One-step sandwich enzyme immunoassay for insulin using monoclonal antibodies

Clin Biochem, Vol. 21, pp. 311-314, 1988

0009-9120:88 $5.~,,5 Copyright © 1988 The Canadian Society of Ciinicat Che~, :~

Printed in Canada. All rights reserved.

One-Step Sandwich Enzyme Immunoassay for Insulin Using Monoclonal Antibodies W.

BORGIJ

M.

B R I N E R , ~ N.

1 D e p a r t m e n t of L a b o r a t o r y 2Boehringer Mannheim GmbH,

FRANKEN, ~ and

A.-CH.

KESSLER

2

Medicine, Kantonsspital Aarau, Switzerland, and Biochemical Research Center, D-8132 Tutzing, FRG

An enzyme-linked immunosorbent assay for the measurement of insulin in human serum has been developed. The test is based on the sandwich technique with two monoclonal antibodies directed against two different epitopes of insulin using coated plastic tubes as the solid phase and horse radish peroxidase as the label. The immunoreactions are completed in one step within 2 h. The horse radish peroxidase activity bound to the tube wall is measured photometrically after an additional 1-h incubation with the substrate. The standards used cover the range from 0 to 260 mU insulin/L. Employingthe Enzymun-Test® System ES 22 modular batch analyzer, the detection limit was found to be 3.7 mU insulin/L. Coefficients of variation (CV's) between 1.4-7.8% for intraassay precision and 5.610% for interassay precision were obtained over the concentration range of 17-107 mU Insulin/L. The correlation between the procedure described here (y) and a commercially available double antibody radioimmunoassay (x) is expressed by the following equation: y = 1.07x + 1.14 mU insulin/L.

KEY WORDS: insulin; enzyme immunoassay; monoclonal antibodies; evaluation. In~oducfion

he clinical picture of diabetes mellitus can be characterized in terms of absolute or relative insulin T deficiency. Determination of insulin secretion characteristics provides an insight into the type of defect in glucose homeostasis and permits clinical classification into insulin-dependent (type I) and non-insulin-dependent (type II) diabetes mellitus (1). Since the first description of radioimmunoassay (RIA) for the determination of insulin in the late 1950s by Berson and Yalow (2), significant improvements have been made in reliability and practicability of the measurement of this analyte. Although RIAs continue to be the most widely used assay technique, alternative labels have been introduced in insulin immunoassays in order to avoid the disadvantages inherent with the application of radio isotopes. Use of enzyme labels with photometric (3), fluorometric (4), and luminometric (5) measurement of activity in both competitive and sandwich assay techniques (4,5) have been reported.

Correspondence: Prof. Dr. med. W. Bttrgi, Department of LaboratoryMedicine, Kantonsspital Aarau, CH-5001 Aarau/ Switzerland. Manuscript received August 17, 1987; revised March 9, 1988, accepted April 6, 1988. CLINICALBIOCHEMISTRY, VOLUME 21, OCTOBER 1988

Those procedures, however, are tedious depending oil the number of incubation steps and the bound-free performance. Furthermore, they suffer from additional drawbacks if they use polyclonal antibodies (6). In contrast, the application of monoclonal antibodies offers a number of advantages associated with their homogeneity, specificity and availability. The present communication describes a rapid and convenient one-step sandwich enzyme immunoassay for insulin quantification in human serum using two monoclonal antibodies. A preliminary report of this test has recently been published (7). Materials a n d m e t h o d s

