WO1994010576A1

WO1994010576A1 - Allergy test kit and apparatus

Info

Publication number: WO1994010576A1
Application number: PCT/GB1992/001995
Authority: WO
Inventors: Patricia Mary Beckett Robertson
Original assignee: Immunodiagnostic Systems Ltd
Current assignee: Immunodiagnostic Systems Ltd
Priority date: 1992-10-30
Filing date: 1992-10-30
Publication date: 1994-05-11
Anticipated expiration: 1995-04-30
Also published as: AU2875192A

Abstract

A method and apparatus for measuring immunoactivity comprising exposure of a leucocyte population to a potential allergen, preferably on a membrane material which is adapted to selectively retain the leucocyte component of a blood sample and the subsequent stressing of the leucocyte population to bring about release of an agent generated by leucocytes as a result of their involvement in an immunogenic reaction; and finally the detection of said agent using a colourimetric agent and measuring means.

Description

ALLERGY TEST KIT AND APPARATUS

The invention relates to two ideas which are so linked as to form the same inventive concept and is particularly, though not exclusively, applicable to a method for evaluating leucocyte activity and a test kit for practising this method.

An allergic reaction occurs after contact, by the patient, with an allergen such as a particular food or chemical or inhalant allergens. This immune response is known as Type 1 Hypersensitivity . There are two types of cell involved in this allergic response, namely basophils, which are a sub-population of leucocytes, and mast cells. The mechanism of the allergic response is similar in each cell type. The allergen interacts with IgE bound to basophil or mast cells and following this cell surface activation, causes the release of various pharmacologically active substances, including histamine, which produce an acute inflammatory reaction.

There are various techniques which can be used to determine the specific substance which elicits an allergic reaction in a patient and two such methods will be described here. - 2-

The classical intradermal test is the Type 1 Wheal and Flare reaction. This is a technique in which samples of a variety of different antigens are introduced into the skin, a positive reaction, namely increased vascular permeability, local oedema and itching, identifies one or more specific allergens to which the patient is allergic. Alternatively, if the allergen is known, the end point titration can be determined by introducing into the skin a range of dilutions of the antigen. This provides an approximate indication of the patient's sensitivity to the allergen.

A second technique is the radioallergosorbent test (RAST), the results of which correlate well with intradermal test results. In this method, known allergens are covalently bound to a cellulose disc (rather than directly to a radioimmuonoassay plate), incubated with the test serum whereby IgE antibodies bind to the allergen. The IgE is bound by a radiolabelled anti-IgE antibody and detected using a gamma-counter, a positive reaction being the detection or radioactivity.

The closest prior art^* known to the applicants is US Patent Number 4 309 184 which describes the cytotoxic food test. An allergen is identified by analysing in vitro for a specific constituent of a leucocyte, for example an enzyme, with an without challenging the leucocyte with the allergen and comparing the results.

There are, however, disadvantages with these techniques. The intradermal skin test is uncomfortable for the patient and can be time consuming. The RAST test requires specialized, expensive equipment and specialist technical staff. The cytotoxic food test takes over 24 hours before results are obtained and, again, requires skilled technical staff to perform the test .

The present invention provides an in vitro method for evaluating leucocyte activity and a test kit for practising this method. The method, being qualitative rather than quantitative, does not require expensive specialist or highly trained technical staff. The method is immediately administrable and provides a satisfactory indication as to whether or not there is an allergen sensitivity present. The kit itself is small, portable and ideal for use in general practice where no specialist equipment or training would be required.

The present invention further provides two alternative methods for use in evaluating leucocyte activity and two alternative test kits for practising the respective methods.

According to a first aspect of the invention, there is provided a method of evaluating leucocyte activity, the method comprising the steps of:

(a) preparing a suspension of leucocytes in plasma and incubating in each well of a multiwell plate the suspension with or without an allergen;

(b) causing the membranes of the leucocytes to be stressed or lysed by the presence of water, either by the allergen being in aqueous

SUBSTITUTESHEET solution or the addition of water to a mixture of a dehydrated allergen and the leucocyte/plasma suspension;

(c) analysing the supernatant of the suspension for a particular constituent of the leucocytes, which is released as a result of the membrane of the leucocytes being stressed or lysed, detection of the cell constituent being by a colour change, which either is visible to the eye or visible using a probe to detect said cell constituent;

(d) comparing the colour change detected in wells containing allergen and leucocyte/plasma suspension with the colour change detected firstly, in at least one well containing leucocyte/plasma suspension with no allergen and secondly in at least one well containing allergen with no leucocyte/plasma suspension.

