WO2000025135A1

WO2000025135A1 - Dip-stick detection system

Info

Publication number: WO2000025135A1
Application number: PCT/GB1999/003500
Authority: WO
Inventors: Michael Frederick Clark; Nigel Frederick Lyons
Original assignee: Horticulture Research International
Priority date: 1998-10-22
Filing date: 1999-10-22
Publication date: 2000-05-04
Also published as: GB9823177D0; AU6354299A; GB2342992A

Abstract

A two-step capillary flow immunoassay is provided where firstly sample with biotinylated antibody specific to the analyte is applied to a wicking strip to flow to encounter an immobilised immunoreactant which is either antibody specific to the analyte or is the analyte, and optionally to flow to an immobilised control antibody, and secondly gold-labelled antibody specific to biotin is applied.

Description

DIP-STICK DETECTION SYSTEM

The present invention relates to a detection system.

Lateral flow or dipstick technology was originally developed as a simple home- use pregnancy test. It has subsequently been adapted for many other uses in veterinary and clinical medicine but the principles of the test system have remained unchanged.

The basic design is a combination of three components, as shown in Figure 1. A is the sample application area, B is the nitro-cellulose membrane to which a thin stripe of specific antibody (usually monoclonal) has been applied and is immobilised, C is the absorbent wick.

In the majority of commercially available devices the same specific antibody (as in B) but coupled to colloidal gold particles (occasionally coloured latex beads), is preloaded onto the sample area and the liquefied sample is applied directly to A. If the target component (antigen) is present in the sample the gold-labelled antibody attaches to it and is drawn with the liquid flow through the nitro-cellulose membrane. When the antigen reaches the stripe of immobilised antibody it is captured and prevented from continuing with the liquid flow into the absorbent wick. As the antigen has been coated with the gold-labelled antibody this is concentrated on the stripe and a coloured line develops. Where no target antigen is present in the sample all the gold-labelled antibody is drawn into the absorbent wick and no line develops.

In practice most devices also have an additional component immobilised as a second stripe on the membrane which interacts either directly with colloidal gold, or through excess gold-labelled antibody. This constitutes a control to indicate the correct functioning of the test. In this case the development of two coloured lines indicates a positive sample. If only the control line becomes visible the test is deemed negative but proof that the test device is working correctly.

For most tests utilising this technology the components are housed in a cassette made of plastic or other impervious material.

The present invention is concerned with a universal test system for use with polyclonal or monoclonal antibodies, or with a mixture of both. The same basic design is used for direct tests in which the presence of the target analyte (antigen) is signalled by the development of a visible, coloured reaction line, and for competition tests in which the presence of the target analyte (hapten) prevents the development of the reaction line. The system is highly sensitive and allows very rapid results (<20 min).

SUMMARY OF INVENTION

The present invention provides a two-step capillary flow immunoassay where firstly sample with biotinylated antibody specific to the analyte is applied to a wicking strip to flow to encounter an immobilised immunoreactant which is either antibody specific to the analyte or is the analyte, and optionally to flow to an immobilised control antibody, and secondly gold-labelled antibody specific to biotin is applied

The present invention provides the assay procedure, and also provides component parts for the assay. In particular, the present invention provides a two-step capillary flow immunoassay kit comprising a wicking strip with an immobilised immunoreactant which is either antibody specific to the analyte or is the analyte, and optionally an immobilised control antibody, a supply of biotinylated antibody specific to the analyte, and gold-labelled antibody specific to biotin.

PREFERRED EMBODIMENTS

There are currently two particularly preferred embodiments, direct antigen detection and competitive hapten detection. Components of the system for direct antigen detection are shown in Figure 2.

The device is in the form of a stick comprising a free-standing (non-enclosed) membrane (commercially available) connected via an adhesive backing strip to a cellulosic wick. The stick has been designated, provisionally, an "EMC-Stick". Purified antibody specific for the antigen to be detected (capture antibody) is immobilised in a stripe on the reaction membrane at a position referred to as the reaction line.

