novel enzyme immunoassay of anti-insulin igg in human serum

6
Journal of Clinical Laboratory Analysis 2:19-24 (1988) Novel Enzyme lmmunoassay of Anti-Insulin IgG in Human Serum Takeyuki Kohno,‘ Eiji Ishikawa,’ Satoru Sugiyama,* and Syuji Nakamura2 Department of Biochemistry, Medical College of Miyazaki, and Heiwadai Clinic, Miyazaki, Japan A novel enzyme immunoassay of anti-in- sulin IgG in human serum is described. A serum sample containing anti-insulin IgG was treated with dextran-charcoal at pH 6.0 to remove endogenous insulin and subsequently incubated with dinitrophenyl biotinyl nonspecific rabbit IgG-insulin con- jugate. The reaction mixture was further incubated with a rabbit (antidinitrophenyl bovine serum albumin) IgG-coated polysty- rene ball to trap the complex formed be- tween anti-insulin IgG and the conjugate. After washing to eliminate nonspecific IgG in the test serum, the polystyrene ball was incubated with dinitrophenyl-L-lysine to elute the complex. The eluate was incu- bated with an avidin-coated polystyrene ball. Finally, the amount of human anti- insulin IgG in the complex trapped onto the avidin-coated polystyrene ball was mea- sured by incubation with rabbit (antihuman IgG (?-chain)) Fab’-peroxidase conjugate. This enzyme immunoassay was 1,000-fold more sensitive than the conventional en- zyme immunoassay, in which an insulin- bovine serum albumin-coated polystyrene ball was incubated with a serum sample containing anti-insulin IgG and subse- quently with rabbit (antihuman IgG (y- chain)) Fab’-peroxidase conjugate. The principle of the novel enzyme immunoas- say can be used to more sensitively mea- sure antibodies for most kinds of haptens and antigens than the conventional en- zyme immunoassay. Key words: insulin, antibody, peroxidase, Fab’, diabetes mellitus INTRODUCTION In the conventional enzyme immunoassay of antibodies in serum, an antigen-coated solid phase is incubated with serum samples to trap specific immunoglobulins and subsequently reacted with anti-immunoglobulin antibody-enzyme conju- gate to estimate the amount of specific immunoglobulins on the solid phase (1). The sensitivity of the conventional en- zyme immunoassay is seriously limited by the nonspecific binding of nonspecific immunoglobulins in serum samples to the solid phase, which cannot easily be eliminated (2,3). This paper describes a novel enzyme immunoassay of anti- insulin IgG in human serum using dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate, in which the non- specific binding of nonspecific IgG was remarkably reduced and the sensitivity was improved approximately 1,000-fold as compared with the conventional enzyme immunoassay . MATERIALS AND METHODS Serum Samples Serum samples were obtained from 23 healthy subjects (16 women and 7 men, aged 26-74 years) with no previous ex- posure to exogenous insulin, and 29 diabetic patients (17 0 1988 Alan R. Liss, Inc. women and 12 men, aged 22-77 years) who had been treated for 0.7-10 months with porcine insulin (6-28 IU/day, Insu- lin Novo Actrapid MC and Monotard MC from Novo In- dustri AIS, Copenhagen, Denmark, and Insulin Velosulin Nordisk and Insulatard Nordisk from Nordisk Gentofte A/S, Gentofte, Denmark) and porcine insulin plus bovine insulin (6-24 IU/day, Insulin Novo Lente MC from Novo Industri A/S , Copenhagen, Denmark). Buffer The regularly used buffers were 0.1 mol/l sodium phos- phate buffer, pH 6.0, containing 5 mmol/l EDTA (buffer A), 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaC1, 1.0 g/l bovine serum albumin (fraction V, Ar- mour Pharmaceutical Co., Kankakee, IL) and 1 .O g/l NaN3 (buffer B) and 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaCl (buffer C). Received August 31, 1987; accepted September 2, 1987. Address reprint requests to E. Ishikawa, Dept. of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan.