(e) comparing the analysed results of the colour change from step (c) with a standard range of colourimetric results, from no or minimum allergic response to maximum allergic response .

In another broad aspect, there is provided a test kit for evaluating leucocyte activity comprising:

(a) a multiwell plate, each plate containing allergen, either in aqueous solution or dehydrated ; (b) a sedimentation tube;

(c) range or pipettes;

(d) a probe to analyse for particular cell constituent ;

(e) a chart of standard colourimetric results;

and with the test kit being easily portable.

Preferably the multiwell plate is sterile packed with each well being covered by a membrane which is easily pierced by a pipette.

Preferably the leucocyte/plasma suspension and allergen solution are incubated for a maximum of 2 hours.

The colour change may be from a colour to an absence of the colour; alternatively the colour change may be from an absence of colour to the presence of a colour. The colour change may be measured using a colourimeter or a reflectometer or some such other means.

The preferred detection method of ascorbic acid by a colour change preferably is unaffected by the presence of other cell constituents. Alternatively, a cell constituent other than ascorbic acid may be detected using such a colour change.

According to yet further aspect of the invention there is provided an alternative method of evaluating an immunogenic response which method comprises: (a) preparing an allergen impregnated filter;

(b) exposing at least a first side of said filter to a sample to be tested;

(c) adding water to the filter so to as to lyse immunogenic cells;

(d) adding reagent to the filter to detect the presence of an immunogenic reaction;

(e) evaluating the nature of the reaction.

In a preferred embodiment the filter is impregnated with allergen using freeze-drying techniques. Further, prior to use, the filter may be rehydrated using a suitable solvent or solution such as isotonic saline solution. Ideally the filter may also be impregnated with said reagent.

In a preferred example, the method also includes the step of allowing time for the sample applied to the filter to be filtered under gravity so as to remove blood proteins and those constituents not involved in immunogenic reactions.

The reagent used in the reaction is preferably a reagent that is coloured and thus undergoes a colour change as a result of the immune reaction. Preferably the reagent is able to detect the presence of ascorbic acid and a suitable reagent would be dichlorophenyl lindotheny1. In yet a preferred method the process of evaluating the reaction comprises comparing the colour change in the chosen reagent with a range of standards and/or at least one control or using a colourimeter/reflectometer . In yet a preferred method there is also provided a membrane which is not impregnated with allergen and in this method it will be understood that the method, in accordance with the invention, when practised, represents a control. Since no allergen is present on the filter one would not expect any change in colour in a reagent used.

In a further preferred method the filter is impregnated with ascorbic acid. Thus, when the method is practised using this filter, since ascorbic acid is present in the filter one would expect to see a colour change in the reagent used and this method therefore represents a second control.

According to a yet further aspect of the invention there is provided a test kit for evaluating an immunogenic reaction comprising:

(a) at least one test well supporting at least one membrane which is adapted to retain at least the leucocyte component of a blood sample and which is further impregnated with at least one pre-selected allergen;

(b) a lysing agent for adding to the membrane once the immunogenic reaction is complete; and (c) a pre-determined amount of reagent for adding to the membrane so as to detect the presence of the immunogenic reaction.

Preferably the reagent is a colourimetric agent which undergoes a change of colour as a result of the presence of the immunogenic reaction. Ideally the reagent detects the presence of ascorbic acid and the reagent is therefore ideally dichlorophenyl lindotheny1.

Preferably the test kit also includes a calibrated colourimetric standards chart for the purpose of evaluating the magnitude of the immunogenic reaction.

