Reversible immobilization of an antibody with a thiol-substituted sorbent

Analytica Chimica Acta, 197 (1987) 229-237 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

REVERSIBLE IMMOBILIZATION OF AN ANTIBODY THIOL-SUBSTITUTED SORBENT Application to Enzyme Immunoassays

J. L. BOITIEUX*,

R. GROSHEMY

WITH A

and D. THOMAS

Laboratoire de Technologie Enzymatique, BP 233,60206 Compikgne (France)

UniversitG de Technologie

de Comp@gne,

F. ERGAN Institut de Recherche en Biotechnologie, Conseil National de Recherche du Canada, HGpital Royal Victoria 687 avenue des Pins ouest, Montreal H3A 1Al (Canada) (Received 10th March 1986)

SUMMARY A new sensor is described for a specific protein; enzyme-linked immunosorbent assays are combined with electrochemical measurements. Specific sensors are proposed based on immobilization of an antibody by ligands onto artificial protein-based membranes, combined with a computerized system, for the determination of various antigens or haptens. Specific antibodies labelled by ribonuclease are reversibly bound to the membrane by using cysteine as the ligand. Two enzymes are used: ribonuclease is used for reversibly linking the immune-complex to the insoluble matrix via disulfide bridges; p-Dglucose oxidase is used for labelling the antigen. The measurement consists of an immunological process and an enzymatic reaction. The protein-based membrane activated with thiol groups is fixed over an oxygen electrode. After incubation of the free antigen and the antigen labelled with glucose oxidase with specific antibodies linked by ribonuclease, the reaction medium is introduced in a continuous flow cell. The oxygen consumption by the enzyme reaction is measured on-line with the electrode in contact with a standard glucose solution. This response is correlated to the antigen concentration of the sample. The signal is directly proportional to the oxygen consumption. The reproducibility with use of the same membrane is <5%. Cleavage between the immuno-complex and the thiolcontaining membrane by dithiothreitol is 98% complete.

Enzyme immunoassay has developed considerably in recent years especially with respect to the design of new sensors [l, 21. Other solid-state electrochemical sensors such as the ion-sensitive field effect transistor (ISFET) and chemical field-effect transistor (CHEMFET) have also been described [ 3, 41. The rates of formation and dissociation of the antigen/antibody complex are relatively small, so an analytical system has been considered which will allow the assay of almost any biologically active material without having to use another support, the specific antibodies being linked reversibly to electrochemical sensors. Some approaches to this technology have already been studied in the area of affinity chromatography [5,6]. The present paper concerns a new sensor for a specific protein that has 0003-2670/87/$03.50

0 1987 Elsevier Science Publishers B.V.

230

been developed by incorporating enzyme immunoassay with electrochemical measurements. The originality of this work consists in fixing a specific antibody on a protein-based membrane via a bridging group to study the reversibility of this immuno-complex. This application derives from biospecific affinity chromatography applied to the determination of antigens or haptens. The reversible immobilization of antigen/antibody complexes labelled with glucose oxidase for development of an automatic computerized system is described. Sulfhydryl groups (from cysteine) are immobilized on the surface of a protein-based membrane by means of a gelatin solution. Cysteine was fixed on a polypropylene film coated with pig-skin gelatin polymerized with glutaraldehyde. It gave excellent reversible fixation of reduced ribonuclease (RNase)-labelled rabbit antibodies. The active membrane is fixed over an oxygen electrode in the measuring cell. To illustrate the application of this device, rabbit IgG is studied as a model antigen. The reaction medium is injected into the cell, after incubation of the antigen-coupled glucose oxidase and of the antigen to be determined with the corresponding antibodies coupled to a pre-established concentration of reduced RNase. The activity of the glucose oxidase is measured with a standard glucose solution. The amount of linked enzyme is inversely proportional to the concentration of antigen in the medium. After the measure-

PROTEIN

"B"BRANE

IMMUNOLOGICAL

+ CYSTEINE

COMPETITION

+

-bFORMATION

OF DISLTLFIDE CYSTEINE

REA‘TU

AND

RED”CSD

BONDS

BETWEEN

RNase

DESORPTION +

-@

ACTIVATED

AGENT (DTT) AND WASHING WITH PHOSPHATE B"FFBR

l

MEMBRANE MEASUREMEN

CYSTEINE

w +

wGL"COSE

REDUCED RNaSe RABBIT IgG GOAT ANTIBODIE.

OXIDAS LABELLED GOAT ANTIBODIES

Fig. 1. Sequence of events for the competitive assay of goat IgG. Ab: Antibody, Ag: Antigen.

