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CIBA FOUNDATION COLLOQUIA ON ENDOCRINOLOGY

VOLUME 14

IMMUNOASSAY OF HORMONES

Editors for the Ciba Foundation

G. E. W. WOLSTENHOLME, O.B.E., M.A., M.B.,M.R.c.P.

and

MARGARET P. CAMERON, M.A.

With 85 Illustrations

LITTLE, BROWN AND COMPANY

BOSTON

1962

CIBA F O U N D A T I O N C O L L O Q U I A

O N E N D O C R I N O L O G Y

V O L . 14


This Page Intentionally Left Blank

CIBA F O U N D A T I O N C O L L O Q U I A

O N E N D O C R I N O L O G Y

V O L . 14


A leaflet giving fuller details of available earlier volurnes in thisseries, and also the CibaFoundation General Symposia, Colloquia on Ageing, and Study Groups, is available froin the Publishers.

CIBA FOUNDATION COLLOQUIA ON ENDOCRINOLOGY

VOLUME 14


Editors for the Ciba Foundation

G. E. W. WOLSTENHOLME, O.B.E., M.A., M.B.,M.R.c.P.

and

MARGARET P. CAMERON, M.A.

With 85 Illustrations

LITTLE, BROWN AND COMPANY

BOSTON

1962

Library of Congress Catalog Card Number: 62-12016

ALL RIGHTS RESERVED

This book is protected urider the Berne Convention. It may not be reproduced by any means, in whole or in part, without permission. Application with regard to reproduction should be addressed to the

Publishers.

Prirrted in Great Britain

THE Ciba Foundation, a unique international institution, owes its inception to the generosity of CIBA Limited, Basle. However, being established under British trust law, it enjoys complete independence in practice and policy.

Under the guidance of its distinguished Trustees, the Founda- tion offers accommodation to scientists from all over the world at its home in Portland Place. Foremost in its activities is the organization of small conferences, the proceedings of which are published in book form in the manner of the present volume. The Foundation convenes many other informal discussions between research workers of different disciplines and different nationalities and each year invites an outstanding authority to deliver a special lecture. An exchange programme between French and British postgraduates is conducted and a library service is available. Furthermore, the Ciba Foundation attempts in every other way possible to aid scientists, whether they be Nobel Laureates or young graduates makmg their first original contribution to research.

The purpose of the Ciba Foundation, which is to promote international co-operation in medical and chemical research, is symbolized in the armorial bearings by five interlaced rings representing the continents, a black sacrificial cock (emblem of Aesculapius) holding a medical caduceus, and three regular hexagons for chemistry. Its domicile in London is indicated by the red sword of St. Paul and the British lion; the wyvern and the crozier, symbols associated with Basle, refer to the sponsoring firm located in this ancient Swiss town.

V

THE CIBA FOUNDATION

for the Prornotiort of International Co-operation in Medical and Chernical Research

41 PORTLAND PLACE, LONDON, W.I.

Trustees

THE RIGHT HON. LORD ADRIAN, O.M., F.R.S.

THE RIGHT HON. LORD BEVERIDGE, K.C.B., F.B.A.

THE RIGHT HON. LORD BRAIN, LL.D., D.C.L.

THE HON. SIR GEORGE LLOYD-JACOB

SIR RAYMOND NEEDHAM, Q.C., F.S.A.

Executive Council

LL.D., D.C.L., Chairman M.P. THE RIGHT HON. LORD BRAIN,

DR. F. GROSS

PROFESSOR A. HADDOW, F.R.S.

SIR ARTHUR VERE HARVEY, C.B.E.,

THE HON. SIR GEORGE LLOYD-JACOB

PROFESSOR F. G. YOUNG, F.R.S.

Director, and Secretary to the Executive Council

DR. G. E . W. WOLSTENHOLME, O.B.E.

Deputy Director

MR. A. V. S. DE REUCK. MSc.

Assistant Secretary

MISS N. BLAND

Scientijc Assistant and Librarian

h41ss M. P. CAMERON, M.A.

Editorial Assistant

MISS MAEVE O'CONNOR, B.A.

Conference Assistant

MRS. NANCY G. SPWFFORD vi

PREFACE

THE Ciba Foundation has found many profitable subjects for discussion in the fields of endocrinology and immunology; to their marriage we could not “admit impediment”, especially as so many lusty offspring were much in evidence, and we eagerly and gratefully accepted suggestions made by Professor F. G. Young, Professor A. R. Currie, and Dr. J. C. Beck, for the realization of this colloquium on “Detection and Assay of Hormones by Immunochemical Means”.

The meeting was the last to be held in the Foundation’s old conference room, now being rebuilt, and the demolition workers moved into that part of our building almost before the sessions ended. It was all the more agreeable to end this period of the Foundation’s life in the presence of people so forward-looking and creative.