REAGENTS Enzymun-Test® Insulin monoclonal test kit was ob.tained from Boehringer Mannheim GmbH, D-6800 Mannheim, FRG. The package contains incubation buffer (40 mmol/1 phosphate, pH 7.0); anti-insulin-antibody horse radish peroxidase (HRP) conjugate (>30 U/l); substrate buffer (100 mmol/1 phosphate-citrate, pH 4.4, 3.2 mmol/1 sodium perborate); di-ammonium 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulphonate) (ABTS ®) (1.9 mmol/1); 5 insulin standards in bovine serum, their concentrations ranging from 0 to 260 mUi 1; 100 plastic tubes coated with anti-insulin-antibody (binding capacity > 100 ng insulin per tube). The work~ ing solutions are prepared according to the manufac. turer's instructions. Human monocomponent insulin (cat. No. 400), hu. man C-peptide (cat. No. 820), porcine insulin and proin-. sulin (cat. Nos. 480 and 710) and bovine insulin (cat~ No. 500) were obtained from Novo Industry, Denmark. Human proinsulin was a kind gift of Ely Lilly, USA. To determine the assay specificity bovine serum wa:~ spiked with these antigens in concentrations of 100 .... 2000 pmol/1. The standards have been calibrated against the In. ternational Reference Preparation of Insulin, humar: for Immunoassay 66/304. APPARATUS

The assay was performed on Enzymun-Test® Syster:~ ES 22, a modular batch analyzer for semi-automated~ 31L

BORGI, BRINER, FRANKEN, AND KESSLER analysis (Boehringer Mannheim GmbH) (8). The system includes a pipetting and rinsing unit with a transport chain, a filter photometer, a flow-through cuvette, and a desk-top computer (Epson HX20).

1.4

SPECIMENS

Venous blood was drawn in Vacutainer ® tubes and allowed to clot. The serum obtained after centrifugation was stored at minus 80°C until used for this study. There was no special selection of the patients. For the assessment of intraassay precisions and daily quality control, the control sera Precinorm ® IM, Precipath ® IM (Boehringer Mannheim GmbH, cat. Nos. 649945 and 649937), CON 6 immunoassay Tri-Level control (Diagnostic Product Corporation) and Omega ® Ligand control sera (Cooper Biochemical) were used.

1.2

1.0

E f"

.8

O

¢i .6

Assay procedure .4

Samples and standards (100 ~l) in duplicate were incubated with 1 ml anti-insulin-conjugate solution in the coated tubes for 2 h at room temperature. The tube contents were aspirated and the tubes rinsed 3 times with tap water. 1000 ~1 of substrate buffer containing ABTS ® was added to the tubes and incubated for 1 h at room temperature. Absorbance was read at 405 n m and insulin concentrations were calculated from the calibration curve. A new calibration curve for each assay was constructed.

Evaluation protocol and statistics Intraassay and interassay precision in h u m a n sera, control sera, and standards were calculated and expressed in m e a n values (~), standard deviations (sd), and coefficients of variation (CV). The lower detection limit of the assay was determined by performing 20 single measurements of the zero-standard and calculating the average of the absorbance plus the two-fold and three-fold standard deviations (9). The comparison with the commercially available radioimmunoassay was carried out using the biometric regression procedure established by Passing and Bablok (10).

.2

o

~ 0

50

100

150

200

250

Insulin concentration [mU/I]

Figure 1--Standard curve of Enzymun-Test® Insulin. Each point represents the mean of 10 single determinations on each standard solutions containing 0, 17.5, 78.5, 164.5, and 266 mU/L of human insulin. The vertical lines indicate +-2 SD.

8-_ .

_ •

Results

e

0

•

0

_

0

Figure i represents the standard curve, which shows a linear relationship between OD and insulin concentration of at least 266 mU/L. The precision of all standards was assayed in one r u n (n = 10). The absorbances of the zero standard showed a CV of 8%, the standards with the concentration of 17.5, 78.5,164.5, and 266 mU/ L were measured with CV's of 3.9, 1.6, 3.4, and 3.8% respectively. The results of the intraassay precision are presented in Figure 2. H u m a n serum pools and commercial control sera spanning the range from 8.5 to 218 m U insulin/L have been determined. With the exception of two sera (CV's of 7%) all specimen have been measured with CV's less t h a n 4.5%.

Figure 2--Intraassay precision of Enzymun-Test® Insulin. Each point represents the mean of 10 single determinations. The insulin concentration is shown on the x-axe, the coefficient of variation are demonstrated on the y-axe.

