WO2000071698A2

WO2000071698A2 - Mixture for neutralizing enzyme inhibitors

Info

Publication number: WO2000071698A2
Application number: PCT/EP2000/003933
Authority: WO
Inventors: Christian Reiter
Original assignee: Connex Gesellschaft Zur Optimierung Von Forschung Und Entwicklung Mbh
Priority date: 1999-04-30
Filing date: 2000-05-02
Publication date: 2000-11-30
Also published as: WO2000071698A3; AU4754300A

Abstract

The invention relates to a mixture of albumin and/or spermidine and at least one antibody, preferably a mixture of albumin, preferably bovine serum albumin, spermidine and an antibody, or a solution containing this mixture. Said mixture can be used for producing a reaction buffer or for coating reaction vessels. As a result, substances that inhibit enzymes used for manipulating nucleic acids and that are normally present in samples, are neutralized. This considerably improves the efficiency of enzymatically catalyzed nucleic acid modifications, for example polymerize chain reactions (PCR), or in the case of certain samples, enables these types of reactions to take place. The invention also relates to a method for producing the inventive reaction vessels.

Description

Mixture for neutralizing enzyme inhibitors

The present invention relates to a mixture of albumin and / or spermidine and at least one antibody, preferably a mixture of albumin, preferably bovine serum albumin, spermidine and an antibody or a solution containing this mixture. This mixture can be used to produce a reaction buffer or to coat reaction vessels. This can neutralize substances that inhibit enzymes used for the manipulation of nucleic acids and are normally present in samples, thereby reducing the efficiency of enzymatically catalyzed nucleic acid modifications, e.g.

Polymerase chain reaction (PCR), is greatly increased or such reactions are only possible with certain samples. Finally, the invention also relates to a method for producing the reaction vessels according to the invention.

In order to be able to enzymatically manipulate nucleic acids, for example genomic DNA, from biological material, for example to be able to amplify them by means of PCR, it is usually necessary to digest the sample, which, for example, involves heating the sample and enriching and purifying the nucleic acid in order to thereby inhibit the enzymatic modification reaction to destroy or remove. Common purification methods are, for example, ion exchange chromatography and digestion with proteases, for example proteinase K. Known inhibitors of such reactions include hemin, humic acids, heparin, gall salts and certain sugar structures. In addition, there are other inhibitors that have not yet been characterized. However, the cleaning methods described above have the disadvantage that they do not always remove the inhibitors to a sufficient extent, which is then a requires further purification of the nucleic acids. The associated disadvantages include not only the high expenditure of time and money, but also the loss or degradation of the nucleic acid to be manipulated (especially with high molecular weight DNA or RNA).

Another common measure is to use only a fraction of the starting material in order to dilute the inhibitors. However, this can lead to insufficient nucleic acid molecules in the sample for the enzymatic reaction. It has recently been described that the presence of BSA or spermidine in the reaction buffer in PCR can increase their efficiency, but the observed effect has not yet been satisfactory (Wan and Wilkins, PCR Methods and Applications (1993), Cold Spring Harbor Laboratory, Cold Spring Harbor , New York; pages 208-210; Deuter et al., Nucleic Acids Res. 23 (1955), 3800-3801; Forbes and Hicks, J.Clin.Microbiol. 34 (1996), 2125-2128).

The invention is therefore based on the object of providing means with which inhibitors of enzymes used in the manipulation of nucleic acids can be effectively neutralized.

The object described above is achieved by the embodiments characterized in the patent claims. It has surprisingly been found that a mixture of albumin and / or spermidine and at least one antibody, preferably a mixture of albumin, spermidine and an antibody, can very effectively neutralize inhibitors of the enzymes used in the manipulation of nucleic acids, synergistic effects being observed, this mixture either being a constituent of the reaction buffer or being in the form of a coating on the reaction vessel. This has the additional advantage that established protocols do not have to be adapted for the desired enzymatic reaction, so that there is no additional effort for the user. The present invention thus relates to a mixture of albumin, spermidine and an antibody. The term "antibody" used here refers to both a polyclonal and monoclonal antibody. The mixture can also contain more than one antibody. The albumin used is preferably bovine serum albumin and the antibody used is an antibody of the IgG class. The experiments carried out showed that the antibody has a clear inhibitory effect, regardless of its specificity, which exceeds that of a normal protein, for example BSA. The observed effect (see Example 5) with regard to the individual components (spermidine, BSA, antibody) is not additive, but synergistic. IgG antibodies have proven to be particularly effective.

The invention further relates to a solution which contains the mixture according to the invention. These are preferably solutions in which the concentration of albumin is 0.1 to 2 μg / μl, of spermidine 0.05 to 1 μg / μl and the antibody 0.05 to 1 μg / μl. Solutions in which the concentration of albumin is 0.4 μg / μl, of spermidine 0.2 μg / μl and of the antibody 0.2 μg / μl are particularly preferred.