Miss Cameron assisted in the organization of the colloquium and has prepared the papers and discussions for publication. She and the Chairman, Professor Young, join the senior editor in hoping that the data and ideas here presented will find sites for attachment in many minds, and that the complexes so formed will be active for years to come.

vii

CONTENTS

PAGE Chairman’s Opening Remarks

Basic immunological considerations

F. G. YOUNG . . . . . . .

byJ. H.HUMPHREY . . . Questions: ARQUILLA, BERSON, FRASER, GRUMBACH, HUMPHREY, KNOBIL,

MOLONEY, RANDLE, WERNER . . . . . . . Some aspects of immunochemical methods for the characterization of protein hormones

by C. H. LI, N. R. MOUDGAL, A. TRENKLE, G. BOURDEL and K. SADRI . . . . . . . . . . .

Questions: ARQUILLA, BECK, BUTT, GREENWOOD, GRUMBACH, HAYASHIDA, KNOBIL, LI, MCGARRY, PEARSE, SELENKOW, WERNER . . .

General Discussion: ARQUILLA, BERSON, COOMBS, FRASER, GREENWOOD, GRUMBACH, HUMPHREY, LI, MOLONEY, WERNER . . . .

Growth Hormorie Experiences with the haemagglutination method of human growth hormone assay

by C. H. READ, S. A. EASH, and S. NAJJAR . . . . Questions: ARQUILLA, GRUMBACH, RANDLE, READ .

Immunochemical studies on human growth hormone: a consideration of the human growth hormone-anti-human growth hormone system and its application to the assay of growth hormone

. . . . . Questions: BRODY, BUTT, CURRIE, GRUMBACH, HAYASHIDA, HUMPHREY,

by M. M. GRUMBACH and S. L. KAPLAN

KNOBIL, RANDLE, READ . . . . . . . . Immunological studies with human growth hormone

by R. F R A S E R ~ ~ ~ M . H A R T O C . . . . Questions: GREENWOOD, HUMPHREY, KNOBIL, READ .

Immunoassay of serum growth hormone in diabetes mellitus by R. M. EHRLICH and P. J. RANDLE . . . . . .

HUMPHREY, MCGARRY, RANDLE, READ, UNGER . . . .

MCGARRY, RANDLE, READ, UNGER, WHITE

Questions: ARQUILLA, CURRIE, GEMZELL, GRUMBACH, HAYASHIDA,

Discussion on Growth Hormone: ARQUILLA, BECK, BERSON, COOMBS, FRASER, GEMZELL, GREENWOOD, GRUMBACH, HAYASHIDA, KNOBIL, LI,

. . . . . ... Vlll

C O N T E N T S ix

PAGE

Itisdiri Immunological studies of insulin

by E. R. ARQUILLA . . . 146

MOLONEY, RANDLE . . 165

by P. J. MOLONEY . . . 169

Questions: ARQUILLA, BERSON, COOMSS, HUMPHREY, MCGARRY,

Endogenous and pancreatic insulins

Questions: ARQUILLA, BERSON, FRASER, Lr, MOLONEY, RANDLE, SAMOLS, WERNER . . =77

Immunoassay of plasma insulin by S. A. BERSON and R. S. YALOW . . . . 182

UNGER . . . . 201

MOLONEY, RANDLE, READ, SAMOLS, YOUNG . . 204

Questions: ARQUILLA, BERSON, KNOnIL, LI, MOLONEY, RANDLE, SAMOLS,

Dircusriort orr h u h : ARQUILLA, BERSON, FRASER, HUMPHREY, Lr,

Glircugon

by R. H. UNGER, A. M. EISENTRAUT, M. S. MCCALL and L. L. MADISON . . . . . . . . 212

Discussion ott Glucagon: BECK, BERSON, FRASER, GRUMRACH, HAYASHIDA, MOLONEY, RANDLE, UNGER . . . . 221

Demonstration of the hormonal status of endogenous glucagon

Th yrotrop iia Some facets of thyrotropin immunoassay

Some studies with antiserum to thyrotropin

by S. C. WERNER . . 22s

by J. C. BECK, J. M. MCKENZIE, J. FISHMAN, L. GOSSELIN and E.E.McGARRY . . . 238

by H. A. SELENKOW, F. M. PASCASIO and M. J. CLINE . . . 248

FRASER, HUMPHREY, RANDLE, SELENKOW, WERNER, YOUNG . . 263

Immunobiological studies of thyrotropin

Discussion or1 Thyrotropirt : ARQUILLA, BECK, BERSON, BUTT, CURRIE,

Corticotropin Studies with antisera to corticotropin (ACTH)

by E. E. MCGARRY, A. BALLANTYNE and J. C. BECK . . . 273

Discussion on Corticotropirr : ARQUILLA, BECK, BERSON, CUR-, GEMZELL, GRUMBACH, HAYASHIDA, Lr, MCGARRY, MOLONEY, RANDLE, READ, SELENKOW, UNGER, WERNER, WHITE, YOUNG . . 287

X C O N T E N T S

PAGE

Gonadotropins Determination of gonadotropins in urine by a haemagglutination inhibition reaction

by L. W J D E ~ ~ C. A. GEMZELL . . . . . . 296 Questions: ARQUILLA, BECK, BERSON, BRODY, BUTT, GEMZELL, GRUMBACH

KNOBIL, LI, MCGARRY, READ, WHITE . . . . . 307

Immunological investigations of human gonadotropins by W. R. BUTT, A. C. CROOKE and F. J. CUNNINGHAM. . . 310

