Quantitative determination of insulin-binding antibodies in human serum

CliniCa Chimica Acta, 45 (1973) 145-151 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

145

CCA 5564

QUANTITATIVE

DETERMINATION

OF INSULIN-BINDING

ANTIBODIES

IN HUMAN SERUM

LJUBEN

M. SIRAKOV

AND

STEFAN

P. DITZOV

Diabetes Research Groufi, Bulgarian Academy of Sciences, bul. Christo Michailov (Received

November

6, Sofia 3 (Bulgaria)

20, 1972)

SUMMARY

Methods are described for the assay of insulin antibodies using lz61-labeled insulin and for separation of free from antibody-bound hormone, by acrylamide gel electrophoresis. The sensitivity of the method depends upon the specific activity of the labeled hormone; a simple method is described for the calculation of this. Prior to use, the labeled hormone is highly purified by gel electrophoresis.

INTRODUCTION

Antibodies to insulin have been demonstrated in the sera of insulin-treated animals and human subjects, but the theory that endogenously secreted insulin can be immunogenic is not commonly accepted1-4. In these studies, various methods for the assay of insulin antibodies have been used 5- l3 but they have been used to detect and assay them in high rather than very low concentrations. In previous studies we have used precipitating techniques to detect insulin antibodies in the sera of diabetic patients who have never received treatment with insulin14. With a view towards reassessing the results obtained in these studies, we have now developed another sensitive and specific method which is described here for use in the detection of antibodies in low concentrations. MATERIALS

AND METHODS

1261-labeled insulin

Bovine (Hoechst) and porcine (Novo) recrystallized insulins were labeled with carrier-free lZsI (Code I-RB-3; Izotope, Hungary) by the Chloramin T method of Greenwood et al.15.It was purified either by passage through small columns of cellulose powder (Whatman) using a modification (a-mercaptoethanol was omitted from the buffer) of the method of Landon et al.ls, or by acrylamide gel electrophoresis. For routine purposes, the iodination mixture was applied to acrylamide gel columns (prepared as described below), and, after electrophoresis, the labeled hormone eluted with buffer containing 0.25% albumin for subsequent storage at -20’ in small

146

SIRAKOV, DITZOV

portions. On the day before each experiment, this material was repurified as follows: a small portion of the preparation (IO ,ul) was added to a solution of sucrose (30 ,ul; 33%, w/v) and applied to the top of the column of acrylamide gel. After electrophoresis (6 mA per gel for 50 min), the gel was cut into thin slices (2-3 mm) and only the slices containing the maximum radioactivity corresponding in mobility to that of unlabeled insulin were used in further experiments. The amount of labeled insulin in the gel slice is calculated simply from the specific radioactivity of [lZ51]insulin. Immunoadsorbents Aminocellulose,

prepared

by the method

of Gurvich

et a1.‘7yls, was diazotized

and coupled with crystalline bovine insulin as follows : 200 mg sodium nitrite was added with constant stirring to a cold solution at o” of IO ml 10% (v/v)cont. HCl. To this solution was further added a suspension of IO ml ~"/b(w/v) aminocellulose, stirring being continued for 30 min. The diazotized aminocellulose was washed twice with 20 ml cold distilled water and 3 times with (20 ml) 0.1 M borate buffer (pH 8.6). Crystalline bovine insulin (200 mg) was then dissolved in z ml 0.1 ,v NaOH and diluted in (2 ml) borate buffer to a final concentration of 50 mg insulin/ml. This solution of insulin was added to the suspension of diazotized aminocellulose to give a final concentration of 20 mg insulin/ml. After standing at 4’ for 24 h, the immunoadsorbant deposit was washed five times with 20 ml distilled water, once with 30 ml 0.1 N HCl, and three times with distilled water. The washed adsorbent was suspended in dilute saline (0.14 M NaCl) and stored at 4’ for use within 1-3 weeks. Serum Venous blood was drawn from human subjects after an overnight fast at least 24 h after the last injection of insulin. The serum was allowed to separate and was stored before use in the frozen state (-20~). Such sera were assayed either in the untreated state or after exposure to the immunoadsorbent. Varying amounts (0.1-1.0ml) of suspension of the immunoadsorbent were centrifuged and the supernatant solution removed. A sample of the serum was diluted in saline or buffer to a final concentation of I part serum : 30 parts diluent. Diluted serum (0.1ml) was mixed with the immunoadsorbent and incubated first at 37O for 2 h and then at 4’ for 24 h. After incubation the immunoadsorbent was removed by centrifugation. To diluted adsorbed or unadsorbed serum (0.05 ml) was added a slice of acrylamide gel containing the highly purified labeled insulin (see above). Incubation of this mixture, first for 2 h at 37O and then at 4’ for 24 h, was carried out in polyethylene or siliconized glass tubes to minimize adsorption of the labeled hormone onto vessel walls. The whole sample, including the small piece of gel, was then placed on the top of other gel for electrophoresis. Electrophoresis on polyacrylamide gel Samples of serum which had been incubated with labeled insulin were electrophoresed on polyacrylamide gel (acrylamide : methylenebisacrylamide ratio, 37 : I, w/w) by the method of Reisfeld et a1.18. The acrylamide gel (7%) was prepared in solutions containing (0.2 M) glycine-Tris buffer (pH 8.6) and (0.33%) EDTA and placed in tubes (7 cm x 5 mm i.d.). The sample (100-150 ,ugprotein) in a solution of

