WO1989000693A1

WO1989000693A1 - Protection of proteinaceous reagents

Info

Publication number: WO1989000693A1
Application number: PCT/GB1988/000586
Authority: WO
Inventors: Donald Tills
Original assignee: FRESENIUS Ltd
Current assignee: FRESENIUS Ltd
Priority date: 1987-07-16
Filing date: 1988-07-18
Publication date: 1989-01-26
Anticipated expiration: 1990-01-16
Also published as: AU2080488A; GB8716826D0

Abstract

This invention relates to the protection or stabilisation of a proteinaceous reagent, for example an antibody, an antigen, or an enzyme, bound to a solid support, such as plastics material. The invention provides a method of stabilising a dried proteinaceous reagent bound to a plastics substrate by applying thereto and allowing to dry thereon a solution of trehalose.

Description

PROTECTION OF PROTEINACEOUS REAGENTS

This invention concerns the protection or stabilisation of a proteinaceous reagent, for example an antibody, an antigen, or an enzyme, bound to a solid support, such as a plastics material. It is particularly applicable to anti¬ bodies bound to substrates such as the wells of microtitre plates for use in solid-phase assays for a wide range of antigens such as red or white blood cell antigens and bacterial and viral antigens .

Dried proteinaceous reagents tend to deteriorate with loss of activity and it is an object of this invention to stabilise such reagants so that they retain their activity on prolonged storage. For this purpose, we have found that dried proteinaceous reagents can be effectively stabilised by means of the sugar trehalose.

According to one aspect of this invention we provide a method of stabilising a dried proteinaceous reagent bound to a plastics substrate by applying thereto and allowing to dry thereon a solution of trehalose.

According to another aspect of this invention we provide a proteinaceous reagent bound to a plastics substrate and protected by trehalose which has been applied as a solution to the substrate carrying the bound reagent and allowed to dry.

The solution of trehalose is preferably aqueous and is preferably at a concentration of 2% .

The reagent forms a chemical bond with the plastics substrate, which is preferably polystyrene or most preferably polyvinylchloride , for example in the form of a multi-well microtitre plate. If necessary, to maximise binding of the reagent, it may be applied to the plastics substrate with a suitable coating buffer (usually phosphate-based) of an ionic strength and pH chosen to suit the particular reagent to be employed. The optimum pH can be determined by simple experiment.

The reagenύ is applied to the substrate in liquid form and may be bound to it by incubation, preferably at a temperature from about 18°C to 3^~°C. Any remaining liquid reagent is then discarded, and the substrate washed thoroughly with a washing solution that will both remove any unbound reagent and also act as a blocking buffer to prevent the substrate from binding any other reagent (e.g. antibody) that it might encounter. The washing solution is then removed and the plate dried. Finally, the dried substrate is coated with a 2% solution of trehalose in distilled water and this is allowed to dry at a temperature between about 18°C and 37°C.

Substrates, such as microtitration plates, prepared in this way can be stored for at least six months at 37°C in an aluminium foil pack containing a dessicant with the reagent remaining stable and active.

Example

ABO and Rh(D) typing of erythrocytes (red blood cells) .

A U-bottomed microtitre plate of plastics material is coated with anti-A, anti-B, anti-A,B and anti- Rh(D) antibodies by applying a solution of the appropriate antibody to the wells of the plate, incubating the plate at 37°C, washing the plate several times with a "Tween" (Reg. Trade Mark for a surfactant which is a polyoxyethylene derivative of a fatty acid partial ester of a sorbitol anhydride) /

albumin/phosphate buffered saline washing solution, dried by shaking and then by banging it on to absorbent paper. A 2% solution of trehalose (50 ul per well) is then applied to the plate and the plate dried at 37°C. The antibodies used can either be monoclonal or polyclonal antibodies which have been affinity-puri ied.

The thus-prepared plate may be stored for at least six months in an aluminium foil pack containing a dessleant.

Before use the coated microtitre plate is removed from its aluminium foil container. A 0.25% suspension of the erthrocytes .under test is made up and 50 ul added to each of the appropriate wells of the plate. If required the erythrocytes can be pre-treated with an enzyme.

The microtitre plates are then centrifuged and the results read over an illuminated mirror. In positives the cells bind to the antibodies fixed t > the plates and so become evenly distributed over the bottom of the plate. In negatives all the cells form a single button at the bottom of the plate. Reading can be made easier by holding the plates at an angle of 80° for two minutes when only the negatives, where the blood cells remain unbound to the plate, will stream.

Claims

CLAIMS : - -.-

1. A method of stabilising a dried proteinaceous reagent bound to a plastics substrate by applying thereto and allowing to dry thereon a solution of trehalose.

2. A proteinaceous reagent bound to a plastics substrate and protected by trehalose which has been applied as a solution to the substrate carrying the bound reagent and allowed to dry.

3. A method according to clai l wherein the solution of trehalose is aqueous.

4. A method according to claim 3 wherein the solution is at a concentration of 2%.

5. A method according to claim 1,3 or 4 wherein the plastics substrate is polystyrene or polyvinylchloride,

6. A method according to any of claims 1 3,4 or 5 wherein the reagent is applied to the plastics substrate with an- ionic strength and pH chosen to suit the reagent to be stabilised.

7. A method according to any of claims 1 or 3-6 in which the reagent is applied to the substrate in liquid form and then bound to it by incubation.

8. A method according to claim 7, wherein incubation is at a temperature of from about 18°C to 37°C.

9. A method according to claim 7 or 8 in which, after incubation, the substrate is washed with a washing solution that will both remove any unbound reagent and also act as a blocking buffer to prevent the substrate from binding any other reagent that it might encounter, the washing solution then being removed and the plate dried, the dried substrate then being coated with an aqueous solution of trehalose.