Questions: BUTT, WHITE . . . . * 324

SHIDA,KNOBIL,LI . . . . . , . 325 Discussion on Gonadotropins: ARQUILLA, BECK, BERSON, BUTT, HAYA-

Short Communication: Determination of human chorionic gonadotropin in biological fluids by complement fixation

. . 329 by S. BRODY and G. CARLSTR~M . . . . Discussion on Gonadotropins ( c o d . ) : ARQUILLA, BERSON, BRODY, BUTT,

GEMZELL, GRUMBACH, LI, MCGARRY, READ, WERNER, WHITE . . 334

Prolactin Immunological studies with pituitary lactogenic hormone (prolactin)

by T. HAYASHIDA . . . . . . . . . 338 Discussion on Prolactin: BECK, BERSON, CHADWICK, GEMZELL, GREENWOOD,

GRUMBACH, HAYASHIDA, KNOBIL, LI, MOLONEY, RANDLE, READ, WERNER, YOUNG . . . . . . . . . 360

Panel Discussion: Intracellular detection of hormones by immunochemical means

Growth hormone by M. M. GRUMBACH. . . . * 373 Other pituitary hormones by E. E. MCGARRY. . . . 37s Other protein hormones by R. H. UNGER . . . . . 378

Discussion: ARQUILLA, BECK, BERSON, CURRIE, FRASER, GEMZELL, GRUMBACH, HAYASHIDA, LI, MCGARRY, MOLONEY, PEARSE, RANDLE, READ, UNGER, WERNER, WHITE, YOUNG . . . * 379

General Discussion: ARQUILLA, BECK, BERSON, BUTT, C u m , GEMZELL. GREENWOOD, GRUMBACH, HAYASHIDA, KNOB=, LI, MCGARRY, MOLONEY, PEARSE, RANDLE, READ, UNGER, WHITE, YOUNG . . . . 392

List of those participating in or attending the Colloquium on “Detection and Assay of Hormones by Immunochemical Means”

zsth-z?th July, 1961

E. R. ARQUILLA

J, C.BECK .

S.A. BERSON .

S. BRODY

W. R. BUTT

A. CHADWICK . . R. R. A. COOMBS . A. R. CURRIE .

RUSSELL FRASER . J.GELZER . . C. A. GEMZELL .

F. C.GREENWOOD . M. M. GRUMBACH .

T. HAYASHIDA . J. H. HUMPHREY . E.KNOBIL .

C. H. LI . .

ELEANOR E. MCGARRY P. J. MOLONEY .

A. G. E. PEARSE .

P. J. RANDLE . C.H.READ .

Dept. of Pathology, University of California

Royal Victoria Hospital, Montreal Radioisotope Service, Veterans Administration

Hospital, Bronx, New York Dept. of Obstetrics and Gynaecology, Karolin-

ska Sjukhuset, Stockholm Dept. of Clinical Endocrinology, Birmingham

and Midland Hospital for Women, Birmingham Dept. of Zoology, University of Leeds Dept. of Pathology, University of Cambridge Division of Pathology, Imperial Cancer Research

Fund, London; Professor Elect, Dept. of Pathology, University of Aberdeen

Postgraduate Medical School, London Kinderspital, Zurich Dept. of Obstetrics and Gynaecology, University

Hospital, Uppsala Imperial Cancer Research Fund, London Dept. of Pediatrics, Columbia University College

Dept. of Anatomy, University of California

National Institute for Medical Research, London Dept. of Physiology, University of Pittsburgh

Hormone Research Laboratory, University of

Royal Victoria Hospital, Montreal Connaught Medical Research Laboratories,

Dept. of Pathology, Postgraduate Medical School,

Dept. of Biochemistry, University of Cambridge Dept. of Pediatrics, State University of Iowa,

School of Medicine, Los Angeles

of Physicians and Surgeons, New York

School of Medicine, San Francisco

School of Medicine, Pittsburgh

California, Berkeley

University of Toronto

London

Iowa City n

xii LIST O F PARTICIPANTS

E.SAMOLS . . . Dept. of Medicine, Royal Free Hospital, London H. A. SELENKOW . . Peter Bent Brigham Hospital, Boston R.H. UNGER . . . Depts. of Medicine, University of Texas South-

western Medical School and Veterans Admini- stration Hospital, Dallas

S. C . WERNER . . Dept. of Medicine, Columbia University College of Physicians and Surgeons, New York

R.G.WHITE . . . Dept. of Bacteriology, London Hospital Medical College, London

F . G . y o u ~ c . . . Dept. of Biochemistry, University of Cambridge

CHAIRMAN’S OPENING REMARKS

PROFESSOR F. G. YOUNG

I AM particularly pleased to assist at this Ciba Foundation Colloquium (technically it will be a Symposium as well) on the

Detection and Assay of Hormones by Immunochemical Means”, since the present time is ripe for consideration of this most important and rapidly developing subject. I hope that it will not be considered inappropriate for the Chairman’s opening remarks to include some comment upon the historical background of our subject.