DETERMINATION

OF INSULIN-BINDING

ANTIBODIES

147

sucrose (0.15 ml; 16%, w/v) containing bromophenol blue (5 ,ul; o.z~/~,w/v) as a tracking dye was layered onto the top of the gel under the electrode buffer and electrophoresed (6 mA per tube) for go min. Under these conditions the dye moved almost completely (go%) along the tube. The gels were then placed in a solution of (0.5%, w/v) Amide Black IOB in (7%, v/v) dilute acetic acid for 20 min. The unfixed dye was removed overnight by soaking in (7%, v/v) dilute acetic acid, and the protein bands scanned on an “Ekstinktionschreiber IR IO” (C. Zeiss, GDR). The gel was cut into thin slices (2 mm) using a device made in our laboratory, and the radioactive content of each slice was measured (well-type scintillation counter, “Vakutronic”, G.D.R.). Radioactive contents are expressed as counts (cpm) in individual gel slices or as ng of [12SI]insulin. RESULTS AND DISCUSSION

Using acrylamide gel electrophoresis, Ramachandran et al.l* demonstrated and assayed antibodies in sera of resistant human diabetics but their method, involving the use of large amounts of labeled insulin (mu), could not be applied for detection of low antibody levels. For this purpose, labeled insulin of high specific activity, known immunoreactivity and low degree of contamination with degradation products is required. Many authors have shown that labeled insulin is degraded during storage and TABLE

I

DEGRADATION

OF LABELED

INSULIN

DURING

ITS STORAGE

AT -20’

Acrylamide gel electrophoresis of labeled insulin was performed at the day indicated as described in the Methods section. Mean values of five different experiments. Days after labeling with l=jI 0

3 6 9 12

15 22 27

Surface under the insulin Peak after electrophoresis (in 0/Oof total radioactivity used) 75-90 75-80 70-75 65-70 60-65 55-60 50-55 45-50

recommend its purification before use in immunoassay21v22. As shown in Table I, approximately 50% of the labeled insulin prepared by our method is degraded during four weeks of storage. For purification, various method (gel filtration, electrophoresis, adsorption on cellulose, etc.) have been used to yield products of 85-goo/o purityIS+. This is insufficient for a sensitive method of antibody assay. By acrylamide gel electrophoresis we have consistently achieved degrees of purity in excess of 95%. In addition the method is rapid and convenient. In our opinion, acrylamide gel electrophoresis offers certain advantages for the separation of free from antibody-bound insulin after incubation of sera with labeled insulin 3y5-7: the medium in which the electrophoresis is carried out does not react with insulin, the antibodies or their complexes which can be separated on the basis of their different molecular weights and electrical chargeslos*a. When highly purified labeled insulin is incubated with normal serum it migrates

SIRAKOV,

I48

DITZOV

on electrophoresis more rapidly than the y-globulins to produce one major peak of radioactivity (free insulin). After incubation with serum containing insulin antibodies, it yields two distinct peaks, one associated with the y-globulins (bound insulin) and the second, described above, representing free insulin. These two peaks, originally described by Yalow and BersonzO, were the only ones observed during studies of sera from 288 healthy and diabetic human subjects (Fig. I) ; i.e. labeled insulin was never bound by any other fraction Using only radioactive that of unlabeled insulin, we as shown in Figs. 2a and 2b,

of the serum proteins. material with electrophoretic mobility corresponding to have not always obtained a homogeneous product. Thus, single and sometimes double peaks have been formed on

re-electrophoresis. Lack of homogeneity, in our opinion, could be due to differences in the degree of iodination achieved. The specificity of the method is shown by the effects of previous immunoadsorp-

cm

I

Globulins

Albumin

Fig. I. Serum from diabetic patient with antibodies after incubation with [*a51]insulin. Acrylamide gel electrophoresis as described in the Methods section. The arrow shows the direction of serum protein migration. -, protein fractions; - - - -, radioactivity.

cpm

I

I

cpm

Fig. 2. Purification of insulin the first day after labeling. Method as described Slices of 3 mm thickness. (B) Slices of r.5 mm thickness. - - - - -, radioactivity.

in the text.

