WO1987002464A1

WO1987002464A1 - Enzyme-labelled assay

Info

Publication number: WO1987002464A1
Application number: PCT/GB1986/000622
Authority: WO
Inventors: Anthony Edward George Cass; Wyndham John Albery; Andrew Ramsay Hopkins
Original assignee: Genetics International (Uk) Inc.
Priority date: 1985-10-14
Filing date: 1986-10-14
Publication date: 1987-04-23

Abstract

An enzyme-labelled assay in which the enzyme catalyses a reaction of at least one reactant at a first location to give at least on e product, and the reactant or the product is determined electrochemically in a flow system at a second location downstream of the first location.

Description

ENZYME-LABELLED ASSAY

Background of the Invention

This invention relates to enzyme-labelled assays.

More particularly, but not exclusively, the present invention relates to techniques and equipment useful in immunoassays. The invention is especially concerned with immunoassays of use for instance in a wide range of diagnostic or investigative techniques for humans or animals, or for example in the investigation and monitoring of food chemistry or process chemistry.

An example of a known enzyme-labelled assay is the heterogeneous immunoassay known as the ELISA (enzyme-labelled immunosorbent assay) technique. In the commonly adopted ELISA sandwich procedure, the technique will typically involve three steps, separated by washes and rinses:

(a) immobilisation of a suitable antibody on a support surface, such as polymer beads or possibly the walls of a plastics vessel or recesses in a plastics dish; (b) contacting the surface carrying immobilised antibody with the sample to be investigated, whereby a specific analyte in the sample can bind to the antibody in a proportion dependent on the analyte concentration; and (c) further contacting the surface with an antibody labelled with an enzyme, this enzyme-labelled antibody binding only to the antigen and thus providing at the surface the enzyme in a measurable concentration corresponding to the analyte concentration in the sample. Usually, in the known procedures, the enzyme measurement is effected with a colour-change on formation of product.

The sandwich technique has certain limitations, most especially in that it can only be used with analyte molecules having at least two epitopes, so as to react both with the immobilised antibody and with the enzyme-labelled antibody.

m the alternative ELISA competitive procedure for assay of a specific analyte, a known concentration of enzyme-labelled specific analyte is added to a sample which is then contacted with a surface supporting an known concentration of antibody, whereby labelled and unlabelled analyte compete proportionately for binding sites, and a subsequent measurement of enzyme concentration will indicate the labelled/unlabelled ratio.

Objects of the Invention

The present invention is concerned with the provision of improved enzyme-labelled assays. More specically, a particular object of the present invention is an improved ELISA technique. Other objects include equipment and reagents for novel enzyme-labelled assays.

Summary of the Invention

The present invention provides an enzyme-labelled assay in which the enzyme catalyses a reaction of at least one reactant at a first location to give at least one product, and the reactant or the product is determined electrochemically in a flow system at a second location downstream of the first location. The present invention allows the use of electrochemical procedures for detecting the reactant or the product as an electroactive species, and thus for detecting the enzyme which is present. In turn this allows the determination of an analyte in a qualitatative or quantitative sense. A relatively sensitive and fast immunoassay in the nanomolar range can be achieved.

By virtue of the enzyme at the first location, the reactant in the flowing stream is converted to product to an extent which can be determined electrochemically at the second location. The reactant and the product are selected so that they have different electrochemical activities. If desired, both the reactant and the product can be electrochemically determined downstream of the first location, but usually it will be the product or a component thereof which is determined.

Preferred Embodiments of the Invention

The assay of this invention is typically an immunoassay. The assay suitably involves a sandwich or competitive procedure. Thus, in a preferred aspect, the assay of this invention is a sandwich ELISA or a competitive ELISA.

For a sandwich assay, a mono- or polyclonal antibody is immobilized at the first location, the sample is contacted with the immobilized antibody, enzyme-labelled antibody is then contacted with the system, and the reactant is flowed past the first location and to the second location for the electrochemical determination at the second location.