It is unfortunate in some ways that for many years the subject of immunology has, by virtue of its birth, been regarded as primarily a branch of bacteriology. It is understandable that this should be, since the phenomena of immunity were revealed by a study of the complex processes whereby men and animals react to invasion by infective organisms, and immunology has therefore naturally been regarded as a study of the means whereby the body protects itself against bacteria and bacterial products. The early studies of Pasteur, ROUX, Bordet and others laid the foundations of the study of immunology, and early this century the fertile mind of Paul Ehrlich, inventing side chains and propounding hghly original chemotherapeutic ideas, did much to foster interest in the possible mechanisms of the immunity reactions which had then been described. The development of our knowledge of blood groups, and their importance in transfusion, brought out a new aspect of the subject-a most productive one- and the studies of Landsteiner, Heidelberger and others, on the significance of carbohydrate in the antigenic molecule in the induction of immunity reaction, fastened attention on the non- protein part of protein-containing antigens. I remember very

4 6

I

2 P R O F E S S O R F. G . YOUNG

clearly being told, about 25 years ago, that every known antigenic protein contained carbohydrate, and that antigenicity probably depended upon the carbohydrate part of the molecule-a fact whch we now know to be incorrect.

In 1934 Collip and his colleagues found that preparations of certain protein pituitary hormones were antigenic under suitable condtions (Collip and Anderson, 1934; Collip, 1934), and much research was subsequently pursued on the subject of “anti- hormones”. Anti-hormones were believed by some to play a part in the normal endocrinological balance in the body but this idea was given little support and was ultimately discarded. In the production of anti-hormones at that time the purity of the only available hormone antigens was, to say the least, doubtful, and how far the extent of precipitin reactions, and other means whereby the antibodies could be detected or measured, could properly be related to the antigenic action of the biologically active material which had been administered rather than to that of non-biologically active antigenic contaminants, was a matter for debate. There was indeed no simple unequivocal experimental solution to these problems at that time, and research on anti- hormones gradually fell out of fashion.

It is clear that the acquisition of knowledge is greatly helped by the development of new methods, and that in turn the development of new methods is fostered, or made possible, by a general advance in knowledge. When a particularly important methodo- logical advance has been made-for example, the invention of partition chromatography and its application to filter paper-the stimulus to the acquisition of knowledge which hitherto has not been accessible competes with that directed towards the refin- ing and development of the method itself. But after a time the almost explosive effect of the discovery of the new method tends to dwindle, and the advance of knowledge may then pro- ceed less rapidly, but nevertheless steadily, until the newly acquired knowledge and its consolidation make possible another

CHAIRMAN’S OPENING REMARKS 3

“methodic mutation”, if I may use that term. In this way evolu- tionary stimuli to the acquisition of knowledge succeed one another.

I believe that today we are early in the stage of the explosive expansion of knowledge in relation to the subject of our discussions. The..origin of this stimulus is not, in my view, simply the result of applying the classical methods of immunology to protein-containing hormones. It has been fostered by a number of other developments in recent years: (I) ’the availability of reasonably large amounts of protein hormones in a high state of physicochemical purity; (2) the development of delicate tests for the quantitative assessment of antigens and antibodies, particularly those dependmg upon the tanning of red blood cells and the use of haemagglutination reactions ; (3) the appreciation of the existence of important chemical differences in the structures of protein hormones from different species of animals in some instances, and a realization that such differences may themselves be sufficient to make possible the production of potent antisera to non-homologous hormones; (4) the use of various adjuvants in the administration of the antigenic hormones whereby the raising of potent antisera with relatively small amounts of the hormone becomes possible.

When I look back at my early researches in this field (Young, 1938; Rowlands and Young, 1939) I suffer quantitative qualms, to say the least of it. In the production of an antiserum to prolactin, IOO mg. or more of what we called purified prolactin was injected into rabbits daily for 16 weeks, whde 200-300 mg. was injected daily into monkeys for 40 weeks! This use of our hard-won prolactin, prepared by ourselves, appears now to have been most extravagant, but at that time there was no reasonable alternative. As we rather expected, all the untreated antisera produced precipitin reactions, not only with the ox pituitary hormones we used as antigens, but also with ox serum. After absorption with ox serum, however, the anti-prolactin sera gave

4 P R O F E S S O R F. G. Y O U N G

apparently undiminished precipitin reactions with prolactin although similar treatment of our anti-thyrotropic sera greatly reduced the precipitin reactions with the thyrotropic preparations that we had used (Strangeways, 1938), whch certainly were not pure. But by and large we thought, and I still think, that we had indeed induced the formation of specific antibodies to our protein hormones. Nowadays when we have what are sometimes called “pure” preparations of these protein hormones, the argu- ment is raised that the satisfactory development of antisera depends on the purity of the hormone antigen. But also, it is alleged, the use of the antisera provides the best criteria for the purity of protein hormones; there is a circle there, which perhaps isn’t entirely vicious. But we shall no doubt learn about such matters in the course of this conference.

