GB2057126A - Immunoassay technique - Google Patents

Abstract

For quantitatively assaying antigen in a fluid, a mixture thereof with further antigen, labelled for example by substitution or addition of a radioactive molecule is caused to flow along a paper wick or other carrier on which antibodies to the antigen have been immobilised, e.g. by activating the paper by application of cyanogen bromide, then bringing it into contact with a solution of the antibodies. The paper wick or other carrier is developed by causing a glycerol/water solution to rise through the wick by capillary action, and there is measured the distance travelled along the wick by the labelled antigen.

Description

SPECIFICATION Assay technique The present invention relates to an assay technique for antigenic or potentially antigenic materials, such as protein hormones, steroids or drugs. An antigen is a substance capable of binding specifically to an antibody.

Immunoassay is a method of determining the quantity of a particular material in a sample, such as a blood or urine sample. The technique is both sensitive (for example, picogram quantities of steroids can be detected with ease) and specific.

Accurate measurement of small quantities in the final stage of the method confers sensitivity, whilst immunological reagent (antibody) selection confers specifically.

To perform an immunoassay for a particular antigen one needs an antibody to that antigen, as well as pure labelled antigen as marker. Labelling can be effected by substituting radioactive atom(s) for non-radioactive atom(s) in the antigen (e.g.

tritium is substituted for hydrogen, or carbon-i 4 is substituted for carbon-12) or it can be achieved by the addition of a radioactive atom (such as iodine-125) or by the addition to the antigen of any material that can be detected by a defined quantifiable property (e.g. an enzyme).

In carrying out the technique, a known quantity of the labelled antigen is mixed with the appropriate antibody and the non-iabelled antigen whose quantity it is required to determine. As an equilibrium is established, a proportion of the labelled antigen molecules bind to antibody molecules, and a proportion of the unlabelled antigen molecules likewise bing to antibody molecules. The relative proportion of each species of antigen bound to antibody, or of the remainder of each species not bound to antibody, will reflect the originally-present quantity of unlabelled antigen. Thus it is necessary to separate the unbound (free) antigens from the bound antigens after which measurement of the labelled antigen present in one or both of the separated fractions enables the quantity of originally present unlabelled antigen to be determined.

Known techniques of immuno-assay have a number of special requirements. There is a requirement for a period of incubation, and also for a method of separating free antigen from antibody-bound antigen. Various separation methods are in use, such as charcoal adsorption of the free fraction, use of a double antibody technique (in which a further antibody is used to combine with bound antigen-antibody complexes), or a said phase technique. An example of this last-mentioned technique is a method wherein the antibody to the antigen is fixed to the inside surface of plastic tubes; after a period of incubation, the free unbound antigen fraction can be decanted off and quantified.

An object of the present invention is to provide an immunoassay method in which there is no need for the above-discussed time-consuming equiprnent-dependent step and which can be carried out by, relatively simple, quick and uncomplicated procedures.

With this object in view the present invention provides a method for the immunoassay of an antigen which comprises causing a fluid mixture containing: a) a known quantity (which may be zero) of labelled antigen, together with b) unlabelled antigen to be assayed is caused to flow over and/or through a base, matrix or substrate in which antibodies to the antigen are immobilised, so that both the labelled and unlabelled antigens are removed by combination with the antibodies, and thereafter determining the distance travelled by labelled antigen in said mixture to obtain a quantitative indication of the unlabelled antigen in said mixture.

The rate at which labelled, and therefore detectable, antigen is removed depends on the amount of unlabelled antigen present, it readily being understood that their movement will approximate to the total antigen and therefore enable the relative proportions in the original mixture to be calculated.

In the method of the invention, the antigen may be labelled as described in the introduction to this specification.

The flow of the mixture of labelled and unlabelled antigen may be produced by a chromatographic technique in which the mixture flows through a wick, for example of paper, on which the antibodies specific to the antigen are immobilised. After the chromatography the distribution of the labelled antigen along the wick can be determined to enable the initial quantity of unlabelled antigen to be calculated.

