Clin. Biochem. 1, 110-117 (1967)

SOME FACTORS INVOLVED IN THE IMMUNOASSAY OF INSULIN*

J. M. MARTIN

The Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada

(Received May 31, 1967)

SUMMARY

1. The basic components in the immunoassay of insulin are the anti-insulin serum, the radioactive-labelled insulin and the unlabelled hormone. The necessary controls of the various conditions these components have to meet are stressed.

2. The immunoassay of insulin has proved to be a highly sensitive and reproducible procedure.

THE DEVELOPMENT OF A SUITABLE METHOD for determination of insulin in blood has been pursued since the discovery of the hormone. Initially, the biological assay methods in vivo (rabbit or mouse hypoglycemia, mouse convulsion test) were--and still remain--standard procedures for the determination of the potency of pancreatic extracts. The sensitivity of these methods is in the order of milliunits of insulin and is above the levels expected in blood. An increased sensitivity was obtained by previous preparation of the animals. It was observed that adreno- demedullated-alloxan diabetic-hypophysectomized rats responded to small doses of insulin (0.1-1 international milliunit). However, the difficulty of the prepara- tion and the high mortality of such animals made this method impractical (1).

In search for a more sensitive procedure, the effects of insulin on isolated tissues incubated in vitro were used instead of intact animals. Glucose uptake or glucose oxidation, as well as fatty-acid or glycogen synthesis by the diaphragm or the epididymal fat pad, were measured. The increment of these parameters as an effect of insulin present in the incubation medium constitutes the basis of these methods. The sensitivity of these bioassay systems in vitro allowed the deter- mination of insulin-like activity in peripheral blood in a more systematic fashion (2). To the natural limitation of the number of samples that can be included in a single assay, other problems of a more serious nature were added. The presence of some interfering substances in plasma, which apparently can be removed by dilution of the sample (3), or by acid-alcohol extraction (6), has produced enormous discrepancies in the results when the isolated diaphragm method is used. Values reported for fasting levels of insulin in human plasma range from

*Supported by the Medical Research Council of Canada.

IMMUNOASSAY OF INSULIN 111

30 to 3,000 microunits per ml (4, 8). The epididymal fat pad method seems to be handicapped by a lack of specificity. Persistent levels of insulin-like activity were demonstrated by this procedure long after complete pancreatectomy in the dog (6, 7). In addition, crystalline serum albumin and egg albumin were able to stimulate glucose oxidation in this preparation (8-10).

By exploiting the immunological properties of insulin, a new chapter in the determination of small amounts of this hormone in biological fluids was opened.

In 1955 Moloney and Coval (11) were able to produce insulin antibodies in guinea pigs injected with beef insulin. In 1956 Berson et al. (12) demonstrated that sera of insulin-treated subjects had the ability to bind 131I-labelled insulin in a complex which migrated between the beta and gamma globulin fractions on paper electrophoresis. Such binding did not occur with normal plasma and the labelled insulin remained at the origin. Almost simultaneously Arquilla and Stavltsky (18) reported an immunohemolysis technique for measuring insulin and insulin antibodies. Skom and Talmage, in 1958, (14) described a method in which they used a second antibody (anti-gamma globulin serum) to precipitate and quantitate the soluble insulin-anti-insulin complex. These four basic pieces of research constitute the groundwork of the various radio-immunoassay methods for insulin which were subsequently developed.

The principle of the immunoassay is based on the competitive inhibition of the binding of radioactive labelled insulin to insulin antibody by unlabelled insulin. This inhibition is quantitative. Therefore, the proportion of labelled insulin bound to a given amount of antibody is inversely related to the amount of unlabelled insulin present in the system. By maintaining the quantity of antibody and of radioactive-insulin constant, it is possible to measure the third variable, the unlabelled hormone. Hence, the addition of increasing amounts of insulin will result in a progressive reduction of radioactivity bound to antibody. Since the displacement of labelled insulin from the antibody sites by unlabelled insulin does not follow a linear or a logarithmic relationship to the concentration of unlabelled insulin added, it is necessary to construct a standard curve with each assay. Unknown samples can be measured against this standard curve.

Following these basic principles, several immunoassay procedures have been described. They differ in the way in which bound and free labelled hormone are separated prior to measuring the amount of radioactivity in each fraction (Table 1).

TABLE 1 METHODS FOR THE SEPARATION OF FREE AND BOUND INSULIN

Physico-chemica] Paper electrophoresis (15) Gel filtration (16, 17) Adsorption to Dextran coated charcoal (18) Adsorption to Talcum powder (19)

Chemical Salt precipitation (~0) Immunological Second antibody (21-23)

In the past six years considerable data have accumulated on the use and applica- tion of the immunoassay of insulin. Therefore, it seemed worthwhile to attempt to review some of the basic factors involved in these methods.