Although insulin has been recognized as a protein for 3 5 years it was only comparatively recently that potent antisera to this hormone were unquestionably produced (Stavitsky and A r q d a , 1953; Arquilla and Stavitsky, 1956; Moloney and Coval, 1955). Insulin was indeed the first on the list of highly purified hormones to which potent antisera became available, but that list is now growing Very rapidly indeed.

I am confident that as the result of our discussions, a significant stimulus to advance in this field of study will accrue. The acquisition of knowledge has once again made possible developments in technique, which are assisting the acquisition of more knowledge. It is desirable that one should have critical discussions from time to time of what is believed to be known, and discussions of this sort on an international basis are a most important part of the activities of the Ciba Foundation; indeed the fostering of these is one of the reasons why the Foundation exists.

In the discussions that will follow the papers, I hope that the members of the Conference will not hesitate to display their ignorance. We have among us both immunologists and those who are primarily concerned with hormones. I do not think that

C H A I R M A N ' S O P E N I N G R E M A R K S 5

anybody can claim to be an expert in every aspect of the work. Sometimes discussion is inhibited by the feeling that if one asks a simple question one may display, perhaps quite truly, one's lack of knowledge about that particular matter. But nevertheless, it is often true that the simple question is the most penetrating, and sometimes one of which the experts had not thought before. So please let us not be modest about displaying ignorance that every one of us must, in greater or less degree, possess about the topic of our meeting.

REFERENCES

ARQUILLA, E. R., and STAVITSKY, A. B. (1956). J . c h i . Invest., 35, 458, 467. COLLIP, J. B. (1934). J. Mt Sinai Hosp., I, 28. COLLIP, J. B., and ANDERSON, E. M. (1934). Lancet, 226,76. MOLONEY, P. J., and COVAL, M. (1955). Biochem.]., 59, 179. ROWLANDS, I. W., and YOUNG, F. G. (1939). J. Ph@f. (Lord.), 95, 410. STAYITSKY, A. B., and ARQUILLA, E. R. (1953). Fed. Proc., 12,461. STRANGEWAYS, W. I. (1938). J. Physiol. (Lond.), 93, 47P. YOUNG, F. G. (1938). Biochem.]., 32, 656.

BASIC IMMUNOLOGICAL CONSIDERATIONS

J. H. HUMPHREY

National Institutefor Medical Research, London

MY first introduction to immunology was as a medical student in Professor (now Sir Charles) Harington’s laboratory before the Second world War. At that time it was known that various hormones, such as pregnant mare’s serum and urinary gonadotropins or thyroglobh, were quite potent antigens in other species, but no one seriously considered attempting to assay hormones by immunological means. Harington and his colleagues had just shown that antibodies could be made in rabbits against thyroxine coupled to proteins and acting as a hapten, and-rather remarkably-that such antibodies could actually neutralize the biological effects of thyroxine in experimental animals (rats). I say remarkably, because it was surprising that the affrnity of antibody for a small molecule should be sufficient to prevent it from getting to the sites where it exerted its effects, which were then (perhaps they are still) unknown. However, the fact that antibodies could interfere with pharmacological activity of small molecules was also shown in quite another system, for Butler, Harington and Yuill(1940) were successfully showing that aspirin- protein conjugates could elicit antibodies in rabbits which would neutralize the antipyretic effect of aspirin in rats which had been passively immunized with the antiserum. This group were turning their attention to making antibodies against oestrogens (an aim later achieved with other steroids by Beiser et al., 1957)~ when the War interrupted their work. I mention this because it provides almost my only claim to be present in the company ofyou who are experts on antibodies against hormones. The work

6

IMMUNOASSA Y OF HORMONES G. E. W. WOLSTENHOLME and MARGARET P. CAMERON

Copyright 0 Ciba Foundation

B A S I C I M M U N O L O G I C A L C O N S I D E R A T I O N S 7

which I did in Prof. Harington’s laboratory, under his direction, was to synthesize derivatives of bovine insulin containing attached carbobenzyloxyglucosidotyrosyl groups, and to show that the conjugated ins&, which retained considerable biological activity, was a moderately good antigen in rabbits. However the antibodies were specific for the attached groups, and did not react, in any way which we could detect, against native bovine insulin. Despite mounting, but somewhat indefinite clinical evidence to the contrary, native insulin was still thought to have no or negli- gible antigenic activity. This was considered to be possibly due to the fact that the insdm molecule contained no carbohydrate. The concept of immunological tolerance had not emerged at the time, and I mention t h s because in the brief discussion of general principles which follows there may be a gap in immunological thought, as yet unrecognized and quite as large as that which was filled by t h s concept.

For the detection and assay of hormones, or of anything else, by immunological means it is necessary to have a specific antiserum. In theory, specifically sensitized animals whch would give an immediate or delayed-type hypersensitivity reaction would also suffice for detection, and even-with enough animals -for assay, but for practical purposes what is needed is the antiserum. Once this is to hand there are a number of ingenious ways in which it can be used, many of which will be discussed in this Colloquium. My task is to discuss what are the chances of obtaining such antiserum, and how to do it. If my remarks are vague and generalized you must make allowances for the fact that I am no endocrinologist, and that I even have to think twice before I can grasp the meaning of some of the abbreviations which you use.

