WO2000066770A2

WO2000066770A2 - Method for producing a chemically and thermally stable solid phase matrix for polymerase chain reaction (pcr) and dna hybridisation assay

Info

Publication number: WO2000066770A2
Application number: PCT/DE2000/001309
Authority: WO
Inventors: Pavel Strohner; Eberhard Schmidt; Wolfgang Klein; Joachim Schreiber; Renate Bienert
Original assignee: Biotez Berlin-Buch Gmbh
Priority date: 1999-05-03
Filing date: 2000-04-28
Publication date: 2000-11-09
Also published as: WO2000066770A3; DE10020885A1

Abstract

The invention relates to a method for producing a chemically and thermally stable solid phase matrix, using an inert, thermally stable support material, onto which a biotinylated cross-linking agent V 1 is applied as an adhering agent, having a molecular weight ≥ 500 KD, and being preferably cross-linked immunoglobulin-gamma or bovine gammaglobulin, in highly alkaline buffers at a pH value ≥ 11.0 or in a highly acidic environment at a pH value ≤ 3.0. Once said agent has been adsorbed into the support, cross-linked streptavidine and/or avidine is applied as a cross-linking product of high molecular weight. The matrix produced by this method is chemically and thermally stable and can thus be used for the combined application of PCR and hybridisation assay.

Description

Process for the production of a chemically and thermostable solid phase matrix for polymerase chain reaction (PCR) and DNA hybridization assay

DESCRIPTION

The invention relates to a method for producing a chemically and thermally stable support coated with crosslinked streptavidin and / or avidin, the layer (V 2) consisting of crosslinked streptavidin and / or avidin via a crosslinked and biotinylated Velcro substance (VI) for which preferably immunoglobulin gamma (IgG) or bovine gamma globulin is used, in strongly basic buffers with pH values> 11.0 or in a strongly acidic medium with pH values <3.0, is bound to the surface of the solid support material. The invention further relates to the coated carriers themselves and their use in solid-phase hybridization techniques and solid-phase PCR.

Surfaces coated with streptavidin or avidin have already been described in DE 197 24 787 AI (BioTeZ GmbH) and in EP 0 331 127 AI (Boehringer-Mannheim GmbH). Loaded plastic surfaces made of polystyrene, nylon, nitrocellulose and the like serve as carrier materials for such coatings. A disadvantage of these carriers, however, is that they have only a low thermal stability, which makes them unsuitable for a number of applications (e.g. for carrying out PCR).

The invention was therefore based on the object of developing supports with coatings which permit use even at higher temperatures (_> 60 ° C. to 100 ° C.) and which also have both thermostability and chemostability at these temperatures.

Surprisingly, it turned out that it is possible to apply a chemically and thermally stable coating on an inert, to achieve a thermostable solid phase matrix if a biotinylated crosslinking product V 1 with a molecular weight> 500 KD is bound to the surface of the carrier material as a Velcro substance underlayer either in a basic buffer at pH> 11.0 or in a strongly acidic medium at pH <3.0 , and then reacted with crosslinked streptavidin (also recombinant streptavidin), crosslinked avidin or streptavidin-avidin crosslinking products. It became a stable streptavidin / avidin coating with significantly increased binding capacity (at temperatures of 60 ° C the binding capacity was still 93%, compared to commercially available carriers, whose binding capacity at 60 ° C was only about 40%) and increased Thermal and chemical stability achieved on the usually uncharged carrier surfaces.

The invention accordingly relates to a process for producing a stable solid phase matrix containing streptavidin and / or avidin, in which the crosslinking products of streptavidin, avidin or mixtures thereof are bound to the surface of the solid support material via the biotinylated Velcro protein V 1.

The process is characterized in that the crosslinking product V 1, preferably a protein, in particular IgG or bovine gamma globulin, is biotinylated after it has been crosslinked using reagents known per se and then with a thermostable, preferably uncharged carrier material in a strongly basic medium at a pH value> 11 or in a strongly acidic environment at a pH <3.0. Carnonate buffer solutions are preferably used as the strongly basic buffer solutions and citrate buffer solutions are preferably used as the strongly acidic environment.

In comparison to the processes known from the prior art, according to which coatings generally do not run above 9.6 in a basic environment, the increase to a pH value of> 11 in the basic environment or also a decrease below pH 3 in an acidic environment to a thermally and chemically stable coating with high binding capacity if a crosslinked and biotinylated Velcro protein VI and a crosslinked protein from streptavidin, avidin or mixtures of both V2 are used, the coating on thermostable, uncharged carrier surfaces made of plastic, such as polycarbonate and polypropylene.

What is particularly surprising here is the fact that a chemically and thermally stable coating can be observed both when reacting in a strongly basic manner and when reacting in an acidic environment.