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Journal of Clinical Laboratory Analysis 2:19-24 (1988)

Novel Enzyme lmmunoassay of Anti-Insulin IgG in Human Serum

Takeyuki Kohno,‘ Eiji Ishikawa,’ Satoru Sugiyama,* and Syuji Nakamura2

Department of Biochemistry, Medical College of Miyazaki, ’ and Heiwadai Clinic, Miyazaki, Japan

A novel enzyme immunoassay of anti-in- sulin IgG in human serum is described. A serum sample containing anti-insulin IgG was treated with dextran-charcoal at pH 6.0 to remove endogenous insulin and subsequently incubated with dinitrophenyl biotinyl nonspecific rabbit IgG-insulin con- jugate. The reaction mixture was further incubated with a rabbit (antidinitrophenyl bovine serum albumin) IgG-coated polysty- rene ball to trap the complex formed be- tween anti-insulin IgG and the conjugate. After washing to eliminate nonspecific IgG in the test serum, the polystyrene ball was incubated with dinitrophenyl-L-lysine to elute the complex. The eluate was incu- bated with an avidin-coated polystyrene ball. Finally, the amount of human anti-

insulin IgG in the complex trapped onto the avidin-coated polystyrene ball was mea- sured by incubation with rabbit (antihuman IgG (?-chain)) Fab’-peroxidase conjugate. This enzyme immunoassay was 1,000-fold more sensitive than the conventional en- zyme immunoassay, in which an insulin- bovine serum albumin-coated polystyrene ball was incubated with a serum sample containing anti-insulin IgG and subse- quently with rabbit (antihuman IgG (y- chain)) Fab’-peroxidase conjugate. The principle of the novel enzyme immunoas- say can be used to more sensitively mea- sure antibodies for most kinds of haptens and antigens than the conventional en- zyme immunoassay.

Key words: insulin, antibody, peroxidase, Fab’, diabetes mellitus

INTRODUCTION

In the conventional enzyme immunoassay of antibodies in serum, an antigen-coated solid phase is incubated with serum samples to trap specific immunoglobulins and subsequently reacted with anti-immunoglobulin antibody-enzyme conju- gate to estimate the amount of specific immunoglobulins on the solid phase (1). The sensitivity of the conventional en- zyme immunoassay is seriously limited by the nonspecific binding of nonspecific immunoglobulins in serum samples to the solid phase, which cannot easily be eliminated (2,3).

This paper describes a novel enzyme immunoassay of anti- insulin IgG in human serum using dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate, in which the non- specific binding of nonspecific IgG was remarkably reduced and the sensitivity was improved approximately 1,000-fold as compared with the conventional enzyme immunoassay .

MATERIALS AND METHODS

Serum Samples

Serum samples were obtained from 23 healthy subjects (16 women and 7 men, aged 26-74 years) with no previous ex- posure to exogenous insulin, and 29 diabetic patients (17

0 1988 Alan R. Liss, Inc.

women and 12 men, aged 22-77 years) who had been treated for 0.7-10 months with porcine insulin (6-28 IU/day, Insu- lin Novo Actrapid MC and Monotard MC from Novo In- dustri AIS, Copenhagen, Denmark, and Insulin Velosulin Nordisk and Insulatard Nordisk from Nordisk Gentofte A/S, Gentofte, Denmark) and porcine insulin plus bovine insulin (6-24 IU/day, Insulin Novo Lente MC from Novo Industri A/S , Copenhagen, Denmark).

Buffer

The regularly used buffers were 0.1 mol/l sodium phos- phate buffer, pH 6.0, containing 5 mmol/l EDTA (buffer A), 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaC1, 1.0 g/l bovine serum albumin (fraction V, Ar- mour Pharmaceutical Co., Kankakee, IL) and 1 .O g/l NaN3 (buffer B) and 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaCl (buffer C).