112 MARTIN

INSULIN ANTIBODIES

The method of Moloney ( / / ) - - i n some cases slightly modif ied-- is used by mos t investigators for the product ion of ant ibody. The guinea pig has proved to be the animal best suited for immunizat ion purposes. In order to elicit the forma- tion of an t ibody in a relat ively short t ime, and to obtain an ant iserum of high titre, the antigen is injected with an adjuvant . Complete Freund 's ad juvan t gives the most consistent results, al though some other preparat ions (24) m a y be a l ternat ively used. Serum an t ibody ti tres of the immunized animals can be increased by repeated injections. However , because of the uncer ta in ty of the pur i ty of the antigen, it is advisable to restrict the immunizat ion to only three injections. This procedure general ly produces an ant iserum with a potency tha t is suitable for immunoassay , and also reduces the risk of inducing the development of antibodies to contaminants which will not specifically react with insulin (28).

In theory, the ideal s i tuat ion would be to use a homologous system, i.e., for the determinat ion of human insulin, an ant iserum developed against the human hormone would be used. However , the well established cross-react ivi ty between insulin and an t ibody from human, ox and pork origin makes this unnecessary in practice (Table 2). I t is known tha t the insulin molecule has various antigenic determinants . I t is quite possible also tha t insulins of different species m a y share

TABLE 2 COMPARATIVE INSULIN RESULTS OBTAINED WITH

ANTI-HUMAN AND ANTI-Ox INSULIN SERA*

Plasma insulin (uu/ml)

Anti-human Anti-ox insulin serum insulin serum

SH

AK

Fasting 52 42 30 min 80 84 60 min 144 152

120 min 168 164 180 min 72 64 Fasting 4 4 30 min 4 2 60 min 2 2

120 min 4 4 180 min 4 2

*Plasma insulin values during a glucose tolerance test in a normal (SH) and untreated diabetic (AK) child. The values were read from a bovine crystalline insulin standard curve. 13q-labelled bovine insulin was used as a tracer (double antibody method).

some common de terminants and have a t the same t ime some distinct ones. Therefore, immunized animals will develop antibodies of different quality. Some serum m a y react most ly against the common type of antigenic de terminants and other serum m a y react be t te r with the dissimilar determinants . The former serum would be sui table for assaying insulin of other species, while the latter

IMMUNOASSAY OF INSULIN 113

would react only with insulin of the same species. The quality of each antiserum can be assessed only by its performance. This matter requires careful control in the selection of the best antiserum to be used in the immunoassay.

RADIOACTIVE-LABELLED [NSUL1N

Insulin labelled with either 13ll or 125I is used as a tracer. It has been noted, however, (26, 27) that there is impairment of the immunological properties of insulin that is proportional to the degree of iodination. This observation is at variance with the report of Izzo et al. (28) who found that the attachment of one atom of iodine per molecule reduces the biological activity by 60%, while the incorporation of as many as six atoms of I did not impair the immunological reactivity of insulin. Regardless of these controversial results, the possible effect of iodination of insulin on its behaviour does not invalidate the use of the labelled hormone for the immunoassay. Furthermore, the use of the radioactive labelled hormone in the immunoassay procedure does not imply that the reactions of the labelled and the unlabelled hormone are identical. The immunoassay only requires that there be an immunological competition between labelled and unlabelled hormone for the antibody sites. This idea was emphasized by Yalow and Berson (26) in the form of a theorem: "A labelled protein may be used other than as a tracer for the parent unlabelled protein, in which case identity of behaviour of labelled and unlabelled proteins may be irrelevant to the validity of application of the labelled protein". Therefore, when assessing the quality of the radioactive labelled insulin, it is important to demonstrate that the tracer and the unlabelled hormone react in the system in an identical manner. Because multiple iodination may interfere with the immunoreactive sites of the insulin molecule, there is general agreement that the degree of iodination must be maintained at a low proportion (1 or 2 iodine atoms/insulin monomer). The use of 125I-labelled insulin is becoming more generalized, particularly with immunoassay methods that do not require a high specific activity of the tracer. The main advantage of this isotope is the longer half life when compared to 131I. The latter is still the radio- nuclide of choice for radiochromato-electrophoresis. The degree of radiation damage to the hormone is not significantly different between the two isotopes.