The first necessity is that the hormone should be a large enough molecule to act as an antigen. It used to be thought that a molecular weight of IO,OOO was about the lower limit, but this seems now to have come down to nearer 5,000 or less, inasmuch

8 J . H . HUMPHREY

as insulin and glucagon have been shown to be antigenic. In parenthesis it may be remarked that insulin may well exist in the blood in an associated form, with greater molecular weight- otherwise it is difficult to see how “free” insulin could escape appearing in the urine. Nevertheless, it can still be stated that really small hormones are unlikely to be antigenic unless they are conjugated as artificial haptens to larger proteins. The reason for the existence of a lower limit of molecular weight is not known.

A second consideration is derived from the observed phenomena of immunological tolerance. It is an experimental fact that animals in general remain immunologically unresponsive to their own proteins or to potential antigens with which their immunologically competent cells come into contact during prenatal and very early postnatal life. To this must be added a rider to the effect that for immunological unresponsiveness to persist indefinitely, contact with the antigen may have to be maintained, or at least repeated at intervals throughout the life of the animal. 1 do not want to be too dogmatic on this point, since I think that the duration of unresponsiveness after single injections of foreign antigens may depend not only on persistence of the antigen in the right place but also upon the number and nature of the potentially antigenic groupings (i.e. groupings which are not common to any of the animal’s own proteins) which are present on the surface of these molecules. Nevertheless it cannot in general be expected that animals will respond by making antibodies against such antigenic groupings as are already represented in constituents of their tissue fluids, or in other materials (e.g. cell proteins, etc.) with which immunologically competent cells are in constant contact.

Ths brings me to a third point, namely that most antigens should not be regarded from the immunological point of view as single substances, however pure and homogeneous they may be, but as antigenic mosaics. That is, they have on their surface not a

B A S I C I M M U N O L O G I C A L C O N S I D E R A T I O N S 9

regular repeating pattern of similar groupings but a mosaic of distinct and different potentially antigenic determinants. It is unlikely that all will be equally capable of eliciting an antibody response, and those groupings which are already represented in the animal's own make-up will not elicit antibody in any case. However, assuming that there are several distinct antigenic determinants in the given material, when it is injected into an animal the antiserum which is produced in response will contain a mixture of distinct antibodies against the separate determinant groups. All the antibodies react with the material in question, but not all against the same sites-and the properties of the antiserum will be the sum of the properties of the component antibodies. Inasmuch as certain determinants are more potent anti- genically than others, it is likely that antisera obtained early in a course of immunization d l contain predominantly antibodies against these major determinants, and that antibodies against minor determinants will only occur to any great extent as immunization continues. Thus the chances of obtaining antibodies which do not cross-react with other related materials decrease the more the process of immunization is prolonged and intensified. Furthermore, when two hormones with different activities share antigenic determinants, it will be more or less inevitable that an antiserum prepared against the one will contain some antibodies against the other. Cross-reacting antibodies can, of course, be removed by absorbing the sera appropriately.

The next point is that animals of the same species apparently vary markedly in their response to the same antigen. I am sure that I do not need to tell you ths! Whether it is due to the past immunological history of the animals (e.g. previous stimulation by or tolerance to some determinant groups but not others) or to hereditary factors, or to soinethg else I do not know. However it may well happen that the major antigenic determinant grouping on an antigen for one animal may not be the same as that for another animal. The result could be two antisera against the same

I 0 J . H . HUMPHREY

antigen, from animals of the same species, which reacted predominantly with different parts of the antigen molecule, and they might have different effects-e.g. one might neutralize hormonal or enzymic activity, if the active area were occluded on interaction with antibody, whereas the other might not. This state- ment is intended not to discourage or confuse you, but because in the case of antisera against bacterial enzymes (e.g. penicillinase) such a situation has been shown to occur.

A fifth point is to know what makes a good antigen-apart from considerations already discussed of size, and of the number of determinant groups not shared with components of the animal to be immunized. It should obviously be in such a form as to be presented as effectively as possible to immunologically competent cells. Since it is unfortunately not certain which these are, nor how the antigen affects them, only certain general principles can be suggested. Thus antigen is wasted if it is lost by excretion in the urine or by catabolism in cells (e.g. liver Kupffer cells) which are probably irrelevant to antibody production. Furthermore it should be presented so as to produce a secondary response, rather than simply a primary response, even with a single dose. Empirical observations have shown that adsorption on mineral adsorbants, injection into sites where a depot is formed which drains to lymph nodes, and, best of all, the use of Freund-type adjuvants are effective ways of doing this. In the case of Freud's adjuvant it appears that the antigen emulsion is carried quite widely round the body, and its constituents cause local granulo- mata to form, in which immunologically competent cells are stimulated to proliferate and differentiate, so that with a supply of antigen ready to hand much of the y-globulin which they produce is antibody. Full advantage of these empirical observations has been taken by workers in the hormone field. There is however one aspect of antigenicity which might repay further study. This is the observation of Sela and his colleagues (see Arnon and Sela, 1960) that addition of small amounts of tyrosine (by coupling

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with N-carboxy-ct-aminotyrosine anhydride under very mild conditions) greatly enhances the antigenicity of a very weakly antigenic material such as gelatin, without apparently destroying its immunological specificity.