Received August 31, 1987; accepted September 2, 1987.

Address reprint requests to E. Ishikawa, Dept. of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan.

20 Kohno et al.

Enzymes Laboratories, Kumamoto, Japan) in N,N-dimethylformam-

Horseradish peroxidase (EC 1.11.1.7) (grade I, lyophi- lized RZ = 3.0) and pepsin from porcine gastric mucosa (EC 3.4.23.1) were obtained from Boehringer Mannheim GmbH, Mannheim, FRG.

Antibodies

Rabbit (antihuman IgG (y-chain)) serum was obtained from Medical and Biological Laboratories Co., Ltd., Na- goya, Japan. Rabbit (antidinitrophenyl-bovine serum albu- min) serum was obtained from Miles Laboratories, Inc., Elkhart, IN. IgG was prepared by fractionation of serum with Na2S04 followed by passage through a column of di- ethylaminoethyl-cellulose (Whatman Chemical Separation, Ltd., Kent, UK) (4). F(ab’)z was prepared by digestion of IgG with pepsin, and Fab’ was prepared by reduction of F(ab’)2 with 2-mercaptoethylamine (4). The amounts of IgG and its fragments were calculated from the absorbance at 280 nm (4).

Assay of Peroxidase Activity

The activity of peroxidase was assayed by fluorimetry at 30°C using 3-(4-hydroxyphenyl)propionic acid (Aldrich Chemical Co., Inc., Milwaukee, WI) as a substrate (5). The fluorescence intensity was measured relative to 1 mg/l qui- nine in 50 mmol/l HzSO4 using 320 nm for excitation and 405 nm for emission in a Shimadzu spectrofluorophotome- ter (RF-510, Shimadzu Seisakusho Ltd., Kyoto, Japan).

Preparation of Protein-Coated Polystyrene Bails

Polystyrene balls (3.2 rnm in diameter, Precision Plastic Ball Co., Chicago, IL) were coated with rabbit (antidinitro- phenyl-bovine serum albumin) IgG (0.1 g/l), avidin (0.1 g/ 1) (Avidin D, Vector Laboratories, Inc., Burlingame, CA), bovine serum albumin (1.0 g/l), or nonspecific rabbit IgG (2.0 g/l) by physical adsorption (6). The amount of avidin was calculated from the absorbance at 282 nm by taking the extinction coefficient to be 1.54 g-’.liter.cm-’ (7). Insulin- bovine serum albumin-coated polystyrene balls and insulin- nonspecific rabbit IgG-coated polystyrene balls were pre- pared by treatment of bovine serum albumin-coated polysty- rene balls and nonspecific rabbit IgG-coated polystyrene balls, respectively, with glutaraldehyde followed by reaction with insulin (3).

ide at 30°C for 30 min. After incubation, the reaction mix- ture was subjected to gel filtration on a column (1.0 x 30 cm) of Sephadex (3-25 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) using buffer A. The average number of maleimide groups introduced per nonspecific rabbit IgG molecule was 16 (4).

2. Preparation of N-biotinyl-2-mercaptoethylamine. An aliquot (0.1 ml) of 44 mmol/l biotin-N-hydroxysuccinimide (Zymed Laboratories, Inc., San Francisco, CA) in N,N-di- methylformamide was incubated with 1.0 ml of 4.4 mmol/l 2-mercaptoethylamine (Nakarai Chemicals, Ltd., Kyoto, Ja- pan) in 0.1 mol/l sodium phosphate buffer, pH 7.0, contain- ing 5 rnmol/l EDTA at 30°C for 30 min. After incubation, 0.1 ml of 1 mol/l Tris-HC1 buffer, pH 7.0, was added to the reaction mixture to eliminate remaining biotin-N- hydroxysuccinimide.