312

CLINICAL BIOCHEMISTRY, VOLUME 21, OCTOBER 1988

0

I 50

0

I 100

INSULIN CONCENTRATION

//'

I 220

(mU/I)

• HUMAN SERA o CONTROL SERA

INSULIN EIA TABLE 1

TABLE 3

Interassay Precision of Enzymun-Test®Insulin

Sensitivity of Enzymun-Test® Insulin

Control sera

N series duplicates

mU/1

sd

CV%

BM 1 DPC BM 2

16 13 16

17.6 22.2 107.1

1.83 1.82 5.96

10.4 8.2 5.6

Experiment No. 1 2 3 4 5

Note: The serial determinations were performed on consecutive working days.

The interassay precision was determined from duplicate determinations of three commercial control sera, over a period of 13 to 16 days (Table 1). Mean values of 17.6, 22.2, and 107.1 m U / L were obtained and the corresponding CV's were found to be 10.4, 8.2, and 5.6%. Dilution experiments using h u m a n sera of different insulin concentrations, control serum, and four standard solutions (Figure 1) revealed the results shown in Table 2. The lowest recoveries were found in the experiments with h u m a n serum diluted with control serum, the other experiments resulted in recovery values between 95.2 and 104.5% which confirms the linearity of the assay procedure. The lower detection limit was assayed in five identical experiments at different times. These determinations revealed m e a n variations of the zero standards corresponding to values between 2.4 and 3.7 m U / L (Table 3). The highest ~ + 3 sd value (3.7 mU/L) resulted from experiment No. 5. Thus, it appears reasonable to assume a lower detection limit of 3.7 mU/L. The studies on the specificity of the insulin assay demonstrated a reactivity with porcine insulin approximately three fold higher t h a n t h a t of h u m a n insulin (Figure 3). H u m a n and porcine proinsulin as well as bovine insulin were found to be almost three fold less reactive t h a n h u m a n insulin. It should be added t h a t

Absorbances zero standard X ( n = 20) sd 0.025 0.034 0.033 0.032 0.033

0.027 0.037 0.037 0.036 0.037

Concentration Insulin [mU/1] X + 2sd X + 3sd 1.6 1.8 1.9 1.9 2.35

2.4 2.8 2.9 3.0 3.7

Note: Each of 5 identical experiments carried out at different times comprised 20 intra-assay insulin determinations of the zero standard.

no immunoreactivity was obtained with h u m a n C-peptide. A correlation study between Enzymun-Test ® Insulin and a commercially available radioimmunoassay was performed assaying 60 h u m a n sera. The correlation was shown to be highly significant (r = 0.96; y = 1.07x - 1.14 (Figure 4). Discussion

The present paper describes a new test procedure for the quantitative determination of h u m a n serum insulin with a total incubation time of 3 h at room temperature. The assay features a one-step sandwich enzyme immunoassay using two different monoclonal antibodies (7). One of these antibodies is bound to the inner tube wall, while the other is conjugated with horse radish peroxidase and solubilized in buffer. The monoclonal antibodies have been shown to be directed against two different epitopes of insulin (11). The mechanization offered by the automatic system Enzymun-Test ® System ES 22 with the computer controlled incubation, pipetting and rinsing steps as well

TABLE 2

Recovery in Dilution Experiments Sample dilution steps Human serum/human serum 14.2 mUll/64.4 mU/1 1 + 0.5 1+ 2 1+ 5 Human serum/human serum 37.3 mU/1/186 mU/1 1 + 0.5 1+ 2 1+ 5 Human serum/control serum 20.7 mU/1/103.6 mU/1 1+ 1 1+ 3 1+ 5 Standards c/d 1 + 1 b/e 1 + 1 d/e 1 + 1

Expected values [mU/1]

Measured values [mU/1]

Recovery [%]

30.9 47.7 56.0

29.6 46.3 54.9

95.8 97.1 98.0

86.9 136.4 161.2

90.8 141.7 165.0

104.5 103.9 102.3

62.2 82.9 89.8

57.8 75.8 89.5

92.9 91.4 99.7

121.5 141.8 215.3

126.0 144.0 205.0

103.7 101.6 95.2

Note: Recovery Studies of Enzymun-Test ®Insulin. The standard solutions used were the same as in Figure 1.