The mixture according to the invention or the solution containing it is advantageously to be used in the manipulation of nucleic acids contained in biological samples, in particular for use in routine diagnostic applications. These are used particularly advantageously when the biological samples are materials which are known to contain high concentrations of inhibitors, for example blood, blood components, soil samples, tissue homogenates, fixed tissue, lavage, cerebrospinal fluid, stool for the detection of Helicobacter pylori and urine, preferably for the detection of chlamydia. In addition, when using the mixture according to the invention, success can be achieved in various fields of medicine in which a diagnosis (based on the problems described above) was not possible at all via PCR, for example when detecting individual tumor cells in

Stem cell preparations for autologous bone marrow transplants, for the early detection of relapses in leukemia or for the detection of single cells of colon tumors in the stool. In addition, the mixture according to the invention can be used in the detection of sepsis pathogens via PCR, which is thereby much faster and also more sensitive than the detection of the pathogens via cultivation.

The present invention thus also relates to the use of the mixture according to the invention or of the solution according to the invention for

Production of a reaction buffer or for coating

Reaction vessels for neutralizing inhibitors of enzymes used in the manipulation of nucleic acids.

The term "enzymatic manipulation of nucleic acids" used here refers to any enzymatically catalyzed reaction in which nucleic acids, preferably DNA or RNA, are enzymatically changed. These include polymerization reactions, for example the polymerase chain reaction (PCR) or the ligase chain reaction (LCR), ligation reaction, phosphorylation reactions , Dephosphorylation reactions, coupling reactions, preferably labeling reactions of nucleic acids with detectable markers, for example the addition of radioactive or non-radioactive labels at the 5 ^' or 3 ^' end of an RNA or DNA, the fragmentation of DNA, for example via restriction reactions, the preferred enzymes Polymerases, ligases, kinases, nucleases, restriction endonucleases and transferases are preferably the mixture or solution according to the invention is used when carrying out a polymerase chain reaction moderate mixture or the solution for neutralizing inhibitors of thermostable DNA polymerases, for example Taq DNA polymerase, is used. The present invention further relates to reaction vessels which are characterized in that they have a coating with the mixture according to the invention or have been coated with the solution according to the invention. The reaction vessels according to the invention can consist of different materials, preferably those materials as are used for the production of reaction vessels that are used for the enzymatic manipulation of nucleic acids. The reaction vessels preferably consist of polypropylene, polyethylene, polycarbonate, polystyrene or borosilicate.

The reaction vessels according to the invention are preferably 0.2 ml-0.5 ml or 0.65 ml reaction vessels in the form known to the person skilled in the art (for example in the form of so-called “eppendorft tubes”), strips with 8 or 12 single-use reaction vessels connected to one another a '200 _μ l, for plates with 12, 24, 48, 96, 192, 384, 768 or 1536 cavities, for glass capillaries or for silicon “wafers” with up to several thousand reaction compartments.

In a preferred embodiment, the reaction vessels according to the invention are additionally containing enzymes such as. B. coated polymerases, ligases or nucleases, which are involved in the desired manipulation of the nucleic acid. This offers the additional advantage that in many cases the user only has to add the sample, the buffer and, if necessary, the necessary dNTPs to the reaction vessel. However, the reaction vessels can, for example, already be coated with NTPs. This is preferably a thermostable “hot start” Taq polymerase, the polymerase activity of which is reversibly inhibited by the occupation of the nucleotide binding region by antibodies or aptamers until the first heat denaturation.

The manufacture of reaction vessels with the coating described above can be carried out by a person skilled in the art using standard methods, depending on the material of the reaction vessel walls. So can for example, proceed as follows:

_β Mixing albumin, spermidine and the antibody in ultrapure water.

. Applying the mixture to the inner walls of the reaction vessels, for example on the bottom of the reaction vessels in the area of the usable space depending on the intended reaction volumes. Depending on the material of the reaction vessel, it may be necessary to first coat the surface of the reaction vessel with an adhesion-promoting agent, e.g. Binding silane to treat.

• Drying the reaction vessels, for example in a climate controlled cabinet at 38 ^° C +/- 3 ° C without vacuum until a residual moisture of 5% (preferably 2-10%) is undershot.

• Packaging of the reaction vessels (moisture-proof).

The following examples illustrate the invention. The explanations for the figures result from the tables shown in the examples.

The examples relate to the use of Panscript DNA polymerase for the PCR. In addition, however, the following polymerases were used in the PCR with comparably good results:

Pan System Taq Oplymerase, PAN-Systems GmbH, Adolf-Braun-Str. 45, 90429 Nuremberg; Röche Taq Polymerase, Röche Diagnostics GmbH, Sandhofer Strasse 1 1 6, 68305 Mannheim; Perkin Elmer AmpliTaq DNA Polymerase, PE Corporation, 761 Main Avenue, Norwalk, CT 06859, USA; MBI Fermentas, MBI Fermentas native with BSA, MBI Fermentas (Rec), FERMENTAS AB, V. Graiciuno 8, Vilnius 2028, Lithuania; Biometra Finnzymes. Finnzymes Oy; Riihitontuntie 14 B, PO BOX 148, F1N-02201 ESPOO, FINLAND; Amersham Taq Polymerase, Amersham Pharmacia, Biotech AB, SE-751 84 Uppsala, Sweden, LTI Taq DNA Polymerase recombinant, Life Technologies Inc., 9800 Medical Center Drive, Rockville, MD 20850, USA; Clontech Advantaq Plus Hotstart, Clontech Laboratories Inc., 1020 East Meadow Circle, Palo Alto, CA 94303 USA; Promega TaqStorage Buffer A, Promega Taq DNA Polymerase Buffer B, Promega Corporation, 2800