For a competitive assay, the sample and an enzyme-labelled analyte are contacted with immobilized antibody at the first location, and the reactant then flowed past the first location and to the second location for the electrochemical determination at the second location.

The nature of the reaction catalysed by the enzyme is not critical, and for example the enzyme can be an oxidoreductase, protease, amidase or esterase. By way of illustration, an enzyme can be adopted where the at least one reactant can be oxidised or reduced. Oxidavtion of a reactant is especially suited for the present invention. Examples of suitable enzymes then include catalases and peroxidases. In certain embodiments, the reactant and product form a redox couple, giving reversible reduction and oxidation.

For enzyme reactions involving oxidation, the oxidant preferably includes hydrogen peroxide, which conveniently can be electrochemically generated. Electrochemical generation of hydrogen peroxide can be achieved efficiently and reproducably using an electrode upstream of the first location. For example, hydrogen peroxide can be generated at the surface of a reticulated vitreous carbon electrode upstream of the first location and connected to a galvanostat.

The reaction system may include the oxidant such as hydrogen peroxide, and a reduced form of substrate.

Suitable substrates are electroactive compounds which readily transfer electrons. Such compounds are widely available.

The prefered substrate in the present invention is a water-soluble ferrocene, such as a ferrocene mono-carboxylic acid. Ferrocene compounds have the advantage that it is readily possible to select suitable compounds where the oxidised form is easily detected by reduction at a potential where little else interferes. In an alternative embodiment of the present invention, the reactant system comprises hydrogen peroxide and Fe(CN) (reduced substrate).

The first location can for instance comprise a bed of immobilised enzyme, or in one preferred aspect the first location comprises the disc of a ring-disc electrode, giving faster and more sensitive assays. With ring-disc electrodes, the final detection step of the assay can be considerably shorter compared to normal ELISA assay times.

The electrochemical determination is conveniently effected using a wall jet electrode, which is especially useful with the ring-disc electrode.

More generally, for use in the assays of this invention there is provided an electrochemical cell comprising in use;

(a) a moving stream of fluid containing at least one reactant;

(b) a first location in the stream at which an enzyme can catalyse a reaction of the reactant to give at least one product;

(c) a second location in the stream, spaced apart from the first location and at which the concentration of one or both of the reactant and product is determined, thereby enabling estimation of the catalytic activity of any enzyme present at the first location.

Typically the second location comprises a region confined within an annulus surrounding the first location, as for example where the apparatus comprises a ring-disk electrode, in which an enzyme is located at the first location upon the disk and the second location comprises the ring.

A particular advantage of the use of a ring-disk electrode is that the site of localisation of the enzyme (the first location) and the electrochemical working electrode of the cell (the second location) may be removed from the cell for cleaning, replacement or renewal. Furthermore the aforementioned parts may be removed for treatment of the ring such as to immobilise the enzyme on the disk.

Although there may be certain embodiments in which the disk comprises an electrode, it is prefered that the disk should be electrically isolated and that the enzyme is immobilised thereupon by a specific binding reaction.

Thus in one further aspect of the invention there is provided an electrochemical cell comprising a ring-disk electrode, wherein the disk is so treated to enable the immobilisation of an enzyme, and the ring comprises a working electrode for the estimation, by a suitable electrochemical reaction, of a chemical species which is or forms part of the reactant or the product.

Furthermore, although the invention has been described with particular reference to a ring-disk electrode it should be understood that the invention is not limited to such conficrurations and extends to those configurations in which the flow system is of another type, such as a tube or column. It should further be understood that the ring-disk electrode may be of the rotating or the stationary type. In prefered embodiments a potentiostat is used to measure at the second location the concentration of the reactant or product, as for example the reduced or oxidised form of the substrate.

