cid:16)(cid:4)(cid:21)(cid:29)(cid:27)(cid:20)
`
`BRUCE W. BRIENT AND ALFRED NISONOFF
`
`955
`
`In these experiments p-(p'-hydroxy)-phenylazobenzoate gave significant inhibi(cid:173)
`tion (47 and 503, respectively, at a concentration of 1.6 X 10-3 M) but p-nitro(cid:173)
`benzoate had no effect on the react