Woods, Hollow Road, Madison, Wl 5371 1-5399, USA; Hybaid proof

Sprinter (Taq / Pwo), Hybaid AGS-Gold-Taq, Hybaid Limited, Action Court,

Ashford Road, Ashford, Middlesex, TW1 5 1 XB, United Kingdom;

Eppendorf Taq (Rec), Eppendorf Vertrieb Deutschland GmbH,

Friedensstrasse 1 1 6, 51 145 Cologne, Germany; Advanced

Biotechnologies Thermoprime Plus DNA-Polymerase, Advanced

Biotechnologies Ltd., ABgene House, Blenheim Road, Epsom, Surrey KT1 9

9AP, United Kingdom; Peqlab SAWADY Taq, peqlab biotechnology

GmbH, Am Weichselgarten 7, D-91058 Erlangen; TaKaRa Taq DNA

Polymerse (Rec), Takara Biomedical Europe S.A., Europarc des

Barbanniers, 6, Place du Village, 92230 Gennevilliers, France; Eurogentec

EuroTaq DNA Polymerase, Eurogentec SA, Parc scientifique du Sart-

Tilman, 4102 Seraing, Belgium; Qiagen HotStarTaq DNA Polymerase,

Qiagen (Rec) HotStarTaq DNA Polymerase with Q-Solution, QIAGEN GmbH

- Germany, Max-Volmer-Strasse 4, 40724 Hilden; Stratagene Taq,

Stratagene (Rec) Taq 2000, Stratagene Inc., 1 101 1 North Torrey Pines

Road, La Jolla, CA 92037, USA; Sigma (Rec) REDTAQ, Sigma (Rec) Taq,

Sigma Corp., 3050 Spruce Street, St. Louis, MO 63103, USA; Eurobiotaq

(Rec) Taq; DNAmp (Rec) Taq, DNAmp Ltd, St. Alban's House, 40

Lynchford Road, Farnborough GU1 4 6EF, United Kingdom; Epicenter

Masteramp Taq, Epicenter Technologies Inc., 1402 Emil Street, Madison,

Wl 5371 3, USA, Appligene Taq, Appligene Oncor SA, Parc d Innovation,

B.P. 72, F-67402 llkirch Cedex, France; MoBiTec Taq, MoBiTec

GmbH, Lotzestrasse 22a, D-37083 Göttingen, Germany;

The antibody NF5E6 was deposited with the DMSZ on January 12, 2000 (accession number DSM ACC2436). With the same results, the antibody EDD1D9 can be used analogously to NF5E6. This antibody was also deposited on January 12, 2000 at the DSMZ with the accession number DSM ACC2435. example 1

The production of coated reaction vessels

The following PCR reaction tubes were used for coating: Advanced Biotechnologies LTD, Cat. #. AB-0350, 0.5 ml PCR reaction tubes.

The following components were mixed in ultrapure water so that the following concentration of the individual components was present in the 0.5 ml reaction vessels:

Antibody: NF5E6 of connex, 5 _μ g / 25 _μ l reaction volume spermidine: Sigma, order no. S-0266, 5 _μ g / 25 _μ l reaction volume BSA: Boehringer Mannheim, order no. 711,454, 10 _μ g / 25 _μ l

Reaction volume This mixture was applied to the bottom of the reaction vessels in the area of the usable space depending on the intended 25 _μl reaction volumes.

The tubes were dried for 40 h in a "Weiss" climatic chamber at 38 ° C +/- 1 ° C. After 24 h there was a relative humidity of <2%. Finally, the reaction vessels were closed and packed in a moisture-proof manner.

Example 2

Detection of a microorganism in stool samples when using coated reaction vessels compared to uncoated ones

Reaction vessels

The following PCR reaction tubes were used: Advanced Biotechnologies LTD, Cat. #. AB-0350, 0.5 ml PCR reaction tubes

The reaction tubes were coated as follows: Antibody: NF5E6 from Connex, 5 μg / 25 μl reaction volume Spermidine: Sigma, order no. S-0266, 5 µg / 25 µl reaction volume

BSA: Boehringer Mannheim, order no. 711454, 10 μg / 25 μl

Reaction volume

Thermo Cycler used: Biometra - Personal Cycler, Vers. 3.01 bc, (c)

Biotron 1995, serial no .: 9510443

Target microorganism used: Helicobacter pylori

Gene area: 16s rRNA gene

Length of the amplified fragment: 330 bp

Primers used: Hp-R994 (5 'GTT GGA GGG CTT AGT CTC TCC 3')

Hp-R1284 (5 'CCT GTT AGC AAC TAA GAA AGG 3')

PCR components used:

Conditions for PCR amplification:

94 ° C 2 min 1 x

94 ° C 1 min

58 ° C 30 sec |> 35 x 72 ° C 45 sec

72 ° C 5 min 1 x

4 ° C ∞

Execution:

Various eight human stool samples (20 mg) were mixed with 40 _μ l of sodium chloride solution (0.9%) and 10 _μ l Helicobacter pylori suspension (Complies 5 x 10 ⁵ cells) was added. (Cat "QIAamp Tissue Kit", 293004) was then using a Qiagen kit DNA extraction carried out. The extraction will based on the Qiagen protocol "Isolation of genomic DNA from faeces". The elution was carried out in 50 _μ l Purified water (autoclaved). 2 _μl per 25 _μl PCR reaction volume was used from the extraction samples. To detect the target microorganism (Helicobacter pylori), a PCR was carried out with specific primers. Each DNA sample was placed in reaction vessels with and without a coating The amplified fragments were detected by means of electophoretic separation in an agarose gel (2%). This electrophoretic separation produces characteristic bands of the same fragment length. These bands were stained with ethidium bromide (Merck) and made visible in ultraviolet light .