An anti-species antibody is similarly immobilised at the control line position.

Purified antibody specific for the antigen to be detected is conjugated with the small water-soluble vitamin biotin, using a commercially available biotinylating reagent (detecting antibody). Any suitable biotinylation system may be used in the present invention. The ratio of biotin molecules to antibody is suitably controlled. The present invention also suitably employs colloidal gold conjugated with antibody specifically reactive with biotin (commercially available - see Example 1).

Aqueous buffer solutions are optimised for detection of the target antigen.

The present antigen detection system is run in two stages. In the first stage an aqueous suspension of the material under test (made using an optimised buffer mixture) is treated with the biotinylated detecting antibody conjugate. If target antigen is present in the aqueous suspension, biotin-labelled antibody will bind to it. The stick is positioned in the container with a measured amount of the mixture of aqueous test material and biotinylated antibody, so that the tip of the reaction membrane is immersed in the mixture of a distance of 2-3 mm. The aqueous mixture wicks up the membrane by capillary action, passing through the reaction line and control line positions. Any antigen in the mixture is captured by an immobilised antibody at the site of the reaction line and both the antigen and its attendant biotin label accumulates. Excess (unreacting) antibody passes through the reaction site and some is captured by the anti- species antibody immobilised at the control line. Liquid continues to wick through the membrane into the cellulosic filter until either all the liquid has been drained out of the container or the EMC-stick is physically removed.

In the second stage, the EMC-stick is repositioned in a second container in which has been placed a measured amount of a suspension of size-optimised (usually 40 nm) colloidal gold labelled with antibody specifically reactive with biotin (commercially available). The anti-biotin gold conjugate wicks up the membrane by capillary action. Where the conjugate encounters biotin - at the reaction line if target antigen is present and the control line, the antibody binds to the biotin with concomitant accumulation of colloidal gold. The is results in the formation of highly visible red lines. Hence a positive reaction is one in which two visible reaction zones appear, at the reaction line and the control line positions. A negative reaction is one in which a red line appears only at the control line position. Excess (unreacting) gold conjugate is drawn through the membrane into the wick.

Components of the system for competitive hapten (e.g. pesticide) detection are the same as those shown in Figure 2.

The device is in the form of a stick comprising a free-standing (non-enclosed) membrane (commercially available) connected via an adhesive backing strip to a cellulosic wick. The stick has been designated, provisionally, and "EMC-Stick". A preparation of hapten molecules, conjugated to an inert carrier protein (e.g. bovine serum albumin) is immobilised via the carrier protein in a stripe on the reaction membrane at a position referred to as the reaction line.

An anti-species antibody is similarly immobilised at the control line position.

Purified antibody specific for the hapten to be detected is conjugated with the small water-soluble vitamin biotin, using a commercially available biotinylating reagent (detecting antibody). The ratio of biotin molecules to antibody is suitably controlled. Colloidal gold conjugated with antibody specifically reactive with biotin (commercially available).

Aqueous buffer solution is optimised for detection of the target hapten.

The hapten detection system is run in two stages. In the first stage an aqueous suspension of the material under test (made using an optimised buffer mixture) is treated with the limiting concentration of the biotinylated detecting antibody conjugate. If target hapten is present in the aqueous suspension, biotin-labelled antibody will bind to it. The EMC-stick is positioned in the container with a measured amount of the mixture of aqueous test material and biotinylated antibody, so that the tip of the reaction membrane is immersed in the mixture for a distance of 2-3 mm. The aqueous mixture wicks up the membrane by capillary action, passing through the reaction line and control line positions. Any hapten in the mixture will have already bound to the capture antibody, thus preventing the biotinylated antibody from reacting with the immobilised hapten-protein conjugate on the membrane. If none, or very little hapten is present in the test material, competition for the biotinylated capture antibody does not occur and the antibody is free to bind to the immobilised hapten-protein conjugate. Excess (unreacting) antibody passes through the reaction site and some is captured by the anti- species antibody immobilised at the control line. Liquid continues to wick through the membrane into the cellulosic filter until either all the liquid has been drained out of the container or the EMC-stick is physically removed.