Preferably the test kit comprises a plurality of test wells, each well supporting a filter which is impregnated with a pre-selected allergen. Thus, in this embodiment of the invention the test kit comprises a means of performing multi-allergen screening so as to determine the sensitivity of an individual to a range of allergens.

Preferably the multi-test kit includes at least ninety six test wells in a single plate.

Preferably each of the aforementioned filters are saturated with allergen.

In a preferred embodiment the filter is impregnated with the reagent and Step (c) above is omitted.

It will be understood that a method and apparatus in accordance with this alternative form of the invention

SUBSTITUTE SHEET comprises a number of advantages which include: the removal of a need to prepare a suspension of leucocytes in plasma before working the method. This is because the filter used in the alternative method and apparatus is capable of filtering those constituents of a blood sample which are not required for the reaction. This means that the method is easier to implement and also less time consuming. The two latter advantages make the new method and apparatus more efficient and easier to use. Further, since the filter is impregnated with allergen and the sample is directly applied to the filter there is a substantial reduction in wastage of allergen and sample. Thus, again, the alternative method and test kit are more cost effective to vise and therefore commercially can be offered for sale at a more competitive price.

The invention will now be described with reference to the following examples which are not intended to limit the scope of the invention but rather to illustrate how the invention can be worked.

In a preferred embodiment of the invention a sample of blood is taken from the patient, for example '5 mis' , and transferred to a sedimentation tube provided in the test kit. The tube contains anti-coagulant and the whole blood is allowed to separate out into its components. After about 30 minutes the layer containing leucocytes is removed and mixed with the plasma layer.

A different allergen is contained in a single well of a multiwell plate. The allergen is either in the form of an aqueous solution or is dehydrated. The dehydrated allergen can be immobilized by an known method, for example a carbohydrate matrix or a paper disc. The multiwell plate can be sterile packed with each allergen being retained beneath a membrane.

Using a first pipette from the test kit a sample drop of the leucocyte/plasma suspension is introduced into each well containing allergen. If a membrane is present over each well, the membrane would be easily pierced by the pipette.

The multiwell plate is pro.vided with wells which act as controls for the allergen test. These controls comprise, for example, at least one well containing no allergen but to which a drop of the leucocyte/plasma suspension is added and at least one well containing allergen to which no leucocyte/plasma suspension is added .

If the allergen is in a dehydrated form, a drop of water is added to each well using a second pipette in the kit. This drop of water is sufficient to stress the cells which causes the release of ascorbic acid.

In the presence of an allergen, in addition in water, the leucocytes are further stressed and may eve lyse, in which case all the ascorbic acid in the cell would be released.

Using a pH probe, which produces a visible colour change, the amount of ascorbic acid released by the "stressed" cells can be determined. Alternatively, an agent may be incorporated into each well, so that when ascorbic acid is released and reacts with the agent, a colour change is observed, either by the production of a colour or the disappearance of a colour.

The colour change is monitored over a period of time, a typical range of time intervals would be 0 minutes, 15 minutes and 30 minutes up to a maximum of 2 hours. The resulting colour changes are compared with a standard range to determine whether or not an allergic response is present. Alternatively, the change may be detected quantitatively using a colourimeter or the like.

In the alternative form of the invention, the alternative method and test kit are used as follows.

A multiwell plate is provided with a plurality of wells, wherein each well is provided with a single membrane. Each membrane is impregnated with a different pre-selected allergen. Ideally impregnation takes place using freeze-drying techniques. Thus, ideally the allergen is in a dehydrated form. Each multiwell plate is sterile and sealed by a suitable membrane.

A sample of blood is taken from a patient, which sample may only be in an amount of 1 to 2 microlitres. The membranes are adapted to filter those constituents of the blood sample which are not involved in the immunogenic reaction thus the membranes selectively retain leucocytes thereon. As the filtering takes place reaction between the impregnated allergen and the blood sample leucocytes takes place. After approximately 30 minutes the immunogenic reaction should be complete.

At this stage a lysing agent is added to the filter which agent may, for example, be water. The lysing agent stresses the leucocytes and as a result intracellular substances are released from the leucocytes.