The results are shown in Table 1 and Figure 1:

Table 1

Result:

All stool samples were detected with coated reaction vessels (100%), but this was not possible when using reaction vessels without a coating.

Example 3

Detection of the DNA of a microorganism

Comparison of several substances for the neutralization of enzyme inhibitors in the presence and

Absence of the inhibitor hemin

The following PCR reaction vessels were used:

Advanced Biotechnologies LTD, Cat #. AB-0350, 0.5 ml PCR tubes

The inhibitor was used as follows:

Hemin: Sigma, 1 μg in 25 ml reaction volume

The neutralizers were used as follows:

Antibody: NF5E6 from Connex, 20 μg in 25 μl reaction volume (addition in liquid form) BSA: Boehringer Mannheim, order number 711454, 20 μg in 25 μl

Reaction volume (addition in liquid form) Spermidine: Sigma, Bestelinr. S-0266, 20 μg in 25 μl reaction volume (addition in liquid form) Mixture: antibody: spermidine: BSA in a ratio of 1: 1: 2, a total of 20 μg in 25 μl

Reaction volume (coating) Thermocycler used: Biozym MJ, Research PTC 200

Target DNA used: Helicobacter pylori DNA

Gene area: 16s rRNA gene

Length of the amplified fragment: 330 bp

Primers used: Hp-R994 (5 ^' GTT GGA GGG CTT AGT CTC TCC 3 ^' )

Hp-R1284 (5 ^' CCT GTT AGC AAC TAA GAA AGG 3 ^' )

PCR components used:

Stocklsg. Manufacturer Conc. Per 25 μl

Reaction volume

10xPCR buffer PAN- 1 xPCR buffer

Systems GmbH

MB-1 101000

MgCl ₂ [50 M] PAN-3 mM

Systems GmbH

MB-9120001 dNTPs [1 2.5mM] PAN system 0.5mM

GmbH

MB-9145001

Hp-R994 [1 pmol / µl] MWG-Biotech 3pmol

GmbH

Hp-R1284 [1 pmol / µl] MWG-Biotech 3 pmol

GmbH

Panscript DNA [5E / A I] PAN-1 E Polymerase Systems GmbH

MB-1 101000

Conditions for PCR amplification:

94 ° C 2min 1x

94 ° C 1 min

58 ° C 30sec 35x

72 ° C 45sec

72 ° C 5min 1x

4 ° C infinite Execution:

For each PCR reaction, 3 ng of DNA, which were isolated from a Helicobacter pylori culture using the "High Pure PCR Template Preparation" kit from Boehringer Mannheim, each with the individual components of the mixture according to the invention or with the mixture used according to the invention in the presence and absence of Inhibitos Hemin tested. The individual components were added in liquid form for the reaction. The mixture according to the invention was used in the form of the previously coated reaction vessels. Specific primers were used to amplify the target area on the Hp DNA. By electrophoretic separation in an agarose gel (2% The amplified fragments were detected by this electrophoretic separation to produce characteristic bands of the same fragment length, which were stained with the aid of ethidium bromide (Merck) and visualized in ultraviolet light.

The results are shown in Table 2 and Figure 2:

Table 2

Result:

When H.p. DNA in the presence of the inhibitor hemin, the neutralizer spermidine shows no positive effect. The neutralizers antibody and BSA show a positive effect. The positive effect of the individual components is significantly exceeded by using the coating with the mixture according to the invention.

Example 4

Detection of the DNA of a microorganism

Absence of the inhibitor heparin

The following PCR reaction vessels were used:

Advanced Biotechnologies LTD, Cat #. AB-0350, 0.5 ml PCR tubes

The inhibitor was used as follows:

Heparin: Ratiopharm, heparin sodium 25000 Ratiopharm, 1/3 I.U. in 25 μl reaction volume

The neutralizers were used as follows:

Reaction volume (addition in liquid form) Spermidine: Sigma, order number S-0266, 20 μg in 25 μl reaction volume (addition in liquid form) Mixture: Antibody: spermidine: BSA in a ratio of 1: 1: 2, a total of 20 μg in 25 μl

Reaction volume (coating)

Thermocycler used: Biozym MJ, Research PTC 200 Target DNA used: Helicobacter pylori DNA Gene area: 16s rRNA gene

Length of the amplified area: 330 bp

Primers used: HPR 994 / HPR 1284

PCR components used: see example 3

Conditions for the PCR amplification: see example 3

Execution:

For each PCR reaction, 3 ng of DNA, which were isolated from a Helicobacter pylori culture using the "High Pure PCR Template Preparation" kit from Boehringer Mannheim, each with the individual components of the mixture according to the invention or with the mixture used according to the invention in the presence and absence of Inhibitors heparin tested. The individual components were added in liquid form for the reaction. The mixture according to the invention was used in the form of the previously coated reaction vessels. Specific primers were used to amplify the target area on the Hp DNA. By electrophoretic separation in an agarose gel (2% The amplified fragments were detected by this electrophoretic separation to produce characteristic bands of the same fragment length, which were stained with the aid of ethidium bromide (Merck) and visualized in ultraviolet light.