In the second stage, the EMC-stick is repositioned in a second container in which has been placed a measured amount of a suspension of size-optimised (usually 40 nm) colloidal gold labelled with antibody specifically reactive with biotin (commercially available). The anti-biotin gold conjugate wicks up the membrane by capillary action. Where the conjugate encounters biotin - at the reaction line if target hapten is absent and the control line, - the antibody binds to the biotin with concomitant accumulation of colloidal gold. This results in the formation of highly visible red lines. Hence a positive reaction is one in which only one visible reaction zone appears, at the control line position. A negative reaction is one in which hapten competition for the biotinylated antibody does not occur and thus two red lines develop, at the reaction line as well as the control line positions. Excess (unreacting) gold conjugate is drawn through the membrane into the wick.

In one embodiment, the invention relates to an immunoassay comprising the steps of:

a) treating a suspension of test material with a biotinylated antibody, such that if a target analyte is present in the suspension, biotin-labelled antibody will bind to it; b) locating the sample produced in a) onto a wicking strip so that the suspension wicks up the membrane by capillary action, beyond an immobilised immunoreactant and any immobilised control antibody; and c) contacting the wicking strip with colloidal gold labelled with antibody specifically reactive with biotin, such that the anti-biotin gold conjugate wicks up the membrane by capillary action, beyond the immobilised immunoreactant and any immobilised control antibody.

The suspension of test material is preferably aqueous or substantially aqueous, and preferably has no or only a low surfactant content, such that antibody binding to the target analyte in the suspension is not hindered.

The use of a premixing stage for the antigen and antibody allows any antibody- antigen interaction to be formed before there is any contact with the wicking solution. This is important as wicking solutions may contain a surfactant component to facilitate the wicking process. We have discovered that surfactants can interfere with the antibody-antigen interaction and, in the case of viral capsids for example, can strip the capsid from the virus, preventing or hindering any antibody-antigen interaction. Once the antibody-antigen reaction has occurred, then levels of surfactant in the wicking solution do not generally disrupt the interaction. The present invention also relates to a wicking strip suitable for use in the present invention, comprising a membrane connected to a wick, the membrane having a sample application area to which a test sample is applied, wherein the sample application area comprises no preloaded antibody component. Preloading of antibody onto the test strip is not required, as the mixing of the antibody with antigen takes place before application to the strip, as described above.

The application of this type of dipstick format is thought to be unique for the detection of plant-derived antigens and haptens.

The present invention is particularly related to identification of antigens derived from Plum pox, Tomato Spotted Wilt virus, Cucumber mosaic virus, Bean Common Mosaic Virus, Prune Dwarf virus, Necrotic Ringspot virus, Arabis Mosaic virus, Xanthomonas campestris pv.campestris, Xanthomonas campestris pv. pelargonii and Xanthomonas fragariae. However, the invention is not limited to the detection of antigens derived from such species and is applicable to any suitable plant-derived antigen.

The use of biotin to label polyclonal and/or monoclonal antibodies specific for plant-derived antigens and haptens, in conjunction with anti-biotin colloidal gold, is unique to the recognition system of this EMC-stick system.

This technique has been developed specifically for use both by non-technical personnel as well as by laboratory-trained personnel.

The sensitivity of the method for bacteria has been evaluated in comparison with an enzyme-linked immunoassay, ELISA, giving about the same sensitivity. For pesticide detection such as metaxyl, a comparison with gas chromatography/mass spectrometry showed equal sensitivity, which was also confirmed using ELISA. The detection was around 100 pg/mg, or 0.1 parts per million. The present invention will now be illustrated further by the following Example and Figures, but is not limited to the specific embodiment shown in the Example or Figures, wherein;

Fig 1 illustrates the dipstick of the prior art; and

Fig 2 illustrates the wicking stick of the present invention.