231

ment, the disulfide bonds that have been formed between the immunocomplex coupled to the reduced RNase and the cysteine are broken by injection of a dithiothreitol (DTT) solution. The principle of the system is shown in Fig. 1. EXPERIMENTAL

Apparatus and reagents The measuring apparatus consisted of an oxygen electrode (Clark electrode, Radiometer G-5404610) and oxygen analyzer (Radiometer pHM-71). Control of this circuit were achieved by an Apple-II microcomputer and a graphics recorder connected to the analyzer. This apparatus has recently been described [7]. A schematic representation of the measuring system is shown in Fig. 2. The gas-selective membrane was a modification of the commercially available model. Glucose oxidase (E.C. 1.1.3.4, type II, specific activity 18400 U g-l), RNase and mercaptoethanol were from Sigma Chemical Co. Pig-skin gelatin came from the Rousselot Laboratory (Ribecourt 60400, France). Glutaraldehyde and other reagents were of analytical grade (Merck). Rabbit IgG (lyophilized) and anti-rabbit IgG (goat) were obtained from Miles Laboratories.

Fig. 2. Schematic diagram of the immunosensor for antigen or antibody determination.

and measuring cell, with a flow system

232

Methods Preparation of the modified protein carrier. Preliminary studies of immobilization of specific antibodies on different types of protein carriers showed that the greatest amount of antibodies was retained on a membrane of pigskin gelatin [8]. This gelatin, when acid-treated, has on its surface a significant number of free amine groups. Pig-skin gelatin was dissolved in 100 ml of 0.1 M phosphate buffer, pH 6.8, by heating for 1 h in a water bath at 50°C. A portion (1 ml) of this solution was spread on a polypropylene film (7 cm X 5 cm, ca. 0.05 mm thick to obtain good mechanical stability). The polypropylene was treated beforehand with lauryl sulfate (0.5% w/v in phosphate buffer) to increase the adhesion of the gelatin. The film was left at room temperature until it was dry. This membrane was immersed in glutaraldehyde (1% in 0.01 M phosphate buffer, pH 5.2) for 5 min. The excess of glutaraldehyde was washed out thoroughly with distilled water. The activated membrane was immersed in an aqueous 0.2 M cysteine solution for 1 h. The majority of the amine groups of cysteine reacted with reactive groups of the membrane. The sulfhydryl groups on each membrane were determined spectrophotometrically as described by Ellman [ 91 after different incubation times. The concentrations of sulfhydryl groups fixed on the membrane are shown in Table 1. Preparation of the IgG/RNase. Three coupling methods were studied, the one-step glutaraldehyde technique described by Avrameas and Guilbert [lo] , the carbodiimide technique [ll] and the p-benzoquinone technique [12]. In the glutaraldehyde technique, 4 ml of reduced RNase (2.4 mg ml-‘) was incubated for 2 h at room temperature with 5 mg of IgG, 0.05 ml of a 1% glutaraldehyde solution and 0.5 ml of 0.1 M phosphate buffer, pH 6.8. After dialysis overnight at 4°C against the phosphate buffer, the conjugate was precipitated by adding ammonium sulphate. Further fractionation on Sephadex G-25 of the dialysed conjugate, equilibrated with 0.01 M phosphate buffer, pH 6.8, was used to eliminate the free enzyme. The carbodiimide technique is derived from the method used by Nakane et al. [ll] for coupling acid phosphatase to IgG. A 4-ml portion of reduced RNase solution (2.4 mg ml-‘) was incubated with 5 mg of goat IgG and 20 mg of l-ethyl-2-(3-dimethylaminopropylcarbodiimide) in 0.5 ml of distilled water. This was followed by dialysis against 0.5% sodium chloride solution at room temperature. The conjugates obtained were isolated by chromatography on Sephadex G-25. The p-benzoquinone technique consisted first TABLE

1

Dependence of sulfhydryl cysteine concentration Incubation Cysteine SH cont.

time (M)

group

concentration

30 min 0.1 0.231

(pm01

ml-’