The results are shown in Table 3 and Figure 3: Table 3:

Result:

The use of BSA or antibodies alone shows no abolition of the inhibitory effect of heparin. The inhibitory effect of heparin on the amplification can only be eliminated by adding spermidine or using the mixture according to the invention. The use of the mixture according to the invention leads to a significantly stronger neutralization of the inhibitory effect of the heparin than the use of the single component spermidine.

Example 5

Detection of the DNA of a microorganism

Comparison of several substances for the neutralization of enzyme inhibitors in the presence and absence of the inhibitor gall salts

The following PCR reaction vessels were used:

Advanced Biotechnologies LTD, Cat #. AB-0350, 0.5 ml PCR tubes The inhibitor was used as follows:

Gall salts: Sigma, order number: B8756, 10 μg in 25 μl reaction volume

The neutralizers were used as follows:

Reaction volume (coating)

Thermocycler used: Biozym MJ, Research PTC 200

Target DNA used: Helicobacter pylori DNA

Gene area: 16s rRNA gene

Length of the amplified area: 330 bp

Primers used: HPR 994 / HPR 1284

PCR components used: see example 3

Conditions for the PCR amplification: see example 3

Execution:

For each PCR reaction, 3 ng of DNA, which were isolated from a Helicobacter pylori culture using the "High Pure PCR Template Preparation" kit from Boehringer Mannheim, each with the individual components of the mixture according to the invention or with the mixture used according to the invention in the presence and absence of Inhibitors tested gall salts. For the reaction, the individual components were added in liquid form Mixture was used in the form of the reaction vessels coated in advance.

To amplify the target area on the H.p. DNA, specific primers were used. The amplified fragments were detected by electrophoretic separation in an agarose gel (2%). Through this electrophoretic

Separation creates characteristic bands of the same fragment length. This

Bands were stained using ethidium bromide (Merck) and ultraviolet

Light made visible.

The results are shown in Table 4 and Figure 4:

Table 4:

Result:

As single components, BSA and spermidine show no neutralization of the inhibitory effect of gall salts. The addition of antibody or the addition of the mixture according to the invention have a neutralizing effect on the inhibitory effect of the gall salts. The neutralizing effect of the mixture according to the invention but is significantly stronger than the effect of the single component antibody.

Example 6

Effect of the coating depending on the concentration of the nucleic acid present in the sample

It has a zero stool sample with a target microorganism is added, so that after extraction of DNA and 2 _μ l sample insert per 25 l PCR mixture _μ corresponding to 0.1 to 10,000 genomes for the PCR reaction are available. It was investigated what influence the mixture according to the invention or the coating has on this increasing DNA concentration.

The coating of the reaction vessels was as follows: Antibody: NF5E6 of connex, 05 _μ g / 25 _μ l reaction volume spermidine: Sigma, order no. S-0266, 05 _μ g / 25 _μ l reaction volume BSA: Boehringer Mannheim, order no. 711,454, 10 _μ g / 25 _μ l reaction volume

Thermo Cycler used: Biometra - Personal Cycler, Vers. 3.01 bc, (c) Biotron

1995, serial no .: 9510443

Target microorganism used: Helicobacter pylori

Gene area: 16s rRNA gene

Length of the amplified fragment: 330 bp

Primers used: Hp-R994 (5 'GTT GGA GGG CTT AGT CTC TCC 3')

Hp-R1284 (5 'CCT GTT AGC AAC TAA GAA AGG 3') PCR components used:

Conditions for PCR amplification:

94 ° C 2 min 1 x

94 ° C 1 min

58 ° C 30 sec ^ 35 x

72 ° C 45 sec

72 ° C 5 min 1 x

4 ° C CO

Execution:

Six human stool samples (each 5 mg) were added with 15 ul Natriumcloridlösung (0.9%), and each 10 _μ l Helicobacter pylori suspension (There was a Helicobacter pylori-dilution series of 1 x 10 ¹ to 1 x 10 ⁶ cells created) was added . Subsequently, using a Qiagen kit ( "QIAamp Tissue Kit", Cat.No. 293004)) conducted a DNA extraction. The extraction will based on the Qiagen protocol "Isolation of genomic DNA from faeces" Elution was carried out in 50 _μ l AE buffer (Qiagen). Of the extraction samples was 2 _μ l used per 25 _μl reaction volume. After the DNA extraction and after use in the PCR, the following Helicobacter pylori genome number was found:

0.1 genome / 25 _μl PCR reaction volume 1 genome / 25 _μl PCR reaction volume

10 genome / 25 _μ l PCR reaction volume 100 genome / 25 _μ l PCR reaction volume 1000 genome / 25 _μ l PCR reaction volume 10000 genome / 25 _μ l PCR reaction volume

In order to detect the target microorganism (Helicobacter pylori), a PCR with specific primers was carried out. Each DNA sample was amplified in reaction vessels with and without a coating and the effect of the coating was checked. The amplified fragments were detected as described in Example 2.

The results are shown in Table 5 and Figure 5.

Table 5

Result:

The coating enables the target microorganism to be detected at lower DNA concentrations (two orders of magnitude earlier than without a coating).