Example 1: Preparation and use of lateral flow dipsticks

A. Antibody purification (Polyclonal antibody or monoclonal antibody specific to target organism)

Antibody in the form of purified IgG is required to enable biotin conjugation. The serum sample must be desalted before purification. This may be achieved either by dialysis against application buffer (2 changes of buffer in 4 hours is sufficient to desalt 3ml of serum) or by using a Bio-Rad Econo-Pac 10DG column (Cat. No. 732-2010). If the column is used the following method applies.

1. Discard the buffer above the top frit of the column.

2. Add 20ml application buffeπ-

NaCl 1.636g

Distilled water 975ml

Adjust to pH 8.0 with ION NaOH (4g in 10ml H₂0) Make up total volume to 1000m 1

3. Snap off the bottom tip to start column flowing and allow the buffer to drain to

4. Add 3ml of serum to the column. If the serum sample is less than 3ml, add application buffer to reach a total sample volume of 3ml. 5. Allow the sample to completely run into the column. Discard the first 3ml eluted.

6. Add 4ml of the application buffer to elute the serum, while collecting the 4ml fraction from the column.

7. Wash the column with 20ml application buffer if the column is to be used again immediately. If the column is to be stored, wash with 20ml distilled water containing 0.02% sodium azide.

After desalting the IgG fraction is prepared using a DEAE Af i-Gel Blue gel column. The column is packed with the gel at the rate recommended with the batch of gel purchased (2.5ml gel/1 ml serum sample is recommended). The following volumes of buffers apply to a 10ml column which will process a 4ml serum sample.

1. Prewash for first time use only: Wash the column with 40ml prewash buffer: - Sodium thiocyanate 12.16g

Distilled water to 100ml

2. If prewash buffer is used follow with 40ml application buffer. If the column has already been prewashed begin with step 3.

3. Equilibrate the column with 30ml application buffer.

4. Apply the serum sample to the column and elute the IgG with 20ml of application buffer. Collect the elute in + 1ml aliquots. Determine the absorbance values of each aliquot at 280nm and combine the fractions with values above 0.9. Measure the absorbance value of the combined fractions and calculate the immunoglobulin content based on an absorbance value of 1.42=lmg IgG/ml. Dilute with application buffer if required or concentrate by free drying. The purified IgG may be preserved with 0.02% sodium azide and stored at -20°C in small aliquots until required.

5. Regenerate the column with 20ml prewash buffer followed by 20ml of application buffer containing 0.02% sodium azide. Store the column containing application buffer with sodium azide at 4°C.

B. Biotinylation Biotinylation is carried out using EZ-Link™ Sulfo-NHS-LC Biotin (Pierce Product code 21335).

1. Dialyse 2ml of 1 mg/ml IgG against 2 changes of distilled water - 3 hours each change and freeze dry.

2. Dissolve the IgG in 1ml of 0.01M phosphate buffered saline pH 7-8.5 in a small tube. 3. Immediately prior to use, dissolve 1 mg of Sulfo-NHS-Biotin in 1ml distilled water. Add 75μl of the solution to the IgG. (NB. It is important to allow the bottle of Sulfo-NHS-LC Biotin to come to room temperature before removing an aliquot of the material. Store the bottle desiccated).

4. Place the tube in ice for two hours.

5. Remove unreacted biotin using an Amicon microconcentrator No. 3. Spin rinse the microconcentrators with distilled water before use to remove glycerol used to lubricate the membrane. Use maximum g-force of 14000 for 95 minutes at 25°C.