3. Preparation of biotinyl nonspecific rabbit IgG. An ali- quot (0.22 ml) of the N-biotinyl-2-mercaptoethylamine so- lution was incubated with maleimide-nonspecific rabbit IgG (10 mg) in 2.0 ml of buffer A at 30°C for 30 min. After in- cubation, 0.05 ml of 0.1 mol/l 2-mercaptoethylamine in buffer A was added to the reaction mixture to block remain- ing maleimide groups, and the mixture was subjected to gel filtration on a column (1 .O x 30 cm) of Sephadex G-25 us- ing 0.1 mol/l sodium phosphate buffer, pH 7.5. The average number of biotin residues introduced per nonspecific rabbit IgG molecule was 9.7, which was calculated from the de- crease in the number of maleimide groups (4).

4. Preparation of mercaptosuccinylated biotinyl nonspe- cific rabbit IgG. Biotinyl nonspecific rabbit IgG (6.7 mg) in 3.2 ml of 0.1 mol/l sodium phosphate buffer, pH 7.5, was incubated with 0.32 ml of 0.14 mol/l S-acetylmercaptosuc- cinic anhydride (Nakarai Chemicals, Ltd., Kyoto, Japan) in N,N-dimethylformamide at 30°C for 30 min. After incuba- tion, the mixture was incubated with 0.32 ml of 1 mol/l Tris- HC1 buffer, pH 7.0,0.20 ml of 0.1 mol/l EDTA, pH 7.0, and 0.4 ml of 1 mol/l hydroxylamine, pH 7.0, at 30°C for 5 min. The reaction mixture was subjected to gel filtration on a col- umn (1.0 x 30 cm) of Sephadex (3-25 using buffer A. The average number of thiol groups introduced per biotinyl non- specific rabbit IgG molecule was 17 (4).

5. Preparation of maleimide-dinitrophenyl-L-lysine. An aliquot (1.0 ml) of 5.5 mmol/l dinitrophenyl-L-lysine.HC1 (Tokyo Kasei Kogyo, Co., Ltd., Tokyo, Japan) in 0.1 mol/l sodium phosphate buffer, pH 7.0, was incubated with 0.1 ml of 5.5 mmol/l N-succinimidyl-6-maleimidohexanoate (Do- jindo Laboratories) in N,N-dimethylformamide at 30°C for 3o min.

6. Preparation of mercaptosuccinylated dinitrophenyl bio-

Preparation of Dinitrophenyl Biotinyl Nonspecific Rabbit IgG-Insulin Conjugate

1. Preparation of maleimide-nonspecific rabbit IgG. Non- tiny1 nonspecific rabbit IgG. An aliquot (0.59 ml) of the specific rabbit IgG (12 mg) in 2.0 ml of 0.1 mol/l sodium maleimide-dinitrophenyl-L-lysine solution was incubated phosphate buffer, pH 7.0, was incubated with 0.2 ml of 27.5 with mercaptosuccinylated biotinyl nonspecific rabbit IgG mmol/l N-succinimidyl-6-maleimidohexanoate (Dojindo (4.4 mg) in 3.1 ml of buffer A at 30°C for 30 min. The re-

Enzyme lmmunoassay of Anti-Insulin IgG 21

with 0.0375 ml of the dextran-charcoal suspension. The mixture was intermittently stirred for 5 min and neutralized by addition of 0.015 ml of 50 mmol/l NaOH. The neutral- ized mixture was centrifuged at 1,500 X g for 15 min, and the supernatant was centrifuged in the same way to com- pletely remove the dextran-charcoal.

