Assay of Protein and Polypeptide Hormones

232 Pages·1970·13.88 MB·English

by  H. Van Cauwenberge, P. Franchimont, P. Alexander and Z. M. Bacq (Auth.)

ASSAY OF PROTEIN A ND
POLYPEPTIDE HORMONES
BY
H. VAN CAUWENBERGE and P. FRANCHIMONT
WITH THE COLLABORATION OF
B. BERDE, G. CESSION, G. HENNEN, P. LEFEBVRE, J. J. LEGROS,
J. C. LIBON, A. LUYCKX, G. MILHAUD, J. P. NAETS and R. WINAND
Institute of Medicine, University of Liege
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First edition 1970
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Introduction
Η. VAN CAUWENBERGE
FOR many years, protein and polypeptide hormones were assayed in­
directly in blood and urine by means of biological tests. Even after the
evaluation of gonadal and adrenal steroids in urine and, later, in blood
had become routine and the assay of catecholamines had become possible,
the direct and specific determination of protein or polypeptide hormone
concentrations in biological fluids still seemed illusory. The problem,
however, was crucial since its solution would pave the way for a better
understanding of insulin, glucagon, parathormone, and the various hypo­
physeal stimuUns. From a physiological as well as a clinical and physio-
pathological standpoint, it was essential to examine and verify a whole
series of concepts which had originally been based on anatomical and
surgical observations, autopsy data, and changes in certain biological
constants in blood and urine.
Bioassay techniques provided valuable information but were often
lacking in specificity, sensitivity, and precision.
The introduction of immunological methods created a new field of
investigation for biologist and clinician alike. Nevertheless, even though
these methods had the advantage of being based on the excellent specificity
of the antigen-antibody reaction, the techniques used to demonstrate this
reaction were often insensitive and imprecise.
This deficiency was remedied by the introduction of radioimmunological
techniques which provided a system that was far more sensitive and at the
same time more resistant to the various serum factors capable of altering
visualization of the immunological reaction.
The distinction of introducing this method for the assay of insulin
belongs to Berson and Yalow. Since then, the majority of protein and
polypeptide hormones have been assayed by their technique or by ana­
logous procedures, with the help of the labeling method developed by
Hunter and Greenwood.
4 ASSAY OF PROTEIN HORMONES
After several years of biological and clinical research in this field, it is
possible today to stand back and view its current status. The purpose of
this book, among others, will be to survey the progress that has been
made.
One fact, however, merits our attention. Although the assay of hor­
mones in biological fluids is unquestionably an important advance, it is
not sufficient to perform these determinations under basal conditions. New
methods have made it possible to determine the response of endocrine
glands during various dynamic tests. Other equally important problems to
be resolved include the metabolism of urinary excretion and the peripheral
and visceral clearance of protein and polypeptide hormones.
CHAPTER 1
Biological Methods
H. VAN CAUWENBERGE, P. LEFEBVRE and P. FRANCHIMONT
THE impossibility of evaluating the protein and polypeptide hormone
content of biological fluids by means of purely chemical or physico-
chemical techniques led to the introduction of biological methods for
assaying these factors. Immunological and radioimmunological assays
have recently been developed for several hormones but, as we shall see,
they have not resolved all the diflBculties.
A feature common to all biological tests is that they attempt to repro­
duce one or more of the characteristic physiological or biochemical actions
of the hormone while determining the quantitative relationship between
the observed efiect and the amount of the substance present in the assay
sample. The effects observed with the unknown sample are then compared
with the results that can be obtained with a series of increasing or decreas­
ing doses of the maximally purified hormone used as a standard.
The type of response depends on the particular test employed: death of
the experimental animal, weight or height changes, changes in the ap­
pearance or weight of certain organs, changes in certain chemical com­
ponents of the blood, metabolic changes in various tissues or organs
studied in vivo or in vitro, etc.
I. Essential Conditions for Bioassay
These conditions are discussed in detail by Borth (1952, 1957), Marrian
(1955), Loraine (1958), Emmens (1962), Loraine and Bell (1966).
The bioassay of protein hormones must satisfy the following essential
criteria:
A. Accuracy
This criterion is defined as being the smallest existing deviation between
7
8 ASSAY OF PROTEIN HORMONES
the measured value and the true value. It is generally estimated by means
of recovery experiments.