Example 7 Use of an antibody from another sub-class in the mixture according to the invention

In this experiment it was shown that the NF5E6 antibody in the mixture according to the invention is interchangeable with other IgG antibodies. For this purpose, different IgG antibodies were mixed with BSA and spermidine in the same concentrations as in the 10 × mixture according to the invention and used in the PCR reaction.

A direct sex determination on human heparinized whole blood without previous DNA extraction was chosen as the test system, since whole blood contains a high concentration of inhibitors. Molecular biological gender determination is based on the detection of a gender-specific gene, which is located on the X or Y chromosome. If only the female gene can be detected, it is a female individual (two X chromosomes), but if both genes are found, it is a male individual (one X and one Y in the male).

Components of the mixture according to the invention with the antibody NF5E6: The following components were mixed in ultrapure water so that the following in the 10 × mixture according to the invention (PCR reaction volume: 20 μl)

Concentrations of the individual components:

Antibodies: NF5E6, Connex GmbH, 2mg / mL

Spermidine: Sigma, order no. S-0266, 2mg / mL

BSA: Boehringer Mannheim, order no., 711454, 4mg / mL

Components for the PCR reaction:

The following PCR reaction vessels were used: Abgene LTD, Cat. # AB-0773, 0.2mL flat caps; Thermo Cycler used: Biometra - Personal Cycler, Vers. 3.01 bc, serial no .: 9510443; Sample material used for DNA amplification: human heparinized whole blood; Blood collection system used: Sarstedt Monovette, Li-Heparin 10-30IE / mL, order no. 06 / 1565.001, Sarstedt cannula, GX1, order no .: 85 / 1,160;

Amplified gene regions: amplification of the X and Y-specific single copy gene amelogenin (AMG); Gene, 167 (1995) 327-332. Length of the amplified fragments: X chromosome 330 bp

Y chromosome 218bp Primers used:

AMG-Universal (AMG-univ., 5 ^' -CAGCTTCCCAGTTTAAGCTCT-3 ^' ) AMG-X (AMG-X, 5 ^' -TCTCCTATACCACTTAGTCACT-3 ^' )

AMG-Y (AMG-Y, 5 ^, -GCCCAAAGTTAGTAATTTTACCT-3 ^, )

PCR master mix: component manufacturer final concentration

10xKCI-PCR-Buffer (PAN-System GmbH MB-1101000) 1 X MgCI ₂ (50mM) (PAN-System GmbH MB-1101000) 2.5mM dNTPs (12.5mM) (PAN-System GmbH MB- 9140001) 300μM AMG-U . (1 pmol/μL) (Metabion GmbH) 100nM AMG-X (1 pmol/μL) (Metabion GmbH) 50nM AMG-Y (1 pmol / μL) (Metabion GmbH) 50nM

10 X mixture according to the invention (Connex GmbH) 1 X

Pan DNA polymerase (PAN-System GmbH MB-1101000) 0.033U / μL

In this experiment, the antibody NF5E6 in the mixture according to the invention was replaced by another IgG antibody. The other IgG antibodies were used in the PCR reactions with BSA and spermidine in the same concentration as the antibody NF5E6.

Mixture "4H5" (final concentration in the PCR mixture):

Antibody lgG1 4H5 # DA990517-1, Connex GmbH (0.2μg / μl)

Spermidine Sigma, order no. S-0266 (0.2μg / μl)

BSA Boehringer Mannheim, order no., 711454 (0.4μg / μl)

Mixture "F4" (final concentration in the PCR mixture):

Antibody lgG2a F4 # DA981110-3, Connex GmbH (0.2μg / μl)

Spermidine Sigma, order no. S-0266 (0.2μg / μl)

BSA Boehringer Mannheim, order no., 711454 (0.4μg / μl)

Mixture "1 B5" (final concentration in the PCR mixture):

Antibody lgG1 1B5 # AR991220, Connex GmbH (0.2μg / μl)

Spermidine Sigma, order no. S-0266 (0.2μg / μl)

BSA Boehringer Mannheim, order no., 711454 (0.4μg / μl)

Mixture "2H10" (final concentration in the PCR mixture):

Antibody lgG2a 2H10 # CL000202-1 Connex GmbH (0.2μg / μl)

Spermidine Sigma, order no. S-0266 (0.2μg / μl)

BSA Boehringer Mannheim, order no., 711454 (0.4μg / μl)

Conditions for PCR amplification

95 ° C 4 30 min 1x

94 ° C 45 min

50 ° C 45 min 8X

72 ° C 1 00 min

93 ° C 30 min 54 ° C: 40 min}> 32X

72 ° C: 45 min J

6 ° C oo

Primer system:

The multiplex polymerase chain reaction is a PCR variant in which two or more loci are amplified simultaneously in one PCR reaction. The current method is based on the amplification of the single copy amelogenin gene (AMG). The gene of the Y allele has a deletion in the first intron compared to that of the X allele. This deletion enables specific PCR reactions to differentiate between X and Y alleles. Amplification with three primers, two of which are allele-specific, allows unique identification of both the X and Y alleles in a single PCR reaction.