A further aspect of the present invention provides an arrangement for the immunoassay of an antigen comprising a base, matrix or substrate in which are immobilised antibodies specific to the antigen, and means enabling a flow of fluid containing both labelled and unlabelled antigens to be induced over and/or through said base, matrix or substrate.

The following exemplifies the invention: Let it be assumed that an antigen to be assayed in oestriol-i 6 a-glucuronide (E3G). This is a steroid whose concentrations are elevated during normal human pregnancy. Changes in these concentrations are related to foetal well being.

Used as an antibody specific for E3G is antioestriol-l 6a-glucuronide (1 :200 titre) prepared by standard immunological methods, and this is immobilised over a base, matrix or substrate in the form of a wick made of paper. To effect this, firstly the paper is activated for reception of the antibody. The paper used is Whatman filter paper and activation is effected with cyanogen bromide.

The paper is cut into strips, for example 8 cm long and 1 cm in width, and these strips are arranged on holders comprising parallel spacedapart screws, of suitable plastics material, whose shanks protrude through respective holes in each strip, the strips being kept separate by washers or spacers on the shanks.

So heid, the paper strips were placed in a vessel containing 2 M-carbonate buffer (pH 11.0), and maintained at4-60C by means of an ice/water mixture. Cyanogen bromide, dissolved in a minimum volume of N-methyl-2-pyrrolidone, was then added dropwise to the paper so that there was no significant increase in temperature. The paper was then left in the buffer/activator mixture for approximately 1 5 minutes, with occasional stirring, prior to transferring the paper to a cooled sinter funnel and washing alternately with a cooled acetone/water (5% v/v) mixture and 0.1 Msodium bicarbonate buffer solution (pH 0.5).

The anti-oestriol-1 6a-glucuronide antibody was mixed with 0.1 M-sodium bicarbonate buffer (pH 9.5, 5 ml) and kept at 40C until the activated washer paper immersed therein. Incubation was then effected at 40C with constant stirring for a minimum of 15 hours. After removal from the antibody solution (which was removed and retained for reuse) the paper was washed exhaustively with a suitable buffer solution (i.e.

until no more frothing was observed after vigorous mixing) to remove non-covalently absorbed protein. The paper was allowed to dry at room temperature and was then stored dessiccated in a screw-top bottle until required for use.

Each paper strip was then divided so that each provided two wicks each of 8 cm length by 0.5 cm width each such wick being marked at 0.5 cm intervals along its length. These strips, of course each have the antibody immobilised thereover. In practice, of course, the wicks would be readyprepared and provided as parts of a kit suitable for use in carrying out the method of the invention.

A solution was then prepared of labelled E3G, for instance by dissolving tritiated oestriol-1 6aglucuronide in methanol so that 5y1 contained approximately 1,000 cpm (corresponding to 14 14 pg of steroid) so that tritium is present as the label in this antigen. This solution, and a predetermined amount of antigen being assayed were then applied to one of the described wicks, either as a mixture or separately e.g. by spotting which may take place in any order without affecting final results.

Now, the wick is arranged in a vertical disposition, and an appropriate solvent, for example glycerol and water mixed in equal parts, is allowed to rise by capillary action from the bottom of the wick to the top. The wick is then removed and dried and it will be understood that the distribution of labelled E3G in the wick reflects the amount of unlabelled E3G initially present.

This was conveniently determined by running the wick again in a suitable buffer solution.

Chromatography is then effected in a counting vial insert, taking about 5 to 10 minutes. After being taken from the vial, the wick was cut into pieces 0.5 cm2, and the radioactivity content of each such piece was determined after addition of NE 260 Scintillation Fluid (4 ml). The total number of counts initiaily applied was determined as the sum of that in the separate parts. The % of the total present in each piece was then calculated, and plotted against its position in the original wick.

It will be appreciated that as the mixed solution of labelled and unlabelled E3G (which may, if desired, have been premixed with the glycerol/water solvent instead of being spotted as described above) moves through the wick, carried by the solvent, both labelled and unlabelled E3G is removed from the solution by combination with the immobilised antibodies, and that the proportion of labelled E3G molecules removed depends on the amount of unlabelled E3G molecules present. For different amounts of unlabelled E3G, a fixed amount of labelled E3G will show an altered distribution along the wick.