cm-2) on incubation

60 min 0.2 0.478

0.01 0.049

0.1 0.274

time and

90 min 0.2 0.572

0.01 0.09

0.1 0.463

0.2 0.567

0.01 0.08

233

of treatment of an antibody with an excess of p-benzoquinone, removal of excess of reagent, and coupling of the “activated” IgG to a marker substance. To 3 mg of rabbit IgG in 0.7 ml of 0.15 M sodium chloride were added 10 ~1 of 1 M phosphate buffer, pH 6.0, and 0.2 ml of ethanol containing 6 mg of p-benzoquinone. The preparation was kept at room temperature in the dark for 1 h and passed through a Sephadex G-25 column (0.9 X 4 cm), equilibrated with 0.15 M sodium chloride. The first coloured fraction containing the activated antibody was collected in a volume of ca. 1 ml. To this first fraction (1 ml), 0.2 ml of a 1% RNase solution in 0.1 M phosphate buffer, pH 6.8, was added, followed by one-tenth of the volume of 1 M carbonate/hydrogencarbonate buffer, pH 9. This mixture was left at 4°C for 15 h and the reaction was then stopped by addition of one tenth of the volume of 1 M lysine, pH 7.5. After 4 h, the mixture was dialysed overnight at 4°C against 0.1 M phosphate buffer, pH 6.8, and centrifuged for 20 min at 40 OOOg. The solution was filtered through a sterile Millipore membrane (0.22 pm) and an equal volume of glycerol was added. Preparation of glucose oxidase-labelled goat antibody. A lo-mg portion of glucose oxidase (16.1 U mg-‘) was dissolved in 1 ml of a 1% glutaraldehyde solution in 0.1 M phosphate buffer, pH 6.8. The mixture was left for 18 h at room temperature. Glucose oxidase was purified by gel filtration on a Sephadex G-25 fine column (0.9 X 60 cm) equilibrated with 0.15 M sodium chloride. The fractions of the first peak containing the glucose oxidase were pooled and concentrated with polyethyleneglycol 2000. To the glucose oxidase solution were added 2 mg of goat IgG and 0.5 ml of 0.1 M carbonate/ hydrogencarbonate buffer, pH 9.5. After a 24-h incubation at 4”C, the functional groups still free were inactivated by 0.5 ml of 0.2 M lysine solution, pH 6.8. The glucose oxidase/antibody conjugates were purified by fractionation on Sephadex G-200 equilibrated with 0.01 M phosphate buffer, pH 6.8, to eliminate the free enzyme. The fractions of the first peak were pooled and distributed in sterile 0.2-ml portions. This solution could be stored at 4°C for several months without significant loss in activity. Assay. Discs (lo-mm diameter, ca. 0.05 mm thick) were cut from the thiolated protein membrane and fixed round the tip of the oxygen electrode in the measuring cell. After incubation of RNase-labelled rabbit IgG (50 ~1 of conjugate diluted 10 times) for 1 h, the mixture was pumped into the measuring flow cell for 5 min. The immuno-complex thus bound to the thiolated membrane was washed with 0.01 M phosphate buffer, pH 6.8, for 30 s. The marker glucose oxidase activity was measured in the presence of a standard glucose solution (5 g 1-l). The consumption of oxygen estimated by the sensor is proportional to the enzyme activity retained on the active membrane and consequently to the antigen concentration. The presence of dithiothreitol (25 nM) enables the disulfide bonds between the RNaselabelled rabbit IgG and the cysteine fixed onto the protein membrane to be broken. The cell was first filled with phosphate buffer (0.01 M, pH 6.8) for 15 s to obtain a steady-state signal which represented the zero reference potential.

234

Spectrophotometric determination of reduced RNase/rabbit IgG complexed to glucose oxidase antibody conjugates. Portions (10 ~1) of RNase/ rabbit IgG solution diluted with 90 ~1 of phosphate buffer (0.01 M, pH 6.8) were incubated with 50 ~1 of glucose oxidase conjugate (diluted 1 + 9) for 2 h at room temperature on the thiolated membrane. After washing, the disks were immersed in 2.9 ml of 5 X lo4 M 3,3’-diaminobenzidine solution containing 0.1 mg of peroxidase, and 100 ~1 of 5 g 1-l glucose solution. Enzymatic activity was monitored at 450 nm. Reduction of RNase. The RNase (10 mg) and 0.48 g of urea were mixed with 0.02 ml of mercaptoethanol in 0.5 ml of distilled water. After dissolution, the pH was adjusted to 8.5 with aqueous 5% (w/v) trimethylamine solution. The mixture was incubated for 4 h at room temperature and the pH was adjusted to 4 with 1 M acetic acid. The reduced RNase obtained was purified by chromatography on Sephadex G-25 equilibrated with 0.1 M acetic acid. The fractions of the first peak were pooled and distributed in sterile 0.2-ml fractions. They could be stored at 4°C for several days without significant loss of activity. RESULTS

Two types of disulfide bridges formed via cysteine bound to the gelatin membrane over the oxygen electrode were studied. The bridges were either with rabbit IgG conjugates or with reduced RNase/IgG conjugates. These reactions are illustrated in Fig. 3. In order to improve this reversible binding, attempts were made to couple rabbit IgG (unreduced) to an enzyme which contains potentially more sulfhydryl groups than the membrane. RNase was chosen; it has 4 disulfide bridges (between amino acids 26 and 84, 40 and

,COOH

SH-CH -CH

*

‘NH*

Fig. 3. Schematic representation system for goat anti-rabbit IgG.