It has already been pointed out that an antiserum generally contains a mixture of antibodies against different determinant groups on the antigen. Whenever experiments have been per- formed to determine how many combining groups are present on well defined antibodies, the number has always turned out to be about two. Furthermore, experiments designed to reveal the presence of combining groups with different specificities on the same antibody molecules have always failed to do so. It is therefore probable, though not proved, that each antibody molecule in the mixture has two similar combining sites. When the antigen interacts with the antiserum in suitable (“optimal”) proportions, according to the Lattice Hypothesis large aggregates should form in which the polyhaptenic antigen molecules are linked to each other at two or more sites by bivalent antibody molecules. This, as is well known, commonly leads to diminished water solubility and to precipitation-probably because some of the more hydro- phihc groups on the antigen and antibody are involved in the interaction and so become masked. However it is not uncommon for a greater or smaller proportion of the antibodies in an antiserum to react with the antigen without resulting in precipitate formation-perhaps because fewer hydrophilic or more hydro- phobic groups are involved. Such non-precipitating antibodies can, of course, be recognized by techniques other than precipitation, especially if the antigen has a radioactive label, but the possibility of their presence must always be borne in mind. In the case of immunization with some of the smaller hormones, which may have very few potentially antigenic groups not shared by the animal being immunized, a situation might arise in which the antigen was effectively univalent. If this were so, in whatever proportions antigen and antibody were mixed it would be

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impossible for large aggregates to be built up; presumably also the combination between antigen and antibody would not be very strong. Perhaps-this is pure speculation-something of the sort could explain why precipitating antibodies against insulin are so rare.

To those accustomed to using protein hormones it may appear trite for an outsider to express wonder that such complex molecules obtained from one species should have similar biological activities in another. This implies that the biologically important parts of these molecules must have very similar, if not identical structures. Furthermore, in the case of those polypeptide hormones whose structure has been worked out, the correspondmg hormones have been found to resemble each other closely in different species in respect of their complete amino acid sequences. Although this does not necessarily mean that the peptide chains are folded in exactly the same way, so as to produce identical surface configurations, it does suggest that the number of possible antigenic determinants in such a hormone from one species which are not already found in another species may be very small. To have the best chance of eliciting antibodies, it may be necessary to attempt to do so in several different species, in the hope of finding a combination in which the differences are maximal. Insofar as the biologically active parts of a given hormone from two species are identical, it would not be expected that this part of the molecule should be able to act as an antigenic determinant, nor would it be expected that antibody against a given hormone would react with the corresponding endogenous hormone in an immunized animal. The fact that guinea pig anti-ox ins& neutralizes extracted guinea pig ins& provides a puzzle about which more will probably be heard later in this Colloquium.

So much for general principles relating to antigenicity. Real antigens, as distinct from the hypothetical antigens discussed above, are often contaminated with traces of other antigenic impurities, despite the exercise of much skill and ingenuity in

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their separation. Often the impurities are better antigens than the main material, and to obtain a really mono-specific antiserum against a protein hormone is the exception rather than the rule. Sonietimes it is possible to remove unwanted antibodies by absorption with small quantities of materials which contain more of the impurities and very little or none of the hormone in question. When the antibody is mono-specific it can be used for any of the immunological techniques (precipitation in agar gel, fluorescent antibody, passive cutaneous anaphylaxis, haemagglutination, haemagglutination inhbition, etc.) with impunity, even if the hormone to be detected or estimated is mixed with large amounts of other materials. The only warning necessary is that one must test that the antibody is specific by as many ways as possible before taking this for granted.

Even when an antiserum is not mono-specific, it can still be a useful tool for the purpose of estimating or detecting a hormone, provided that there is available a pure, or nearly pure preparation of the hormone to use in conjunction with it. The immunological technique must in this case however be an indirect one, which depends upon competition between the antigen to be estimated and known amounts of the pure antigen for the particular antibody in the antiserum. The chromatoelectrophoresis technique of Berson and Yalow and the haemagglutination inhibition- test are excellent examples. A basic assumption in such techniques is that the antigen to be estimated and the pure antigen preparation are in the same form and can compete freely with one another.

Having spent a good many years in the Department of Biological Standards at the National Institute for Melcal Research, I have been well and rightly trained to regard with suspicion any assay methods whose results have not been shown rigidly to correlate with those obtained by bioassay. While admitting that immunochemical techniques have been shown to possess a degree of sensitivity not obtained by bioassay, I had intended to insist upon the need for constant vigilance in this

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respect. After reading the precirculated abstracts of this Collo- quium, I realize that the participants are well aware of this. However since the use of your techniques is hkely to become in- creasingly widespread, there may well be a case for introducing some novel biological standards, in the form of well characterized mono-specific sera and pure hormone preparations which could be used as a further yardstick against which others may measure their own results.

REFERENCES

ARNON, R., and SELA, M. (1960). Biochem.]., 75, 103. BEISER, S. M., AGATE, F. J., ERLANGER, B. G., and LIEBERMAN, S. (1957).