B. Precision
This factor is assessed by reproducibility experiments, where the same
measurement is repeated a certain number of times and the standard
deviation with respect to the arithmetic mean is calculated. Precision
may also be tested by the method of Snedecor (1956).
C. Specificity
According to this criterion the effect observed must be attributable to
the hormone under study and to it alone. This very important considera­
tion is often ignored in bioassays of protein and polypeptide hormones.
D. Sensitivity
This factor has been defined by Borth (1957) as "the smallest single
result which, with some assurance, can be distinguished from zero, or, in
statistical terms, as the smallest single result whose fiducial limits for, say,
ρ = 0.05, do not include zero". For many polypeptide hormones, current
biological methods are not sensitive enough to permit determination of
the very small quantities of these substances which are present in biological
fluids such as blood or plasma.
These four criteria are fundamental. However, in order for a large
number of samples to be handled in practice, it is also necessary that the
techniques be neither too time-consuming nor too costly and that they
be feasible in laboratories which are well equipped without being highly
specialized.
II. Basic Factors in Bioassay
A. Experimental Animals
Biological assays require the use of pure strains of animals raised in
standardized conditions. It is also necessary to ensure that the animals
receive an unvarying diet and that they display no seasonal variations in
reactivity. Even under these conditions, laboratories may obtain divergent
results because of differences in the strains of animals used.
BIOLOGICAL METHODS 9
It is equally essential to standardize the conditions under which samples
are drawn and, where appropriate, the manner in which the animals are
sacrificed.
B. Standard Preparation
The results of assays performed with unknown samples must be com­
pared with those obtained with the use of standard preparations.
The ideal solution is to employ internationally estabHshed standards
such as those supplied by the NIH in the United States and by the NMRC
in Mill-Hill, England. Similar standards are in the process of being
prepared in France.
When international standard preparations are not available, each
laboratory must rigorously define the standard it utilizes. Even with well-
defined standards, of course, it can happen that animals of the same strain,
raised in identical conditions, will give difiering responses.
C. Evaluation Techniques Used in Bioassays
1. In the great majority of cases, a quantitative effect is sought. Several
groups of animals are tested, each at a different dose level. As Emmens
(1962) has pointed out, it is best to select experimental conditions such that
the results can be plotted in a linear segment of the logarithmic scale, even
if the usable segment of the curve happens to be relatively short.
An example of this technique is the insulin bioassay method which
utilizes epidydimal adipose tissue from the rat (Renold et al, 1960).
2. In certain instances, one is looking for a qualitative response
("quantal effect" or "all or none effect" of Gaddum, 1933). This is the
case, for example, in the bioassay of insulin which is based on the test for
convulsions in the mouse.
D. Experimental Design
The reader is referred to the classic article of Gaddum (1953) for a
fuller discussion of this subject. Three types of experimental design are in
routine use:
1. Three-point assays
Three groups of animals are used; two receive the standard preparation
and the third receives the unknown sample.
10 ASSAY OF PROTEIN HORMONES
2. Four-point assays
Four groups of animals are used. The first two receive the standard,
the other two receive the unknown sample. This method makes it possible
to test the parallehsm of the dose-response curves of the standard and the
unknown sample. If no parallehsm can be demonstrated, the assay is not
valid.
3. Six-point assays
Six groups of animals are utilized. Three receive different doses of the
standard while the other three receive the unknown sample at varying
dose levels. The parallehsm of the curves may thus be determined with more
certainty.
E. Statistical Analysis of Results
A detailed review of statistical analysis was published by Emmens in
1962. The two most widely used methods are based on the determination
of fiducial limits and the index of precision.
1. Determination of fiducial limits
For a normal distribution, the fiducial limits may be determined by
classical procedures: calculation of the variance, the standard deviation,
the standard error of the mean. Student's parameter i, and by finding the
probabihty in the t tables.
2. Index of precision
This term was originally proposed by Gaddum (1933). The index of
precision is calculated by dividing the standard deviation by the slope of
the curve relating the observed response to the log of the utilized dose.
The most precise bioassays have an index of precision less than or equal
to 0.3.