After fixation to the support surface, an upper layer of cross-linked streptavidin, avidin or a mixture of these is applied to this underlayer. According to the invention, the preferably uncharged carrier surface consists of plastic, preferably of polycarbonate or polypropylene. The carrier is a coated solid phase on a thermostable plastic surface, preferably made of polycarbonate, polypropylene, silicate, oxidic or else metallic and semiconductor surfaces, as well as combinations of the carrier materials mentioned.

The invention furthermore relates to the stable solid carriers thus prepared, coated with streptavidin and / or avidin per se, which consist of a thermostable, generally uncharged carrier material, a crosslinked and biotinylated protein, preferably IgG or bovine gamma globulin, as an underlayer and crosslinked streptavidin and / or avidin as the upper class.

The new carrier produced according to the invention provides a coated solid phase on a thermostable plastic surface, preferably made of polycarbonate, polypropylene, silicate, oxidic or else metallic and Semiconductor surfaces and combinations of the carrier materials mentioned and has the following advantageous properties:

high binding capacity on the surface (at 60 ° C still 93%), sufficient chemical stability, also against detergents, universal usability and necessary sensitivity of the determination method, - high thermal stability up to 96 ° C, (at 80 ° C still at least 50% Binding capacity)

The solid support described is therefore advantageously suitable for carrying out molecular biological solid-phase reactions and solid-phase hybridization techniques, in particular for hybridization at temperatures> 60 ° C., which was not possible with the previously known streptavidin coatings, and for the first time enables all possible variants of solid-phase reactions in which higher temperatures are required, such as DNA displacement assay, hot start PCR, allele-specific PCR with labeled primers or primer extension assays with labeled dideoxy nucleotides and the like. a ..

The invention is then explained in more detail using exemplary embodiments, to which, however, it should not be restricted.

example 1

Preparation of the Velcro Protein V 1:

120 mg of γ-globulin (cattle) are dissolved in 13 ml of 0.1 M phosphate buffer (pH 7.5). 7.5 mg of disuccinimidyl suberate (DSS, PIERCE), dissolved in 620 μl of dioxane, are added dropwise to the γ-globulin solution over the course of 25 min with stirring. After a reaction time of 4 hours with stirring at room temperature, 6.0 mg of sulfo-NHS-LC-biotin (F. PIERCE) are introduced into the reaction solution in solid form, the biotinylation reagent dissolving immediately. A previous separation by gel filtration can be carried out, but is not necessary, and the reaction is continued immediately. This second synthesis step is also carried out with stirring at room temperature. The reaction time is initially 2.5 hours. The reaction mixture is stored in the refrigerator overnight.

The mixture is worked up by dialysis, first four times against water, then against 0.05 M PBS (pH 7.4) / 0.05% sodium azide. The largely clear retentate is expediently pressed through a 0.6 μm Sartorius filter before the spectrophotometric measurement.

Example 2

Thermostable coating of a solid support

(Microtiter plates):

For the basic coating of the cavities (wells) of a microtiter plate (MTP) made of thermostable polycarbonate, 50 - 250 μl of highly cross-linked biotinylated γ-globulin (cattle) according to Example 1 in a concentration of 10-20 μg / ml in 0.1 M carbonate buffer are added to each well (pH> 11) pipetted and let stand at 25 ° C for 12 hours.

To build up the top layer, the wells are washed with distilled water with the addition of 0.9% NaCl for 12 hours at 25 ° C. with a solution of the highly crosslinked streptavidin (V 2) in 0.05 / 0.15 M PBS buffer (pH 7.2) treated. After washing again with distilled water with the addition of 0.9% NaCl and blocking with distilled water with the addition of 0.1% casein and 0.9% NaCl, the layer is washed with Denhardt's solution in 0.1 M Mc Ilvaine. Buffer (pH 6.0) preserved. After drying, the coated MTP (with desiccant) can be sealed in plastic bags and refrigerated be stored.

Example 3

Determination of the biotin binding capacity

The example describes the determination of the biotin binding capacity of thermally stable coated MTP after different temperature loads. The different molecular sizes of biotin, biotinylated oligonucleotides or other biotinylated compounds, but also chemical influences lead to different results. A competitive enzyme immunoassay using biotin (biotinylated compounds) and biotinylated horseradish peroxidase (biotin-POD) is used as the determination method for the biotin binding capacity of the streptavidin coating. The binding yield is determined by measuring the difference in the liquid free phase. The biotin binding capacity of the carrier layer can be calculated from the difference between the total value (enzyme activity of the biotin-POD used) and the unbound enzyme activity in the protruding free phase of the assay.

To characterize the thermal stability of the coated plates, the wells are filled with a PCR buffer mixture (0.01 M Tris / HCl, 0.15 M NaCl, 0.002 M MgCl ₂ ; pH 7.9) and exposed to fixed temperatures for a fixed period:

25 ° C, 60 ° C, 80 ° C, 90 ° C and 96 ° C.