An aliquot (0.095 ml) of the dextran-charcoal-treated serum sample was mixed with 0.015 ml of 30 mmol/l so- dium phosphate buffer, pH 7.0, containing 3. l mol/l NaC1, 3 g/l bovine serum albumin and 3 g/l NaN3, and 0.02 ml of nonspecific rabbit IgG (0.3 mg) in buffer B and incubated with dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate (30 fmol) in buffer B (0.02 ml) at 37°C for 8 h and at room temperature overnight. The reaction mixture (0.15 ml) was further incubated with a rabbit (antidinitrophenyl- bovine serum albumin) IgG-coated polystyrene ball, which had been treated with nonspecific rabbit IgG (3), at 20°C for 4 h. After incubation, the polystyrene ball was washed twice by addition and aspiration of 2 ml of buffer C and incubated with 1 mmol/l dinitrophenyl-L-lysine in 0.15 ml of buffer B containing nonspecific rabbit IgG (0.3 mg/tube) at room temperature overnight to elute the complex of anti-insulin IgG and the conjugate. After removal of the polystyrene ball, the eluate was incubated with an avidin-coated polystyrene ball, which had been treated with nonspecific rabbit IgG (3), at 20°C for 3 h. After incubation, the polystyrene ball was washed twice with buffer C as described above and incu- bated with rabbit (antihuman IgG (y-chain)) Fab'-peroxi- dase conjugate (50 ng) in 0.15 ml of 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaCl and 1.0 g/l bovine serum albumin at 20°C for 3 h. Finally, the poly- styrene ball was washed twice with buffer C, and bound per- oxidase activity was assayed for 10 min as described above.

action mixture was subjected to gel filtration on a column (1.0 x 30 cm) of Sephadex G-25 using buffer A. The av- erage number of dinitrophenyl groups introduced per mer- captosuccinylated biotinyl nonspecific rabbit IgG molecule was 7.3, whiclh was calculated from the absorbance at 360 nm by taking the molar extinction coefficient to be 17,400 mol-'.liter.cni-' (8).

7. Preparation of maleimide-insulin. Porcine insulin (40 IU in 1.0 ml, Actrapid MC, Novo Industri A/S) was incubated with 0.1 ml of 4.4 mmol/l N-succinimidyl-6-maleimido- hexanoate in N,N-dimethylformamide at 30°C for 30 min, and subjected to gel filtration on a column (1 .O x 30 cm) of Sephadex (3-25 using buffer A. The average number of mal- eimide groups introduced per porcine insulin molecule was 0.23 (4). The amount of insulin was calculated from the ab- sorbance at 280 nm by taking the extinction coefficient and molecular weight to be 0.9 g-'.liter.cm-' (3) and 5,778 (9), respectively.

8. Preparation of dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate. Maleimide-insulin (1.2 mg) in 0.3 ml of buffer A was incubated with mercaptosuccinylated dini- trophenyl biotinyl nonspecific rabbit IgG (0.73 mg) in 0.2 ml of buffer A at 4°C for 20 h. (The average number of thiol groups per mercaptosuccinylated dinitrophenyl biotinyl nonspecific rabbit IgG molecule was 7.) After incubation, the reaction mixture was subjected to gel filtration on a column (1.5 X 45 cm) of Ultrogel AcA 34 (LKB, Stockholm, Swe- den) using 0.1 mol/l sodium phosphate buffer, pH 6.5. The average number of insulin molecules conjugated per dinitro- phenyl biotinyl nonspecific rabbit IgG molecule was 5.8. The amount of dinitrophenyl biotinyl nonspecific rabbit IgG was calculated from the absorbance at 360 nm, and the num- ber of insulin molecules introduced was calculated from the absorbance at 280 nm.

Preparation of Rabbit (Antihuman IgG (y-chain)) Fab'-Peroxidase Conjugate

Rabbit (antihuman IgG (y-chain)) Fab' was conjugated to horseradish peroxidase using N-succinimidyl-6-maleimi- dohexanoate (Dojindo Laboratories) (10). The amount of the conjugate was calculated from peroxidase activity (4).

Preparation of Dextran-Charcoal The dextran-charcoal was prepared by the method of

Dixon (11) with the following substitutions. Bovine serum albumin (fraction V, Armour Pharmaceutical Co.) and Norit A (Nakarai Chemicals, Ltd., Kyoto, Japan) were substituted for human serum albumin and Norit NK (Norit GSX), re- spectively. Thie dextran-charcoal suspension contained 60 mg of charcoal in dry weight per ml.