In order that the present invention may be further illustrated it will be explained by way of example and with reference to the accompanying drawings wherein;

Description of the Drawings

Figure 1 shows apparatus for putting the present invention into practise;

Figure 2 shows a detailed view of an electrode configuration for the apparatus of Figure 1;

Figure 3 is a graph of disc current i_D in microamps less a background current i_bg, against human IgG concentration in nanomoles per dm^-3, measured in Example 2 by a sandwich assay with horseradish peroxidase ("HRP") as enzyme label;

Figure 4 is a graph showing ring current in nanoamperes against concentration of human IgG in nanomoles per dm^-3 as measured in Example 4 by a sandwich immunoassay using a glassy carbon disc of a wall jet ring-disc electrode as the binding location;

Figure 5 shows the results of an investigation in Example 4 similar to that of Figure 4 but with product detection at the disc itself; and

Figure 6 is a graph of the ring current measured in nanoamps before and after addition of known catalase concentrations in nanomoles per dm^-3, measured in

Example 7 on a wall-jet ring-disc electrode. Examples of the Invention

Turning to Figure 1, there is shown a wall-jet electrode assembly (1) comprising a ring-disk electrode (2) disposed within a suitable container (3). A jet of working fluid (4) is directed against the central disk (6) of the wall jet electrode by a nozzle (5). The fluid passes across the annular electrode (7) and out of the container through port (8).

The fluid is supplied under pressure by pump (9) after being drawn from reservoir (10). Before it reaches the electrode assembly (1) it is exposed to a treatment electrode (11) for the generation of hydrogen peroxide. In the embodiment shown, the treatment electrode (11) comprises a reticulated vitreous carbon electrode upstream of the electrode assembly and connected to a galvanostat (12), in order that the hydrogen peroxide may be generated quantitatively.

The apparatus further comprises a potentiostat (13) for the estimation of one or more species at the annular electrode (7) by a suitable electrochemical method and a waste line (14) draining the chamber from port (8).

Figure 2 shows a more detailed view of an electrode suitable for putting the present invention into practice. The electrode (20) comprises a non-conducting reactant (21) having a circumferentially disposed annular electrode (22). A suitable material for the annular electrode (22) is platinum. The electrode (20) further comprises a glassy-carbon disk (23) disposed centrally. It should be noted that although glassy carbon is conducting, it is not necessary for any electrical connection to be made to the glassy carbon disk . In an exemplary method, the disk (23) has an antibody immobilised thereupon by a suitable technique such as the well-known carbodiimide method. The antibody is selected to bind with the analyte which is to be assayed. A drop of sample suspected of containing the analyte is placed on the disk and left for 30 minutes. The electrode is washed in buffer. A drop of antibody (anti-analyte) labelled with peroxidase is placed on the disk and left for 10 minutes. The electrode is washed again in buffer.

A solution of potassium ferrocyanide, K₄Fe(CN)₆, is passed from the reservoir through the treatment electrode and directed at the ring-disk electrode. Hydrogen peroxide is generated at the surface of the treatment electrode (11) and acts as an oxidant on the ferrocyanide. The rate of oxidation of the redox couple is altered in the presence of the enzyme upon the disk.

The extent of alteration of the oxidation states of the substrate is determined downstream of the enzyme. In this instance, the ferricyanide concentration is determined at the ring electrode by reduction to ferrocyanide. In this way, the activity of the enzyme and thus the concentration of the analyte may be determined.

After measurement is complete, the electrode is treated with glycine buffer at pH 2.2 for about 10 minutes, and then washed, enabling further measurements to be made.

It has been determined that the analyte concentration range gives linear results from 0.5nM to 50nM. Example 1

Feasability study:

Horseradish Peroxidase on Sepharose Beads.

Cyanogen bromide-activated Sepharose (0.5g, Sepharose is a trade mark) was washed with HCl (1mM, 200 ml).

Horseradish peroxidase ("HRP", Boehringer) (0.6rag) was dissolved in sodium bicarbonate (0.1M, 2.5ml) containing sodium chloride (0.5M) and added to the washed sepharose. This mixture was tumbled in a sealed container at 4°C overnight. The HRP was immobilised on the Sepharose. It was thereafter filtered, washed with NaHCO₃/NaCl (0.1M/0.5M) and TRIS (0.1M, pH 8.0) to block any remaining active sites. Finally, the beads were washed with three cycles of acetate buffer (0.1M acetate, 0.5M NaCl, pH 4.0) and TRIS buffer (0.1M TRIS, 0.-5M NaCl, pH 8.0) and stored in phosphate (0.1M. pH 7.21) at 4°C. The beads, were assayed for HRP activity by a standard method, for instance as described in U. L. Bergmeyer "Methods in Enzymatic Analysis", Verlag Chemie, Basel 1981.