III. Current Status of Biological Assays
When immunological and especially radioimmunological methods for
assaying protein hormones were first introduced, there was some specu­
lation that biological assay techniques would no longer have any place in
BIOLOGICAL METHODS 11
physiology or pathology. However, as the later chapters of this book will
demonstrate, this has not occurred.
Biological methods have in fact proved indispensable for developing
and ensuring the specificity of various radioimmunological techniques.
This has been the case for somatotropin (STH), the gonadotropins, para­
thormone, and thyrocalcitonin. The similarity of the results obtained with
biological and radioimmunological methods argues for the validity of
radioimmunological techniques in assaying these hormones.
In certain fields, bioassay methods have even sharpened and broadened
our perspectives, for example in the assay of insuHn. While the significance
and value of radioimmunological techniques for assaying insuUn are indis­
putable, bioassay techniques have shown that circulating blood also
contains an "Insuhn-Uke Activity" (ILA) which is not suppressible by
antibodies (NS-ILA) and which might have considerable biological
importance (Power, 1967).
Similarly, Sokal and Ezdinli (1967) were able to demonstrate, by means
of a bioassay technique, that excessively high glucagon levels had been
obtained for peripheral plasma with radioimmunological methods. An
analysis of the discordant results revealed that the overestimation of the
original value was due to an artefact of technique: a fraction of the
P^^-labeled glucagon was destroyed or damaged during incubation
with plasma and was therefore unable to react with the corresponding
antibodies.
References
BORTH, R. T. Ciba Found, Coli Endoer., 1952, 2, 45.
BORTH, R. Τ. Vitamins Hormones, 1957,15, 259.
EMMENS, C. W. In: Methods in Hormone Research (R.I. Dorfman, Ed.), Vol. I, p. 3.
Academic Press, New York, 1962.
GADDUM, J. H. Spec. Rep. Ser. Med. Research Coun. No. 183. London, 1933.
GADDUM, J. H. Pharmacol. Rev., 1953, 5, 87.
LORAINE, J. A. The Clinical Application of Hormone Assay. Livingstone, Edinburgh, 1958.
LORAINE, J. A. and BELL, E. T. Hormone Assays and their Clinical Application. Living­
stone, Edinburgh, 1966.
MARRIAN, G. F. In: Proceedings of the Third International Congress of Biochemistry,
Brussels, 1955, p. 205.
POWER, L. Lancet, 1967, 1, 1138.
RENOLD, A. E., MARTIN, D. B., DAGENAIS, Y. M., STEINKE, J., NICKERSON, R. J. and
SHEPS, M. C. /. Clin. Invest., 1960, 39, 1487.
SNEDECOR, G. W. Statistical Methods, 5th ed. State University Press, Iowa, 1956.
SoKAL, J. E. and EZDINLI, E. Z. /. Clin. Invest., 1967, 46, 778.
CHAPTER 2
Immunological Methods
Η. VAN CAUWENBERGE, P. FRANCHIMONT and A. LUYCKX
THE introduction of immunological techniques into the field of endo­
crinology constituted a considerable advance. With these techniques, it
became possible to assay in vivo SL series of protein hormones which are
intimately involved in regulating the body's metaboUc processes.
These methods are all based on the same fundamental principle: the
reaction between a specific antigen, i.e. the hormone to be assayed, and its
corresponding antibodies. Certain criteria must be satisfied in order to
perform a valid immunoassay; these will be discussed in the first part of
this chapter. It is also essential to make use of sensitive techniques for
visualizing the immunochemical reaction between antigen and antibody.
These techniques will be taken up in Part II. Among the immunological
methods currently available, radioimmunoassay is considered the method
of choice. Lastly, we will examine the reasons for the superiority of this
technique as well as the conditions essential for its use.
I. Essential Conditions for Immunoassay
A. Antigenicity of the Hormone
The injection of a protein hormone obtained from one species of animal
readily induces the formation of antibody in an animal of another species
(usually the rabbit or guinea pig), provided the hormone has a molecular
weight of more than 10,000. This condition is satisfied by the anterior
hypophyseal hormones, chorionic gonadotropin, and placental lactogenic
hormone. The chemical structure of these hormones presents a species-
specificity which is very helpful in the production of antibodies; the
precise degree of species-specificity varies with the hormone considered.
12