b

and biochemical structure of the reversible bienzymatic

235

95, 65 and 72 and 58 and 110) and therefore, after reduction, 8 sulfhydryl groups. Three techniques were evaluated for coupling RNase to IgG. The conjugate obtained by glutaraldehyde coupling is named conjugate A. Conjugate B was obtained by coupling previously reduced RNase to IgG by the carbodiimide technique. Conjugate C was prepared with unreduced RNase binding via the carbodiimide technique. The concentrations of free sulfhydryl groups found for l-ml solutions of each of these conjugates are shown in Table 2. The conjugate prepared from unreduced RNase showed a higher concentration of sulfhydryl groups than those prepared with previously reduced RNase. This is certainly due to the fact that there is a concomitant reduction and partial denaturation of IgG during the coupling with RNase. A dramatic decrease of sulfhydryl group concentration was observed after a 15-day storage, which decreased further after 30 days for each type of conjugate. Reduced RNase that had not been coupled to IgG had an average sulfhydryl concentration of 7 X lo-’ mol ml-‘, which is more than in reduced IgG (2.6 X lo* mol ml-‘). Immunological tests through immunodiffusion of these immuno-complexes showed that there was no alteration of the immunological sites. To evaluate these properties, double diffusion tests were made on agar-coated slides by the method of Campbell et al. [13]. They showed an important precipitation reaction between glucose oxidase-labelled goat antibodies, anti-rabbit IgG and conjugate A. For conjugates B and C the precipitation reactions were less important. Calibration graphs were obtained for different dilutions of each glucose oxidase. Labelled goat antibody conjugates were measured amperometrically with the oxygen electrode. This electrode was coated with the thiolated membrane which had been saturated with RNase-labelled rabbit IgG. Figure 4 shows that the variation of the electrode signal after 1 min for the different conjugates was proportional to the enzyme activity, and consequently to the antigen concentration, i.e., the goat antibodies. Measurements of the glucose activity of the conjugate were reproducible for the same membrane, with a relative standard deviation of 0.3% (n = 7) at a rate of oxygen consumption of 1.46 mm Hg s-‘. Studies done through the spectrophotometric measurement of the glucose oxidase activity of these immuneTABLE 2 Free sulfhydryl bonds of RNase as achieved by various conjugation techniquesa Time

On preparation After 15 days After 30 days

SH concentration A 102.4 29.4 14.7

%ee text for identification of conjugates A-C.

(rmol ml-‘) B 6.34 1.98 1.91

C 29.4 17.2 7.35

236

oz-

Fig. 4. Effect of dilution of goat anti-rabbit IgG labelled with glucose oxidase, on the electrode signal for the conlugates produced by the three methods: (1) conjugate A; (2) conjugate B; (3) conjugate C.

complexes with reduced IgG showed that the amount of reduced IgG immobilized on a membrane was 35%. Amperometric measurements of the glucose oxidase activity of the immuno-complexes showed that 35% of conjugate A was fixed on the membrane; 17% of conjugate B and 42% of conjugate C. The results are summarized in Table 3. Conjugates A and C gave the best overall results, better than those obtained when RNase reduction increased the binding of the IgG/RNase complex to the thiolated membrane. The choice between conjugates A and C is difficult; conjugate C seems better because it generally showed higher glucose oxidase activity. The breaking of the disulfide bridges formed between cysteine bound to the protein membrane on the electrochemical sensor and RNase-labelled rabbit IgG was achieved by 25 mM dithiothreitol. The rate of disulfidebridge breaking was such that 72% of the immuno-complex was uncoupled TABLE 3 Immunological properties of different conjugates (for identification, RNase/IgG conjugate Presence of free thiols Immunodiffusion test Glucose oxidase activity (mm Hg s-‘) RNase/IgG conjugate bound to membrane (%) Spectrophotometry Amperometry a+ + + very positive, + + positive, + slightly positive.

A +++ Z7 35 35

see text)s B + + 0.42

C + + 0.70

17 17

42 44

237

after 2 min, 98% after 7 min and 100% after 15 min. These results are very encouraging. The electrode can be used continuously over many months without apparent loss in response. Repeated assays (n = 7) done with the same sample of goat antibody and the same membrane for each type of RNase/IgG complex (coupling and uncoupling) in the same run gave standard deviations of ca. 5%. However, the variation between different membranes was much higher (20%), but this is not a handicap, except that it necessitates recalibration after each membrane change. This computerized enzyme immunosensor allowed 20 sample measurements per hour. The measurement time included the washing step between samples, the sampling step and the oxygen measurement. Conclusions The use of reversible systems based on disulfide-bridge formation and rupture in order to establish competitive enzyme immunological assays of antigenie substances by means of amperometric measurements has given encouraging preliminary results. The measurement technique is not yet fully tested but a fully computerized system has been achieved, based on the indirect reversibility of immuno-complexes which will allow for decreased measuring time and provide a large number of measurements with the same protein carrier, without having to change the membrane after each assay. This work 858006).

was made possible

with the help of INSERM

(Contract

no.

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