The described technique is a very simple, quick and convenient method for the assay of the antigen. The method eliminates the need for an incubation period, and for separation of free from bound antigens. These steps are replaced by the described chromatography.

The method of the invention can be used in connection with a wide range of antigens, such assteroids, progesterones, hormones, and insulin. In contrast with electrophoretic methods, it can be used with electrically-uncharged antigens, and it can be employed with small antigens. The invention, because of its simplicity and particularly the avoidance of a need to separate free from bound antigens at any stage, provides a method which its easily automatable. A further advantage is that it is a simple matter to combine several assays for different antigens in a single operation.

In this last mentioned method a single wick might be used with different sections having antibodies corresponding to the different assays. It is to be noted that the parameter to be measured is not enzyme activity, radio activity or other qualitative property arising from the labelling; the parameter to be measured is the distance travelled along the matrix.

In carrying the invention into effect, the labelling of the labelled antigen may be achieved in ways other than tritiation. For instance it could be coupled to an enzyme or a light-emitting enzyme such as luciferase or to a fluorescent agent, so that evaluation thereof may be effected by photometric techniques. It may even be feasible to incorporate an electronic sensor capable of switching transistors or gates incorporated into the matrix to enable the result to be derived directly from the matrix. Other variations are possible.

Although the method of the invention has been described as being carried out by use of a wick, other arrangements, for example, a strip or tube coated with a suitable polymeric substance may be used as the matrix for the immobilisation of the antibodies.

Claims (14)

1. A method for the immunoassay of an antigen which comprises causing a fluid mixture containing: a) a known quantity (which may be zero) of labelled antigen, together with b) unlabelled antigen to be assayed is caused to flow over and-or through a base, matrix or substrate in which antibodies to the antigen are immobilised, so that the labelled and unlabelled antigens are removed by combination with the antibodies, and thereafter determining the distance travelled by labelled antigen in said mixture to obtain a quantitative indication of the unlabelled antigen in said mixture.

2. A method as claimed in claim 1 wherein the labelling is effected by substitution of radioactive atom(s) for non-radioactive atom(s) in the antigen, or by the addition of a radioactive atom to the antigen, or by addition of a material detectable by photometric or electromic techniques.

3. A method as claimed in claim 1 or 2 wherein the base, matrix or substrate is in the form of a wick.

4. A method as claimed in claim 3 wherein the wick is of paper.

5. A method as claimed in claim 3 or 4 wherein immobilisation of the antibodies on the wick is effected by firstly activating the material of the wick by application thereto of cyanogen bromide, then applying antibody solution thereto, and subsequent drying.

6. A method as claimed in claim 5 wherein the activation is effected by immersion of the buffer and thereafter adding the cyanogen bromide, in solution, dropwise.

7. A method as claimed in claim 6 wherein, after activation, the wick is washed alternately with a cooled acetone/water mixture and a sodium bicarbonate buffer solution.

8. A method as claimed in claim 5, 6 or 7 wherein antibody application is effected by immersion of the activated wick in a solution of the antibody.

9. A method as claimed in any of claims 5 to 8 wherein the wick, after removal from the antibody solution, is washed with PBSM to remove noncoralently absorbed protein.

10. A method as claimed in any preceding claim wherein flow of the antigen mixture along the wick is effected by way of a solution of glycerol and water.

1 A method as claimed in claim 10 wherein the antigen mixture is spotted onto the wick and the wick is then arranged vertically with its iower end dipping into the glycerol/water solution.

12. A method for the immunoassay of an antigen as claimed in claim 1 and substantially as herein described by way of example.

13. For use in performing an immunoassay of an antigen as claimed in any preceding claim, a base, matrix or substrate, in the form of a matrix on which antibodies to the antigen have been immobilised.

14. A matrix as claimed in claim 13, which is in the form of a paper wick.

1 5. A paper wick as claimed in claim 14 and substantially as herein described by way of example.