Present Enzyme lmmunoassay A 0.075 ml aliquot of human serum samples was mixed

Conventional Enzyme lmmunoassay

The dextran-charcoal-treated serum sample was diluted 5.3 X lo4-fold with buffer B, and the diluted serum sample (0.15 ml) was incubated with an insulin-bovine serum albu- min-coated polystyrene ball at 37°C for 3 h. After incuba- tion, the polystyrene ball was washed twice with buffer C and incubated with rabbit (antihuman IgG (y-chain)) Fab'- peroxidase conjugate (50 ng) at 37°C for 3 h as described above. Finally, the polystyrene ball was washed twice with buffer C and bound peroxidase activity was assayed for 10 min as described above.

Improved Conventional Enzyme lmmunoassay

The dextran-charcoal-treated serum sample was diluted 1.6 X lo4-fold with buffer B and the diluted serum sample (0.15 ml) was incubated with insulin-nonspecific rabbit IgG- coated polystyrene ball, which had been treated with non- specific rabbit IgC, at 20°C for 3 h in the presence of non-

with 0.015 ml of 0.2 mol/l HCl to adjust pH-to 6.0 and then specific rabbit IgG (0.3 mg/tube) (3). After incubation, the

22 Kohno et al.

polystyrene ball was washed twice with buffer C and incu- bated with rabbit (antihuman IgG (y-chain)) Fab ‘-peroxi- dase conjugate (50 ng) at 20°C for 3 h as described above. Finally, the polystyrene ball was washed twice with buffer C and bound peroxidase activity was assayed for 10 min as de- scribed above.

RESULTS AND DISCUSSION

The present enzyme immunoassay is schematically shown in Figure 1. Human serum containing anti-insulin IgG was treated with dextran-charcoal to remove endogenous insu- lin. The dextran-charcoal-treated serum was incubated with dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conju- gate and subsequently with a rabbit (antidinitrophenyl bo- vine serum albumin) IgG-coated polystyrene ball in the presence of nonspecific rabbit IgG. In this step, the complex formed between anti-insulin IgG and dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate was trapped onto the polystyrene ball. After washing to eliminate nonspecific human IgG in the test serum, the polystyrene ball was in- cubated with dinitrophenyl-L-lysine to elute the complex. After removal of the polystyrene ball, the eluate was incu-

Insulin

Rb IgG - DNP

Biotin

I I

Dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate

Incubate with test serum (Anti-insulin IgG), nonspecific rabbit IgG and rabbit (anti-dinitrophenyl bovine serum albumin)lgG-coated polystyrene ball (Anti-DNP-PS Ball) I

1 Insulin - - - Anti-insulin IgG

Rb IgG - DNP - - - Anti-DNP-PS Ball

Biotin

I

I

After washing, incubate with dinitrophenyl-L-lysine and nonspecific rabbit IgG, and remove Anti-DNP-PS-Ball

Insulin - - - Anti-insulin IgG

Rb IgG - DNP

Biotin

I

I

Incubate with avidin-coated polystyrene ball (Avidin-PS Ball) 1 Insulin - - - Anti-insulin IgG

Rb !gG - DNP

Biotin - - - Avidin-PS Ball

After washing, incubate with rabbit (anti-human IgG ( r-chain)) Fab-peroxidase conjugate

(Rb (anti-H IgG) Fab-POD)

I

I

1 Insulin - - - Anti-insulin IgG - - - Rb (anti-H IgG) Fab-POD

Rb IgG - DNP

Biotin - - - Avidin-PS Ball

I

I

Fig. 1. IgG in human serum.

Procedure of the present enzyme immunoassay for anti-insulin

bated with an avidin-coated polystyrene ball in the presence of nonspecific rabbit IgG to trap the complex. After wash- ing, the avidin-coated polystyrene ball was incubated with rabbit (antihuman IgG (y-chain)) Fab’-peroxidase conjugate to estimate the amount of human anti-insulin IgG in the complex on the avidin-coated polystyrene ball.