6. Store the biotinylated IgG at 4°C until ready for use.

C. Construction and use of dipsticks.

The dipstick is constructed of a sheet of nitrocellulose membrane attached to a reinforced backing and an absorbent cellulosic wick. A line of non-biotinylated IgG antibody (Polyclonal antibody or monoclonal antibody specific to target organism) at a concentration of 1 mg/ml is applied as the reaction line. A second line of non- biotinylated anti-species IgG at a concentration of 1 mg/ml is applied as the control line. The lines are allowed to air dry and the membrane is then blocked by immersion in 1% bovine serum albumin for 5 minutes before air drying. Individual 6mm wide dipsticks are then cut from the sheet and stored desiccated at 4°C.

For use diseased plant material is ground in extract buffer to release antigen:- 0.3M glycine (adjusted to pH 8.3) 0.01M MgCl₂ 0.4% Triton X-100 0.2% Tween 20 0.1%) polyyinyl alcohol (preserved with 0.02%) sodium azide)

The tip of a dipstick is immersed in a small aliquot of the plant extract mixed with an equal volume of specific biotinylated antibody until the liquid phase is drawn partway into the absorbent cellulosic wick. The dipstick is then immersed in a small quantity of anti-biotin conjugated gold suspension:-

a. Buffer

0.15M NAC1

0.4% Triton X- 100

0.2% Tween 20

0.2%) Bovine serum albumin

(preserved with 0.02%) sodium azide)

b. Working solution

1 part immunogold goat anti-biotin (British BioCell International BA.GAB40)

2 parts buffer

(store refrigerated at 4°C)

The wicking continues until this reagent is drawn across both lines of immobilised antibodies. Where the gold conjugate encounters biotin - at the reaction line if target antigen is present and at the control line, the antibody binds to the biotin with concomitant accumulation of colloidal gold. This results in the formation of highly visible red lines. Hence a positive reaction is one in which two visible reaction zones appear, at the reaction line and the control line positions. A negative reaction is one in which a red line appears at only the control line. Excess (unreacting) gold conjugate is drawn through the membrane into the wick.

Claims

1 A two-step capillary flow immunoassay where firstly sample premixed with biotinylated antibody specific to the analyte is applied to a wicking strip to flow to encounter an immobilised immunoreactant which is either antibody specific to the analyte or is the analyte, and optionally to flow to an immobilised control antibody, and secondly gold-labelled antibody specific to biotin is applied.

2 An immunoassay according to claim 1 , when in the form of a competitive hapten immunoassay.

3 An immunoassay according to claim 1 or 2, wherein a preparation of analyte molecules conjugated to an inert carrier protein is immobilised via a carrier protein in a stripe on the reaction membrane.

4 An immunoassay according to claim 1 , when in the form of a direct antigen immunoassay

5 An immunoassay according to any preceding claim, for the detection of plant- derived antigens and haptens.

6 An immunoassay according to any preceding claim, for the detection of plant pathogens.

7 An immunoassay according to any preceding claim, wherein the analyte is derived from Plum pox, Tomato Spotted Wilt virus, Cucumber mosaic virus, Bean Common Mosaic Virus, Prune Dwarf virus, Necrotic Ringspot virus, Arabis Mosaic virus, Xanthomonas campestris pv. campestris, Xanthomonas campestris pv. pelargonii and Xanthomonas fragariae.

8 An immunoassay according to any preceding claim, wherein the sensitivity of the method is substantially the same as the enzyme-linked immunoassay method for a given analyte.

9 An immunoassay according to any preceding claim, wherein the wicking strip is in the form of a stick comprising a non-enclosed membrane connected via an adhesive backing strip to a cellulosic wick.

10 A wicking strip suitable for use in the immunoassay as described in any of claims 1-8, comprising a non-enclosed membrane connected to a wick, the membrane having a sample application area to which a test sample is applied, wherein the sample application area has no preloaded antibody component.

11 A two-step capillary flow immunoassay kit comprising a wicking strip with an immobilised immunoreactant which is either antibody specific to the analyte or is the analyte, and optionally an immobilised control antibody, a supply of biotinylated antibody specific to the analyte, and gold-labelled antibody specific to biotin.