STANDARDS AND UNKNOWN

As pointed out before, the insulin content of unknown samples is determined from the ability of the unlabelled insulin to inhibit competitively the binding of

t he labelled hormone to antibody. This inhibition is then compared to that of standard samples of crystalline insulin. For this purpose a standard curve is constructed with increasing amounts of crystalline insulin. If the results of this assay are to be valid, it is necessary that the three insulins present in the system ; namely, the standard crystalline insulin, the radioactive-labelled insulin and the insulin of the sample, react identically with the anti-insulin serum. Therefore, the use of insulins of other species--both as standards and tracer--for the assay

114 MARTIN

of human insulin assumes that the equivalent cross-reactivity between the various insulins with the antibody has been demonstrated (Table 3). In spite of the con- siderable amount of evidence that such cross-reactivity between human and bovine

"FABLE 3 PERCENTAGE OF RADIOACTIVITY DISPLACED FROM

THE INSULIN-ANTIBODY COMPLEX BY THE ADDITION OF CRYSTALLINE INSULIN*

Radioactivity (% displaced) 1 Insulin added

(/~u/ml) Human insulin 2 Bovine insulin s

1.5 7.10 8.50 3. O 13.10 12.50 6.0 31.00 27.3

12.0 50.3 49.6 25.0 65.6 65.6 50.0 79.4: 79.8

100.0 89.0 87.7

*The antiserum was obtained from guinea-pig immunized with bovine insulin (GP-9-64) at a dilution of 1:160,000. Herbert's method.

1Percentage decrease of radioactivity (cpm) was calculated from the number of counts in the 125I- insulin-anti-insulin complex when only the labelled insulin was present in the system.

2Lilly, lot 493-10GP-99. 3Connaught, lot 979.

or porcine insulin exists, it is necessary to establish its existence in each new system. Two main factors may alter the cross-reactivity: the quality of the antiserum and the labelling of the hormone. When these factors are optimum, if the plasma sample is assayed at various dilutions, the displacement of radioactive insulin from the insulin-anti-insulin complex should parallel the slope of the standard curve. It is a wise procedure to routinely assay every sample at two dilutions. This control rules out any possible distortion of the results due to a lack of parallelism and permanently monitors the conditions of the system. Concentrated plasma produces a certain degree of damage to the labelled hormone during the incubation (18). This damage is likely produced by proteolytic enzymes in the plasma and can be minimized by dilution of the samples. There- fore, it is the general practice to dilute the plasma for the assay. The limits of dilution are determined by the sensitivity of each assay system.

SEPARATION PROCEDURES

T h e var ious aspects discussed above are appl icable to all r ad io - immunoas say methods . In all of t h e m the an t i - i n su l in serum, the radioact ive- label led hormone a n d the un labe l led insu l in ( s t anda rd and u n k n o w n ) are i nc uba t e d for a var iable length of t ime according to the me thod used. Th i s i n c u b a t i o n period (from 2 hours a t 37 ° to several days a t 4 °) proceeds un t i l the reac t ion be tween insu l in a nd ant i- b o d y reaches the equ i l ib ra t ion poin t . For each procedure, as well as for each

IMMUNOASSAY OF INSULIN 115

antiserum, this time has to be pre-determined. Once the reaction reaches the steady state, separation of antibody-bound and free insulin should be performed. The various possible methods are listed in Table 1. I t would be beyond the scope of this review to discuss them separately. Although each method has advantages and disadvantages, it ig important that when properly controlled, all give reliable results. Furthermore, results obtained in many laboratories around the world, regardless of the particular method employed, are comparable within remarkably narrow limits (29). Once the separation of bound and free hormone is achieved, the radioactivity is counted in one or both fractions. Most of the procedures include a correction for the number of counts not bound to antibody due to hormone damage. After correction, the proportion of total labelled hormone combined to antibody is established for the insulin standards and the unknown samples. This proportion is expressed either as a ratio between free and bound insulin (15) or as the ratio between the radioactivity bound to the antiserum in the absence of unlabelled insulin and the radioactivity bound when standard or unknown insulin is present in the reaction tube (22). In a co-ordinate system, the Tatter gives a linear relationship for the standards, while the former is plotted as a descending curve.

GENERAL REMARKS

The slope of the standard curve determines the precision of the assay. The most precise assays result when the standard curve is very steep, indicating a high avidity of the antiserum to bind insulin. I t is obvious that the concentration of insulin (labelled and unlabelled) in the system should be in excess of the anti- body concentration. Only in this way will both insulins compete for the antibody sites. In addition, the sensitivity of the method may be increased at the low range by reducing the concentration of the tracer. As a rule the amount of labelled hormone added should be at a concentration approximately equal to the desired lower limit of sensitivity. In our laboratory, with a labelled hormone concentra- tion of 50 ~gg/ml, the method has a sensitivity as low as 1.5 gu/ml.

The inclusion of a control ("blank") for the standards and for each sample has proved extremely important in our hands. Such a "blank" contains the labelled insulin, the plasma sample or the standard, but the antiserum is omitted. There- after, it is processed in the same manner as the test samples. This "blank" must be included in methods employing charcoal or talcum for the separation (18, 19). In the double-antibody system, it has been demonstrated useful in ruling out any non-specific precipitation produced by the second antibody.