For comparison, the conventional enzyme immunoassay was performed as follows. An insulin-bovine serum albu- min-coated polystyrene ball was incubated with a serum sample at 37°C and after washing, with rabbit (antihuman IgG (y-chain)) Fab‘-peroxidase conjugate to estimate the amount of anti-insulin IgG bound to the polystyrene ball.

Necessity of Dextran-Charcoal Treatment in the Present and Conventional Enzyme lmmunoassays

In the present and conventional enzyme immunoassays, serum samples were treated with dextran-charcoal at pH 6.0. The detection limit of anti-insulin IgG in human serum was 1.5 to 2-fold less than that obtained without the dextran- charcoal treatment. The dextran-charcoal treatment at pH 2.5-5.5 of serum samples from healthy subjects caused a higher bound peroxidase activity, that is, a higher back- ground, which may have been due to an increased nonspe- cific binding of partially denatured nonspecific IgG to the solid phase.

Assay Variation

In the present enzyme immunoassay, the variation coeffi- cients of within-assay and between-assay for serum samples from healthy subjects were 5.9% and 9.2%, respectively, and the variation coefficients of within-assay and between- assay for serum samples containing anti-insulin IgG were 10.0-10.4% and 8.4-12.0%, respectively (Table 1) .

Measurement of Anti-Insulin IgG in Human Serum Samples by the Present and Conventional Enzyme I m m u noassays

Serum samples from 23 healthy subjects and 29 diabetic patients, who had been treated with porcine insulin and por- cine insulin plus bovine insulin for 0.7-10 months, were subjected to the present and conventional enzyme immu- noassays (Fig. 2). Eighteen serum samples (62%) out of 29 gave higher bound peroxidase activities than those obtained with 23 serum samples from healthy subjects by the present enzyme immunoassay. However, only 2 serum samples (7%) out of 29 (the same diabetic patients) were shown to contain anti-insulin IgG by the conventional enzyme immunoassay.

Comparison with the Previously Described Enzyme lmmunoassays

In order to more exactly compare the sensitivity of the present enzyme immunoassay with that of the conventional

Enzyme lmmunoassay of Anti-Insulin IgG 23

TABLE 1. Within-Assay and Between-Assay Variations

Fluorescence intensity Coefficient

for bound of Serum No. of peroxidase variation

Assay no. determinations activity (%)

Within- 1' 15 10.7 k 0.63 5.9

2 15 36.1 f 3.6 10.0 3 15 65.5 rt 6.8 10.4

Between- 4' 15 10.2 + 0.94 9.2

5 15 19.1 f 2.3 12.0 6 15 55.7 & 4.7 8.4

assay

assay

'Serum samples from healthy subjects.

2 40 co I- 0 L I t

I 0 0

0-0 - - 0- - - - - - - - - - - - _

0

' 0

00

8 0 U 0

0 I 0 I 10 (month )

lo Healthy (month 1 Subjects

Hea It h y Subjects

Period of Treatment with Insulin Period of Treatment with Insulin

Fig. 2. Measurement by the present (A) and the conventional (B) enzyme imrnunoassays of anti- insulin IgG in serum samples from 23 healthy subjects and 29 diabetic patients who had been treated with insulin for 0.7-10 months. Open and closed circles indicate values obtained for serum samples from 29 diabetic patients who had been treated with porcine insulin and porcine insulin plus bovine insulin, respectively.