With the 5'-primer (HAMG-univ, 5'-CAGCTTCCCAGTTTAAGCTCT-3 ') an amplification of the X and the Y-AMG allele is possible. The 3 'primers, on the other hand, are specific for both the X and the Y allele. The 3 'X-specific primer (HAMG-X, 5'-TCTCCTATACCACTTAGTCACT-3') is derived from the sequence deleted in the Y chromosome and the 3'Y-specific primer (HAMG-Y, 5'- GCCCAAAGTTAGTAATTTTACCT-3 ' ) covers the area that connects the two deletion endpoints. The 330 bp (X chromosome) and the 218 bp (Y chromosome) PCR products are then analyzed using agarose gel electrophoresis. Usually three primers are used simultaneously in one reaction in a reaction vessel.

Test execution:

Heparinized whole blood (heparin concentration 10-30 IU / ml) was used for the PCR reaction. The nucleated cells present in the blood served as a DNA resource; the heparinized blood was also a complex mixture of inhibitors (eg: hemoglobin, heme, heparin, etc.). The blood was diluted with H ₂ 0 (0, 1: 5, 1: 10, 1: 20) and 1 μL of each was added to 19 μL master mix. An approach without inhibitors served as a positive control; 10ng of human genomic DNA (male) were used as the template. For each PCR reaction, a direct comparison was made with the mixture according to the invention, H ₂ O (without the mixture according to the invention) and one of the mixtures listed above with further IgG antibodies. The amplified fragments were detected by electrophoretic separation in an agarose gel (2%). The characteristic band pattern was stained with ethidium bromide (Merck).

Results:

Direct comparison of the mixture according to the invention with antibody NF5E6 and mixture “4H5”:

When using the mixture according to the invention with antibody NF5E6, a complete inhibition of the PCR reaction was found when using a 1: 1 and a 1: 5 blood dilution. With a 1:10 blood dilution, only the DNA fragment which is assigned to the Y-chromosomal allele was amplified. An amplification of both alleles could already be achieved with a 1:20 blood dilution.

When using the 4H5 mixture, an amplification with a 1:10 blood dilution and a complete amplification of both alleles with a 1:20 blood dilution were also already shown.

Without adding the mixture according to the invention with antibody NF5E6 or the mixture 4H5 to the reaction mixture, the PCR reaction was completely inhibited at each of the blood concentrations used. The results are shown in FIG. 6:

M markers

1 neg. Control with the mixture according to the invention

2 Pos. Control with mixture according to the invention

3 1 μl of heparinized blood with the mixture according to the invention

4 1 μl from 1: 5 dilution (blood) with the mixture according to the invention.

5 1 μl from 1:10 dilution (blood) with the mixture according to the invention.

6 1 μl from 1:20 dilution (blood) with the mixture according to the invention.

7 neg. Control without the mixture according to the invention. 8: Pos. Control without the mixture according to the invention.

9: 1 μl of heparinized blood without the mixture according to the invention.

10 1 ul from 1: 5 dilution (blood) without the mixture according to the invention. 11 1 μl from 1:10 dilution (blood) without the mixture according to the invention. 12 1 μl from 1:20 dilution (blood) without the mixture according to the invention. 13 neg. Control with antibody 4H5 14 Pos. Control with antibody 4H5 15 1 μl heparinized blood with antibody 4H5 16 1 μl from 1: 5 dilution (blood) with antibody 4H5 17 1 μl from 1:10 dilution (blood) with antibody 4H5 18 1 μl from 1:20 dilution (blood) with antibody 4H5

Direct comparison of the mixture according to the invention with antibody NF5E6 and mixture “F4”:

When using the mixture according to the invention with antibody NF5E6, a 1: 1, 1: 5 and 1:10 blood dilution was found to be complete

Inhibition of the PCR reaction. From a 1:20 blood thinning one could

Amplification of both alleles can be achieved.

When using mixture F4, a complete amplification of both alleles was shown from a 1:10 blood dilution.

Without addition of the mixture according to the invention with antibody NF5E6 or the

Mixture F4 to the reaction mixture, the PCR reaction was completely inhibited at each of the blood concentrations used. The results are shown in FIG. 7:

M: marker

Neg. Control with the mixture according to the invention.

Control with mixture according to the invention.

1 μl of heparinized blood with the mixture according to the invention

1 μl from 1: 5 dilution (blood) with the mixture according to the invention.

1 μl from 1:10 dilution (blood) with the mixture according to the invention.

1 μl from 1:20 dilution (blood) with the mixture according to the invention. 7 neg. Control without the mixture according to the invention.

8 pos. Control without the mixture according to the invention.

9 1 μl of heparinized blood without the mixture according to the invention.

10 1 ul from 1: 5 dilution (blood) without the mixture according to the invention.

11 1 μl from 1:10 dilution (blood) without the mixture according to the invention.

12 1 μl from 1:20 dilution (blood) without the mixture according to the invention.

13 neg. Control with antibody F4.

14 Pos. Control with antibody F4.

15 1 μl of heparinized blood with antibody F4.

16 1 μl from 1: 5 dilution (blood) with antibody F4.

17 I ul from 1:10 dilution (blood) with antibody F4.

18 1 μl from 1:20 dilution (blood) with antibody F4.

Direct comparison of the mixture according to the invention with antibody NF5E6 and mixture “1B5”:

When using the mixture according to the invention with antibody NF5E6, a 1: 1 and a 1: 5 blood dilution was found to be complete

Inhibition of the PCR reaction. Even with a 1:10 blood thinning one could

Amplification of both alleles can be achieved.