The specificity of the method seems to be demonstrated by the virtual absence of immunoassayable insulin following pancreatectomy (30). In juvenile diabetes, extremely low values of plasma insulin are found during glucose tolerance tests (81). In most instances, fasting values alone are of little help from the diagnostic standpoint. Table 4 illustrates the findings in a patient with an insulinoma. Diazoxide, which has a blocking effect on the release of insulin (32), counteracts the stimulatory action of glucose. In children with idiopathic hypoglycemia, higher levels of circulating immunoreactive insulin are demonstrable following

116 MARTIN

TABLE 4 PLASMA INSULIN VALUES OBTAINED BY HERBERT'S METHOD (18) DURING

A GLUCOSE TOLERANCE TEST IN A PATIENT WITH AN INSULINOMA

Plasma insulin (tm/ml)

Fasting 30 rain 60 rain 90 min 120 min

Before treatment 27.5 250.0 273.5 200.0 154.5 On treatment with

Diazoxide 27.0 20.5 35.0 25.0 30.0

intravenous tolbutamide administration (33). I t has been found that all the immunoreactive insulin present in the plasma of normal subjects is readily available to react with insulin antibodies (10, 34). This observation is in sharp contrast to results obtained by biological procedures in which, by dilution or extraction, a great increase of insulin-llke activi ty could be demonstrated.

The discrepancy between insulin values determined by bioassay and immuno- assay methods raises some doubts as to whether the immunoreactive insulin represents all the insulin present in plasma. The observation of Rasio et al. (35) tha t comparable levels of insulin in lymph could be measured by both bioassay and immunoassay suggests tha t the immunoassay detects all the insulin able to leave the intravascular space. Therefore, the immunoassayable insulin would represent at least that fraction of the hormone with biological act ivi ty while the nature of the insulin-like material remains to be determined.

Because of the sensitivity of the method and the essential agreement of the results obtained, increasing confidence is at tached to its usefulness in clinical research. I t is the consensus among investigators working in this field tha t the immunoassay procedure cannot be considered a routine method as yet. A high efficiency and the best of its possibilities are only achieved through constant control of the various factors involved. I t was the aim of this review to outline some of the most important of these factors.

REFERENCES

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2. GOETZ, F.C. Concerning the validity of different methods for the assay of insulin in blood. In On the Nature and Treatment of Diabetes, LEIBEL, B. S. & WRENSHALL, G. A., eds. Excerpta Medica Found. N.Y. 1965. pp. 181-193.

3. RANDLE, P. J. ~: TAYLOR, K.W. fn Hormones in Blood, GRAY, C. n . ~: BACHARACH, A. L., eds., Academic Press, New York, 1961, pp. 11-47.

4- DAVIDSON, J. K., HAIST, R. E. • BEST, C. H. Studies employing a new method for the recovery of biologically active insulin from acid alcohol extracts of pancreas and blood serum. Diabetes, 12, 448-453 (1963).

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6. SIREK, A., SCHOEFFLING, I~. B. & DITSCHUNEIT, H.A. Serum insulin-like activity in Houssay dogs (abstr.). Diabetes, 12, 352 (1963).

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IMMUNOASSAY OF INSULIN 117

8. ALP, H. & RECANT, L. Effect of the insulin-inhibitory albumin fraction from normal and diabetic subjects on adipose tissue. Metabolism, 13, 609-619 (1964).

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23. GOETZ, F. C., GREENBERG, B. Z., ELLS, J. & MEINERT, C. A simple immunoassay for insulin: Application to human and dog plasma. J. Clin. Endocr. Metab. 23, 1237-1246 (1963).

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27. ARQUILLA, E. R., OOMS, H. & FINN, J. Genetic differences of combining sites of insulin antibodies and importance of C-terminal portion of the A chain to biological and immuno- logical activity of insulin. Diabetologia 2, 1.-13 (1966).

28. Izzo, J. L., RONCONE, A., Izzo, M. J. & BALE, W.F . Relationship between degree of iodina- tion of insulin and its biological, electrophoretic and ilnmunochemical properties. J. Biol. Chem. 239, 3749-3754 (1964).

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its hyperglycemic effects. Diabetes 15, 143-148 (1966). 23. EHRLICH, R. M. ~z MARTIN, J . M . Tolbutamide tolerance and plasma insulin response in

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676-690 (1960). 35. RASlO, E., CAHILL, G. F., JR. & SOELDNER, J . S . Comparison of insulin-like activity (ILA)

and immunoreactive insulin (IRI) in serum and lymph. Diabetes (Abstr.) 14, 463 (1965).