(A)

DETERMINATION OF INSULIN-BINDING

ANTIBODIES

I49

tion. If, before incubation with labeled insulin, serum is first exposed to sufficient insulin conjugated aminocellulose, the proportion of added labeled hormone bound by y-globulins is greatly reduced (Fig. 3) ; insulin antibodies are adsorbed from the serum by the immunoadsorbent.

9 I

cpm .z e

1.5-1 I i

Globulins

Albumin

S 1.0s \ \

o--.,. 0 2 4 6 Unlobelled

, 10 14 20 insulin added (plJ)

Fig. 3. Acrylamide gel electrophoresis of diabetic serum. -, protein fractions: - - - - -, radioactivity after incubation with [‘*“I]insulin; . . . . ., also radioactivity but the serum was treated with immunosorbent prior to the incubation with labeled insulin. The arrow shows the direction of serum protein migration. More details are given in the text. Fig. 4. Standard dose-response curve in radioimmunoassay. -, the first standard curve; - - - - - -2 the second curve with different amount of labeled hormone only. The details are described in the text.

For the quantitative assay of antibodies in an anti-insulin serum, labeled insulin of known specific activity is essential. We have directly assayed the preparations of labeled insulin using unlabeled hormone for reference. In one experiment, a fixed volume of antiserum was incubated with a known quantity of labeled and increasing amount of unlabeled hormone. In a second experiment, run in parallel, the same volume of antiserum was incubated with increasing amounts of the labeled hormone; no unlabeled insulin was present. For convenience, the amount of trace labeled insulin used in the first experiment is used as the base amount (in cpm) used in the second, multiples of this amount being added ( x 2, x 3, x 4, x 5, etc.) The B/F (bound to free labeled insulin ratio) values are calculated for each point of the experiments, and the values plotted for the first against amounts of added unlabeled insulin. The values for the second experiment are superimposed onto the first curve (Fig. 4). If the affinities of the antibodies for labeled and unlabeled insulin are the same, the intercepts of abscissa given by every single point in the second experiment should be equal. Thus the immunoreactivity of the labeled hormone can be estimated. In this way one can determine the effective speci$c activity of the labeled insulin preparation used for antibody assay. The sensitivity of the method depends largely upon the specific activity of the

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DITZOV

insulin used, and for the assay of sera binding IO to 20 PU insulin per ml serum we found that labeled insulin had to have a specific activity of 300 to 600 mCi per mg. The precision of the method, expressed as the coefficient of variation for samples analyzed more than twice, was 14.8’~/~. The following scheme might be recommended as a routine: a standard volume of diluted serum (0.05 ml; I : 30 or more) was incubated with a fixed small dose of labeled insulin (0.02 to 0.2 ng; ca. 5000 cpm). If 80% of this small dose of radioactivity was recovered in the peak of free insulin and no radioactivity could be detected in the gamma glubulins (not more than z-3-fold of the background value), the serum was considered normal; it contained no antibodies. If radioactivity was observed in the y-globulin fraction (Fig. I), the assay was repeated using increasing amounts of labeled insulin (up to IO ng or more). For calculation of serum insulin binding capacity, the proportion of labeled insulin bound by the antibodies should not exeed 10% of total radioactivity added. In addition, the dilution of serum should be such that only trace amounts of endogenous insulin are present. In practice, incubation of diluted serum with several amounts of labeled antigen over a IO-fold range usually gives several points in an experimental curve from which the binding capacity can be calculated (Fig. 5).

Fig. 5. The saturation effect of different doses of labeled insulin. Two kinds of serum are shown : hyperimmune serum of guinea pig: x -.-.-x , the free insulin peak; O---O, the globulinbound peak; and (b) a serum of diabetic patient: 0 . . . . . .o, free insulin peak: 0- - -0, globulinbound peak. It is clear that the hyperimmune serum is still unsaturated and needs more labeled insulin, or alternatively higher dilution, while the second serum is saturated at very low dose of [ra51]insulin. (I) a

This technique has been used to study sera from diabetic and normal subjects, some of whom received treatment with insulin. The results of these studies will be published elsewherez3. ACKNOWLEDGEMENTS

This work was partially supported by International Atomic Energy Agency, Vienna, Austria, Research Contract 1223. Grateful thanks are due to Mrs. E. Bojadzieva and Mrs. S. Michovska for their skilful technical assistance.

DETERMINATION

OF INSULIN-BINDING

ANTIBODIES

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