CLINICALBIOCHEMISTRY, VOLUME 21, OCTOBER 1988

313

BIJRGI, BRINER, FRANKEN, AND KESSLER

2.25

Porcine Insulin

2.0 1.75

Human Insulin

1.5

E

etO O q¢

1.25

d 6

1.0

Porcine Proinsulin j

0.75

Human Proinsulin Bovine Insulin

0.5

as evaluation of measured absorbances allows assay of 150 samples in one r u n performed by one person. In separate experiments three different sera were analyzed in duplicate by the m a n u a l procedure. The precision was found to range between 4.1 and 6.9% CV. When the same sera were analyzed by the ES 22, the corresponding CV's were from 2.7 to 4.9%. With the present sandwich enzyme immunoassay, lower detection limit of 3.7 m U / L was found. This value represents a m e a n of five separate experiments. Values determined by the enzyme immunoassay (y) and by double antibody radioimmunoassay (x) correlated highly (r = 0.96, y -- 1.07x - 1.14; p < 0.05, N = 60) which demonstrates the reproducibility in different techniques. References

Figure 4--Comparison of Enzymun-Test® Insulin (y) with RIA insulin (x) in 60 human sera.

1. Rausch-Stroomann JG. Die Bestimmung der Insulinsekretion und ihre diagnostische Bedeutung. Diagnose und Labor 1986; 36: 133-9. 2. Berson SA, Yalow RS. Assay of plasma insulin in human subjects by immunological methods. Nature (London) 1959; 184: 1648-9. 3. Yoshioka M, Taniguchi H, Kawaguchi A, et al. Evaluation of a commercial enzyme immunoassay for insulin in human serum and its application. Clin Chem 1979; 2511: 35-8. 4. Imanaga M, Hashida S, Ishikawa E, et al. A highly sensitive sandwich enzyme immunoassay for insulin in human serum developed using capybara anti-insulin FA'horseradish peroxidase conjugate. Analytical Letters 1983; 16: 1509-23. 5. Strasburger CJ, Fricke H, Gadow A, Klingler W, Wood WG. Luminescence immunoassays--Alternativen zum Radioimmunoassay. Artzl Lab 1983; 29: 75-82. 6. Hubbuch A. Enzyme-immunoassays: a review. In: Progress in clinical biochemistry and medicine. Vol. 4. Pp. 109-143. Berlin, Heidelberg: Springer Verlag, 1986. 7. Franken N, Haug H, Deeg R, Wahlefeld AW. A new onestep enzyme immunoassay for insulin. Clin Chem 1986; 32: 1066. 8. Meyer HD, Braun SL. Mechanisierung von heterogenen Enzym-immunoassays mit einem modular aufgebauten Batch-Analyzer. Arztl Lab 1985; 31: 308-12. 9. Kaiser H. Zum Problem der Nachweisgrenze. Z Analyt Chem 1962; 209: 1-18. 10. Passing H, Bablok W. A new biometric procedure for testing the equality of measurements from two different analytical methods. J Clin Chem Clin Biochem 1983; 21: 709-20. 11. Schroer JA, Bender T, Feldmann RJ, Kim KJ. Mapping epitopes on the insulin molecule using monoclonal antibodies. Eur J Immunol 1983; 13: 693-700.

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CLINICAL BIOCHEMISTRY, VOLUME 21, OCTOBER 1988

0.25 ~ 120

C-peptide 500

1000

1500

2000 pMol/I

Figure 3--Crossreactivity of different insulins with Enzymun-Test ®Insulin.

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50I,u 4 0 -

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2010-

0

I I I I 10 20 30 40

I I I I 50 60 70 80

i 90 100

RIA INSULIN [mUll]