The beads were then used to form a small packed-bed (volume 0.8 ml) of HRP/Sepharose which was interposed between the two electrodes (11) and (1) of Figure 1. The amount of enzyme present on the bed was known from the HRP assay, and could be varied by diluting the HRP beads with Sepharose CL-4B containing no enzyme.

Phosphate buffer (0.1M, pH 7.21) containing ferrocenemonocarboxylic acid (5mM) was pumped through the system at a controlled flow rate (2.45 ml min^-1). The electrode (11) was galvanostatted at -1.4mA to reduce dissolved oxygen to hydrogen peroxide. Any ferricinium ion produced by the HRP activity in the presence of the hydrogen peroxide was detected downstream at the electrode (1) at -60 mV versus Ag/AgCl. Response times were approximately 100 seconds.

The results for the interrogation of the bed demonstrate that the technique is adequate for the assay of nanomolar quantities of HRP.

Example 2

Sandwich Immunoassay for Human IgG Using the Packed-bed Method

The three components of a sandwich assay were employed as follows:

(a) Agarose-anti-human IgG (Sigma) was diluted into Sepharose CL-4B (final volume 0.8 ml) in phosphate buffered saline ("PBS"; 0.1M phosphate, 0.5M NaCl, pH 7.21) containing Tween 20 (0.02%, Tween is a trade mark) to give an approximate anti-human IgG concentration of 300 nM in the bed.

(b) The bed was exposed to PBS Tween 20 containing varying amounts of human IgG (0 to 9 nM) and left for 24 hours.

(c) The bed was washed with PBS Tween 20 and exposed to a stock solution of antihuman IgG-HRP conjugate

(250 nM) for a further 24 hours.

The bed was then interrogated as in Example 1. Blanks were also run, using the bed plus conjugate only. A relatively high background current, measured from the bed plus conjugate only, is due to adventitious binding of the conjugate to the Sepharose beads. The results are shown in Figure 3.

The results show excellent linearity and the measured i_D values suggest a high level of binding of both the human IgG and the conjugate.

Example 3

Immobilisation of Human IgG on a Glassy Carbon Disc

The glassy carbon disc (23) of electrode (1) was polished and electrochemically cleaned in nitric acid (10%). The disc was switched to +2.2V versus Ag/AgCl for 60 seconds, removed from the acid and washed. The electrode was placed in acetate buffer (50 mM, pH 4.6) containing water-soluble carbodiimide (50mM) for 30 minutes at room temperature. After washing the electrode was placed in a stirred solution of human IgG (lmg/ml) in acetate buffer (50mM, pH 4.6) for 2 hours at room temperature. The electrode was washed and stored in PBS 20 at 4°C. Thereafter, to establish the rate of binding, the electrode was incubated with goat anti-human IgG-HRP conjugate.

For optimal use of immunoassay tests, it is important to know the extent of binding of the antigen and antibody at a given time (that is, the rate of binding). The resultant binding curve for goat-anti-human IgG-HRP conjugate to immobilised human IgG is of the standard type, indicating that after 45 minutes about 50% of the material was bound. It was for this reason that 45 minutes was chosen for the period of incubation in the assay. Adventitious adsorbtion of the conjugate appeared to be low. Example 4

Immunoassay of Human IgG

The component steps of a sandwich immunoassay using the apparatus of Figure 1 were as follows:-

(a) Goat anti-human IgG (Sigma) was immobilised on the glassy carbon disc (23) of electrode (1), as in Example 3.

(b) The disc (23) was dipped into PBS Tween 20 containing known amounts of human IgG for 45 minutes at room temperature.