A reagent, that is preferably an amount of dichlorophenyl lindothenyl, is then added to the membrane. This is a colourimetric agent which is sensitive to the presence of ascorbic acid, thus in the presence of ascorbic acid the colour of this reagent changes from a dark blue through to pink and finally to a clear colour. The use of a reagent which changes from a coloured reagent to a non-coloured reagent is extremely advantages in the practising of the invention since it enables individuals who may be colour blind to monitor the reaction.

The multiwell plate is provided with at least one well which includes a membrane which has not been impregnated with allergen. The addition of a blood sample to this membrane will not result in an immunogenic reaction and therefore the addition of reagent to this membrane will not result in a change of the reagent. Thus the addition of a reagent to this membrane will not result in a colour change.

The multiwell plate is also provided with at least one well including a membrane which is solely impregnated with the substance to which the reagent is sensitive. For example, a membrane solely impregnated with ascorbic acid will be provided. When the reagent is subsequently applied to this membrane, because the reagent is sensitive to ascorbic acid, it will undergo a colour change. This well is therefore a useful guide as to whether the reagent is still functional.

SUBSTITUTE SHEET The colour change is monitored over a period of time, a typical range of time intervals would be 0 minutes, 15 minutes and 30 minutes, and the resulting colour changes are compared with a standard range on a chart so as to determine whether or not an allergic response is present.

It can therefore be seen that the methods and apparatus used in the invention represent significant advancements in primary health care allergen identification. Notably because the invention provides a simple and straightforward assay which quickly produces unambiguous results.

SUBSTITUTESHEET

Claims

1. A test kit for evaluating an immunogenic reaction comprising:

(b) a lysing agent for adding to the membrane once the immunogenic reaction is complete; and

(c) a pre-determined amount of reagent for adding to the membrane so as to detect the presence of the immunogenic reaction.

2. A test kit according to Claim 1 wherein the reagent is a colourimetric agent which undergoes a change of colour as a result of the presence of the immunogenic reaction.

3. A test kit according to Claim 2 wherein the reagent detects the presence of ascorbic acid.

4. A test kit according to Claim 3 wherein the reagent is dichlorophenyl lindothene.

5. A test kit according to any preceding Claim wherein there is also provided a calibrated colourimetric standards chart for the purpose of evaluating the magnitude of the immunogenic reaction.

6. A test kit according to any preceding Claim wherein the kit comprises a multitest plate including a plurality of test wells.

7. A test kit according to Claim 6 wherein each well supports a filter which is impregnated with a pre¬ selected allergen.

8. A test kit according to Claim 7 wherein each well supports a filter which is impregnated with a different allergen.

9. A test kit according to any of the preceding Claims wherein each filter is saturated with allergen.

10. A test kit according to any preceding Claim wherein the filter is impregnated with dehydrated allergen.

11. A test kit according to Claim 10 wherein the filter is impregnated with dehydrated allergen using freeze-drying techniques.

12. A test kit according to any of Claim 6 to Claim 11 wherein at least one well includes a filter which is not impregnated with allergen.

13. An test kit according to any of Claim 6 to 12 wherein at least one well includes a filter which is impregnated with a substance to which the reagent is sensitive.

14. A test kit according to Claim 13 wherein the substance is ascorbic acid.

15. A method of evaluating an immunogenic response which method comprises:

(a) preparing an allergen impregnated filter;

(b) exposing at least a first side of said filter to a sample to be tested;

(c) adding a lysing agent to the filter so as to lyse immunogenic cells;

(e) evaluating the nature of the reaction.

16. A method according to Claim 15 wherein the filter is rehydrated prior to beginning the reaction by exposing the filter to a suitable solution.

17. A method according to Claim 16 wherein the solution is isotonic saline.

18. A method according to any of Claim 15 to 17 wherein the sample is allowed to filter through the membrane under gravity so as to remove blood proteins and those constituents not involved in the immunogenic reaction.

19. A method according to any of Claim 15 to 18 wherein the reagent undergoes a colour change as a result of the immunogenic reaction.

20. A method according to Claim 15 wherein the process of evaluating the reaction comprises comparing the colour change in the chosen reagent with a range of standards and/or at least one control.