When using mixture 1B5, an amplification with a 1: 5 blood dilution and a complete amplification of both alleles with a 1:10 blood dilution were also already shown.

Mixture 1 B5 to the reaction mixture, the PCR reaction was completely inhibited at each of the blood concentrations used. The results are shown in FIG. 8:

M: marker

Neg. Control with the mixture according to the invention

Control with mixture according to the invention

1 μl of heparinized blood with the mixture according to the invention

1 μl from 1: 5 dilution (blood) with the mixture according to the invention. 5: 1 μl from 1:10 dilution (blood) with the mixture according to the invention.

6: 1 μl from 1:20 dilution (blood) with the mixture according to the invention.

7: Neg. Control without the mixture according to the invention.

8: Pos. Control without the mixture according to the invention.

9: 1 μl of heparinized blood without the mixture according to the invention.

10: 1 μl from 1: 5 dilution (blood) without the mixture according to the invention.

11: I ul from 1:10 dilution (blood) without the mixture according to the invention.

12: 1 μl from 1:20 dilution (blood) without the mixture according to the invention.

13: Neg. Control with antibody 1 B5

14: Pos. Control with antibody 1B5

15: 1 ul heparinized blood with antibody 1 B5

16: 1 μl from 1: 5 dilution (blood) with antibody 1 B5

17: 1 μl from 1:10 dilution (blood) with antibody 1 B5

18: 1 μl from 1:20 dilution (blood) with antibody 1 B5

Direct comparison of the mixture according to the invention with antibody NF5E6 and mixture “2H10”:

When using the mixture according to the invention with antibody NF5E6, a complete inhibition of the PCR reaction was found when using a 1: 1 blood dilution. With a 1: 5 blood dilution, only the DNA fragment which is assigned to the Y-chromosomal allele was amplified. An amplification of both alleles could be achieved from a 1:10 blood dilution. When the mixture 2H10 was used, complete amplification of both alleles was shown from a 1:10 blood dilution.

Without adding the mixture according to the invention with antibody NF5E6 or the mixture 2H10 to the reaction mixture, the PCR reaction was completely inhibited at each of the blood concentrations used. The results are shown in FIG. 9:

M: marker

1: Neg. Control with the mixture according to the invention

2: Pos. Control with the mixture according to the invention 1 μl of heparinized blood with the mixture according to the invention

1 μl from 1: 5 dilution (blood) with the mixture according to the invention.

1 μl from 1:10 dilution (blood) with the mixture according to the invention.

1 μl from 1:20 dilution (blood) with the mixture according to the invention.

Neg. Control without the mixture according to the invention.

Control without mixture according to the invention.

1 μl of heparinized blood without the mixture according to the invention.

1 μl from 1: 5 dilution (blood) without the mixture according to the invention.

1 μl from 1:10 dilution (blood) without the mixture according to the invention.

1 μl from 1:20 dilution (blood) without the mixture according to the invention.

Neg. Control with antibody 2H10

Pos. Control with antibody 2H 10

1 μl of heparinized blood with antibody 2H10

1 μl from 1: 5 dilution (blood) with antibody 2H 10

1 μl from 1:10 dilution (blood) with antibody 2H 10

1 μl from 1:20 dilution (blood) with antibody 2H10

Claims

claims

1. Mixture containing albumin and / or spermidine and at least one antibody.

2. Mixture according to claim 1, which contains albumin, spermidine and an antibody.

3. Mixture according to claim 1 or 2, wherein the albumin is bovine serum albumin.

4. Mixture according to one of claims 1 to 3, wherein the antibody is an IgG antibody.

5. Solution containing the mixture according to any one of claims 1 to 4.

6. Solution according to claim 5, wherein the concentration of albumin is 0.1 to 2 μg / μl, of spermidine 0.05 to 1 μg / μl and that of the antibody 0.05 to 1 μg / μl.

7. Solution according to claim 6, wherein the concentration of albumin is 0.4 μg / μl, of spermidine 0.2 μg / μl and that of the antibody is 0.2 μg / μl.

8. Use of the mixture according to one of claims 1 to 4 or the solution according to one of claims 5 to 7 for the preparation of a reaction buffer or for the coating of reaction vessels for neutralizing inhibitors of enzymes used in the manipulation of nucleic acids.

9. Use according to claim 8, wherein the manipulation of nucleic acids is polymerization reactions, ligation reactions, phosphorylation reactions, dephosphorylation reactions, restriction reactions and coupling reactions and the enzymes used are ligases, kinases, nucleases, restriction endonucleases and transferases.

10. Use according to claim 8 or 9, wherein the enzyme used is a thermostable DNA polymerase.

11. Use according to claim 10, wherein the DNA polymerase is Taq DNA polymerase.

12. Reaction vessel, characterized in that it has a coating with a mixture according to one of claims 1 to 4 or has been coated with a solution according to one of claims 5 to 7.

13. A reaction vessel according to claim 12, wherein the coated material of the reaction vessel is polypropylene, polyethylene, polycarbonate, polystyrene or borosilicate.

14. Reaction vessel according to claim 12 or 13, which is additionally coated with enzymes.

15. A method for producing a reaction vessel according to one of claims 12 to 14, which is characterized by the following steps:

(a) mixing albumin and / or spermidine and at least one antibody in a solution;

(b) applying the mixture to the inner walls of the reaction vessel; and

(c) drying the reaction vessel.