BUTLER, G. C., HARINGTON, C. R., and YUILL, M. E. (1940). Biochem.]., 34, Bull. N.Y. A d . Med., 33,655.

103.

QUESTIONS

Werner; If immunological tolerance is acquired because the antigen is present in prenatal or early postnatal life, does this imply that if a substance does not produce an antibody it must therefore have been present at that same time in development ? I have in mind the fact that certain hormones, such as luteinizing hormone, theoretically appear somewhat later in life-also thyrotropin which I am not certain is present prenatally-and yet evoke no immune response.

Humphrey: That is a very interesting idea. Assuming that the hormone in question were a reasonably good antigen in another species (i.e. it is not, for some reason, a poor antigen as is gelatin) it seems reasonable to think that if such a hormone, which would normally not appear until adolescence, were not capable of eliciting an immune response, then the animal was tolerant to it as a result of prenatal contact. Of course you would have to demonstrate the existence of the hormone in the mother and passage across the placenta into the foetus; however a good many things do cross in small amounts from the mother to the foetus.

Moloney: Is it fair to suggest that insulin when serving as an antigen has a molecular weight of 6,000 ?

QUESTIONS IS

Humphrey: You know more about this than I do, Dr. Moloney. With physicochemical techniques at pH 4.8 or so it appears to have a molecular weight of 6,000, but I think that it is probably much larger than that when it travels around the body. This is something which I hope somebody else can answer for me, because I am really basically ignorant on hormone structure.

Moloney: I had assumed that the molecular weight of unaltered insulin at pH 7 - 5 is 36,000 to 48,000. On the other hand, insulin which has been sulphated (sulphonated) has a smaller molecular weight at pH 7.5, in the order of 6,000 to 12,000. We had hoped that this altered insulin might not induce antibodies but unfortunately it does.

Humphrey: Is glucagon thought to exist in an associated form ? Moloney: I don't know. Humphrey: It is another hormone with a low molecular weight

which seems to be antigenic. From another field-it is known that fragments of silk fibroin, which are fairly small, are still antigenic, provided one uses Freund's adjuvant.

Berson: I think that the molecular size of insulin depends on the conditions under which one is using it. In the usual ultracentrifugation analysis one uses a moderately high concentration of insulin, and the tendency to polymerization would be of course much greater. From E. Fredericq's values for the free energy of polymerization (1956. Arch. Biockem., 65, 218), one can calculate that at the very low concen- trations in which insulin exists in the circulating fluids of the body, this tendency would be very much diminished. We have some evidence on the molecular size of insulin as it circulates in the plasma, by means of ultracentrifugal analysis. We can say only that it sediments at a very much lower rate than serum albumin, which of course does not set a lower limit to the sedimentation velocity of endogenous plasma insulin, but indicates at least that the insulin is not in a very highly aggregated state.

You suggested that because insulin does not appear in the urine, it might be aggregated. Firstly, I am not at all sure that insulin does not appear in the urine. Secondly, if we inject 1311-labelled insulin it disappears from the circulation very rapidly, which indicates that it has a molecular size under about 35,000 or 40,000, which seems to be the borderline for rapid transfer out of the circulation. Also, minute

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amounts of 1311-labelled insulin do appear in the urine; so that it may be that small amounts of endogenous insulin do appear in the urine.

You speculated also on the univalency of insulin as an antigen. W e too have thought that that may account for the lack of precipitation in most systems. We have analysed the sedimentation velocity of insulin-antibody complexes in the region of very marked antibody excess-conditions under which the tendency for insulin to combine with more than one antibody molecule would be greatest-and have found that the sedimentation velocity of 1311-labelled insulin-antibody complexes is only very slightly greater than that of labelled y-globulin alone. Our calculations, based upon the assumption that the partial molar volumes are essentially unchanged in the complex from those in the free insulin and antibody molecule, indicate that but one insulin molecule is combined with a single antibody molecule; the molecular weight of the complex is calculated to be about 12,000 more than that of the antibody alone. Whether this is because the insulin is present as a dimer, or whether this is the error of the determination we’re not sure, but the complex is at least never present as a large aggregate, even in the presence of very marked antibody excess.

Hcimphrey: I think that is a most interesting observation. People have tried studying the behaviour of monovalent antigens, using con-

jugated haptens; but in order to elicit much of avid antihapten antibody one usually has to use proteins with as many hapten groups attached as possible. It is rather difficult to obtain for testing a protein-hapten conjugate which contains only molecules with a single hapten group attached; ifyou have a clear answer on this point, it would be important from the theoretical point of view.

Bcrson: The finding of a complex containing only a single antibody molecule of course does not prove that insulin is univalent in the sense that it has but a single antigenic group ; it simply indicates that regard- less of how many antigenic groups it has, it cannot react with inore than one antibody molecule. It could, for example, have two antigenic groups, but because the insulin molecule is so small compared with the antibody molecule, steric restrictions might prevent two antibody molecules combining with the single insulin molecule.

Arquih: Precipitating antibodies have been demonstrated against very small molecules that have been used as haptens, molecules of

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