(c) The electrode was then washed and placed in a solution of goat anti-human IgG-HRP conjugate (0.3-μM) for 15 minutes.

The disc (23) of the electrode (1) was then interrogated for enzyme activity as in Example 1 using the apparatus shown in Figure 1. Any ferricinium ion produced by enzyme on the disc is detected on the ring (held at -60 mV versus Ag/AgCl). Response times were fast at about 30 seconds. Adventitious binding of the conjugate to the disc seems small and constant. After interrogation, the electrode was dipped in glyσine/HCl buffer (0.2M, pH 2.2) to clean away any bound material, allowing the electrode to be re-used.

The results are shown in Figure 4.

Figure 4 shows the test is linear over the concentration range, under the conditions employed.

Similar experiments may be conducted with product detection at the disc (-60 mV versus Ag/AgCl). These results are displayed in Figure 5. The currents detected are larger, but the noise is much greater and thus sensitivity is lost at low concentration.

Example 5

Immunoassay of Human IgG in a Standard Serum

Assays were performed using Precinorm U standard serum (Boehringer). A range of IgG concentrations, based on the data supplied with the sample, were prepared by dilution into PBS Tween 20. The analyses were then performed exactly as previously.

Results for the immunoassay of human IgG in Precinorm U are shown in the following Table.

The measured values agree well with those from Figure 7, indicating little or no interference from other serum constituents.

Example 6.

Catalase assay using a rotating ring disc electrode

The disc of a glassy carbon-platinum ring disc electrode was poised at -630 mV versus a standard calomel electrode ("SCE") to reduce the dissolved oxygen in an oxygen-saturated phosphate buffer (0.1M, pH 7.21) to hydrogen peroxide. Hydrogen peroxide was then detected at +700 mV versus SCE on the ring, while rotating at 5 Hz.

Aliquots of catalase (Sigma) were then added to the solution and the decrease in the ring current (i_R) measured. These data were then plotted to establish a working range for the enzyme.

Example 7

Catalase assay using a wall jet ring disc electrode

A glassy carbon/gold disc-ring wall jet electrode was employed. The disc was poised at -680 mV versus Ag/AgCl, producing hydrogen peroxide from the dissolved oxygen in the flowing buffer (phosphate 0.1M pH 7.21, V_f=2,45 ml min^-1). The ring was cycled between 0 and +1.0 V versus Ag/AgCl at 10 mV s^-1 with the i_R for hydrogen peroxide oxidation being measured at +920mV Ag/AgCl. Aliquots of catalase (Sigma) were injected via a Rheodyne injection port and the effect on i_R noted. The results are shown in in Figure 6.

The results of Examples 6 and 7 demonstrate the viability of using catalase as a marker enzyme for the nanomolar range. From Figure 6 the currents were low and reproducibility was good.

Claims

1. An enzyme-labelled assay in which the enzyme catalyses a reaction of at least one reactant at a first location to give at least one product, and the reactant or the product is determined electrochemically in a flow system at a second location downstream of the first location.

2 . An assay according to Claim 1, wherein the assay is an immunoassay.

3. An assay according to claim 2, wherein the assay is a sandwich ELISA or a competitive ELISA.

4. An assay according to Claim 1, wherein the reactant is oxidised or reduced.

5. An assay according to Claim 4, wherein the enzyme is a peroxidase or a catalase.

6. An assay according to Claim 5, wherein the enzyme is a peroxidase and the substrate is a reduced form of a potentially electroactive compound.

7. An assay according to Claim 6, wherein the reactant system includes hydrogen peroxide as oxidant for the reduced form of substrate.

8. An assay according to Claim 7, wherein the hydrogen peroxide is electrochemically generated.

9. An assay according to Claim 1, wherein the first location comprises a bed of immobilized enzyme.

10. An assay according to Claim 1, wherein the first location comprises the disc of a ring disc electrode.

11. An assay according to Claim 1, wherein the electrochemical determination is effected using a wall jet electrode.

12. An assay according to Claim 1, wherein the product or: a component of the product is determined.