enzyme immunoassay, a serum sample from a diabetic pa- tient, who had been treated with porcine insulin plus bovine insulin for 4 years, was diluted with pooled serum from healthy subjects to various extents and was subjected to both the present and conventional enzyme immunoassays. The detection limit was taken to be the maximal dilution of the test serum with pooled serum from healthy subjects, which gave a bound peroxidase activity significantly in excess of that observed with serum from healthy subjects (the back- ground). A significant difference from the background was confirmed by thle t-test (n = 5 , p < 0.001). The sensitivity of the conventional enzyme immunoassay was seriously lim-

ited by the nonspecific binding of nonspecific human IgG to the polystyrene ball (2,3). In the present enzyme immunoas- say, the nonspecific binding of nonspecific IgG in test serum was remarkably reduced by transfer of the complex formed between anti-insulin IgG and dinitrophenyl biotinyl nonspe- cific rabbit IgG-insulin conjugate from the (antidinitro- phenyl bovine serum albumin) IgG-coated polystyrene ball to the avidin-coated polystyrene ball. As a result, the sensi- tivity of the present enzyme immunoassay was improved ap- proximately 1,000-fold as compared with the conventional enzyme immunoassay in terms of the dilution of serum sam- ple containing anti-insulin IgG with serum from healthy sub-

24 Kohno et al.

t P ._ 2 ,Ot

e u a V

0 m

d A

P

-. ~

lo3 lo2 10 I W

Dilution of Patient's Serum with Normal Serum (-fold 1

Fig. 3. Dose-response curves of anti-insulin IgG in human serum by the present enzyme immunoassay (circles), the conventional enzyme immu- noassay (triangles), and the improved conventional enzyme immunoassay (squares).

jects (Fig. 3). In the conventional enzyme immunoassay for anti-insulin

IgG in guinea pig serum, the nonspecific binding of nonspe- cific guinea pig IgG was reduced by incubation of insulin- nonspecific rabbit IgG-coated solid phase (instead of insu- lin-bovine serum albumin-coated solid phase) with guinea pig anti-insulin serum in the presence of nonspecific rabbit IgG at 20°C (instead of 37°C) (3). The same method was applied to the measurement of anti-insulin IgG in human serum. However, the sensitivity was improved only 10-fold, and this improved conventional enzyme immunoassay was 100-fold less sensitive than the present enzyme immunoas- say (Fig. 3).

The sensitivity for anti-insulin antibodies in human serum was remarkably improved by measuring insulin bound to anti-insulin antibodies (12). Human serum containing anti- insulin antibodies was incubated with insulin, and the com- plex formed between insulin and anti-insulin antibodies was separated from free insulin by dextran-charcoal treatment and by polyethylene glycol precipitation. The precipitated complex was treated with acid to inactivate anti-insulin an- tibodies and dissociated insulin from anti-insulin antibodies. The dissociated insulin was measured by sandwich enzyme immunoassay. This enzyme irnmunoassay is 30-fold more sensitive than the present enzyme assay in terms of the di- lution of human anti-insulin serum with serum from healthy subjects. However, this sensitive enzyme immunoassay suf- fers from three disadvantages as compared with the present enzyme immunoassay. First, the class of anti-insulin im-

munoglobulins cannot be identified because insulin in the complex with anti-insulin antibodies is measured. In the present enzyme immunoassay, anti-insulin antibodies in the complex are measured using anti-immunoglobulin Fab'-per- oxidase conjugate. Second, antibodies for antigens that partly or largely lose antigenicity by acid treatment cannot be mea- sured with high sensitivity. In the present enzyme immu- noassay, there is no process of acid treatment. Third, antibodies for antigens that cannot be separated from anti- gen-antibody complexes by dextran-charcoal treatment or polyethylene glycol precipitation cannot be measured with high sensitivity. Although free antigens can be separated from antigen-antibody complexes by precipitation with anti- immunoglobulin antibodies or by trapping on a column of anti-immunoglobulin antibody-coupled Sepharose, the sep- aration is not so complete as by dextran-charcoal treatment and polyethylene glycol precipitation (unpublished). As a result, the background is enhanced to lower the sensitivity. By contrast, the present enzyme immunoassay can sensi- tively measure antibodies for most kinds of haptens and an- tigens with identification of immunoglobulin classes.

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