For comparison, known MTP of polystyrene with a standard coating (biotinylated bovine serum albumin in 0.1 M carbonate buffer (pH 9.6) and streptavidin or avidin) are exposed to the same temperature regime.

The chemothermal stability is determined by loading at 25 ° C with 0.15 M NaOH, at 65 ° C with 0.1 M NH ₄ OH and at 85 ° C with PCR buffer. The chemothermal load on the streptavidin layers is ended by washing the wells with distilled water with the addition of 0.9% NaCl and 0.1% Tween 20 at room temperature and the binding capacity still available for biotin or biotinylated compounds with the aid of a competitive biotin EIA certainly.

In this competitive biotin EIA, reaction mixtures come from various concentrated solutions of d ( ⁺ ) biotin or biotinylated compound (biot oligonucleotide, biot IgG) and a solution of a constant concentration of biotin POD in 0.005 M PBS buffer (pH 7 , 4 / 0.1% BSA). These reaction mixtures (50 - 200 μl) should react with the coated wells of the chemothermally loaded MTP for at least 2 hours at 25 ° C. The biotin-POD content is determined photometrically in the liquid phase using an enzyme immunoassay standard curve of the extinctions of the substrate reaction with o-phenylenediamine (OPD). The extinction of the bound to the solid phase (B) is determined as the difference between the total value of the extinction (T) and the extinction measured in the free phase of the reaction supernatant (F) (B = T-F). Instead of the extinction values, the binding rates B / T are used for the further evaluation by dividing the extinction of the bound phase by the extinction of the total value. After the above experiment has been carried out, enzyme immunoassay standard curves can be drawn up, in which the resulting B / T values are shown as a function of the biotin concentrations used, as shown in the figure below. The biotin binding capacity of the surface layer can be determined with the aid of a curve fit using the Hill model. The differences in the biotin binding capacity between the surfaces coated with thermostable streptavidin according to the invention and the surfaces coated with streptavidin / avidin according to the standard are shown in the following tables:

Claims

00/66770 PATENT REQUIREMENTS

1. A method for producing a chemically and thermostable solid phase matrix for the combination of PCR and hybridization assay, characterized in that a biotinylated crosslinking product V 1 with a molecular weight> 500 KD as a Velcro substance with an inert, thermostable carrier material in a strongly basic buffer at a pH > 11.0 or in a strongly acidic environment at a pH <3.0 and after fixation on the carrier surface, crosslinked avidin, crosslinked streptavidin or crosslinked recombinant streptavidin or mixtures of these are applied as high molecular weight crosslinking products V2 via the biotin bridge become.

2. The method according to claim 1, characterized in that the crosslinking product V 1 is a protein, preferably bovine gamma globulin or immunoglobulin gamma of another species.

3. The method according to claim 1 or 2, characterized in that the surface of the carrier material consists of up to 100 ° C thermostable plastic, preferably of polycarbonate or polypropylene, or that as a carrier also silicate, oxidic or metallic and semiconductor surfaces as well as combinations of the above Carrier materials are used.

4. The method according to any one of claims 1 to 3, characterized in that

Crosslinking of the Velcro substance V 1 and the crosslinking products V 2 by methods known per se using bifunctional linkers known per se, preferably disuccinimidyl suberate (DSS).

5. Chemo- and thermostable solid phase matrix for carrying out PCR and hybridization assays, characterized in that it consists of an inert, thermostable support material and a thermostable layer, which consists of a biotinylated crosslinking product V 1 with a molecular weight> 500 KD in combination with a high molecular weight Crosslinking product V 2 is composed.

6. Solid phase matrix according to claim 5, characterized in that the crosslinking product V 1 is a protein, preferably bovine gamma globulin or immunoglobulin gamma of another species.

7. Solid phase matrix according to claim 5 or 6, characterized in that the crosslinking product V 2 crosslinked avidin, crosslinked streptavidin or recombinant streptavidin or mixtures of the substances mentioned.

8. Solid phase matrix according to one of claims 5 to 7, characterized in that the inert, thermostable carrier material has a plastic surface, preferably made of polycarbonate or polypropylene.

9. Solid phase matrix according to one of claims 5 to 8, characterized in that the carriers are silicate, oxidic, metallic or semiconductor materials and also combinations of the carrier materials mentioned.

10. Use of a solid phase matrix according to one of claims 5 to 9 for PCR and hybridization assays, which are also carried out as a combination on the same solid phase matrix.

11. Use according to claim 10, characterized in that biotinylated oligonucleotides, biotinylated DNA or RNA fragments are fixed both chemically and thermally stable on their surface.

12. Use of the solid phase matrix according to one of claims 10 to 11, characterized in that all variants of solid phase hybridization techniques are carried out on it.

13. Use of the solid phase matrix according to claim 5 to 12, characterized in that hybridization, denaturation, chain extension or other reaction steps also take place at temperatures> 60 ° C.