DE20111308U1 - Locking device for multiple reaction rooms - Google Patents

Description

t ft ♦·♦··*

Closure device for multiple reaction chambers Description

Many areas in clinical research and diagnostics, pharmacological drug testing, veterinary medicine and food analysis require the exact concentration of selected proteins in a sample to be analyzed. In addition to chemical analysis methods, enzyme immunoassays (ELISAs) are often used for this purpose, which reach a lower detection limit of approx. 1-10 ng analyte protein per ml. To detect lower values, so-called radioimmunoassays (RIAs) are used, in which radioactively labeled antibodies are used.

1H

This achieves a lower detection limit of approximately 10" mol protein. This method, although extremely sensitive, has the disadvantages that the use of RIA technology entails considerable health risks for the user and that large quantities of radioactively contaminated waste are generated, which must be detected and disposed of at great expense. A novel approach to solving the problem is the use of the principle of the immuno-polymerase chain reaction (iPCR), which combines the high specificity of the antigen/antibody reaction with the high sensitivity of the polymerase chain reaction (PCR) (Fig. 1). Potential applications of the technology include the development of ultra-sensitive, non-radioactive assays for determining extremely low protein concentrations such as gliadins in dietary foods or endocrinological tests for measuring serum hormone concentrations (e.g. leptin, growth hormone).

The current state of iPCR technology assumes that the resulting final PCR products are still detected using conventional detection techniques, in particular agarose gel electrophoresis combined with subsequent imaging procedures (Sano T. et al, Science 1992, 258; 120-122, Zhou H. et al, Nucl. Acids Res. 1993, 21: 6038-6039, Chang T.C. et al, J. Immunol. Meth. 1997, 208: 35-42, Case M. et al, Biochem. Soc. Trans. 1997, 25: 374, Saito K. et al, Clin. Chem. 1999, 45: 665-669). This is usually time-consuming and involves numerous possibilities for error. More modern detection techniques, so-called real-time (RT) PCR product detection methods, however, allow PCR reaction and fluorescence detection of the resulting products to be carried out simultaneously in one step (Köhler T. et al., J. Lab. Med. 23: 408-414). The associated analysis technology is available from PE Biosystems (Weiterstadt, Germany) in the form of the ABI PRISM™ 7700 Sequence Detection System or GeneAmp® 5700 Sequence Detection System, which work according to the so-called TaqMan® principle, and from Roche Molecular Biochemicals (Penzberg, Germany) in the form of the LightCycler™

available. After analyzing the recorded raw data, these devices can immediately create a calibration curve using defined standards, which can be used to automatically calculate the concentration of the analyte in the sample. Attempts to combine iPCR and real-time detection are therefore obvious.

The main problem with implementation so far has been that the TaqMan® technology requires special polypropylene reaction vessels which - sealed gas-tight with laser-permeable "optical caps" - allow amplification, fluorescence excitation and measurement simultaneously and directly in the tube. These reaction vessels, which were specially designed for RT-PCR, have the disadvantage that they are primarily suitable for measuring nucleic acids, thus only allowing very low protein binding and are therefore largely unsuitable for iPCR applications. In addition, the consumables associated with TaqMan® are relatively expensive and generally not suitable for automatic processing (e.g. with a microtiter plate washer). The use of a combined real-time iPCR technique (RT-iPCR) using more suitable microtiter plates or strips with high protein binding capacity has therefore not been possible so far.

The object of the invention was therefore to eliminate the disadvantages of the prior art. The problem was solved by using conventional PCR-suitable, protein-absorbing microtiter plates or strips (multiple reaction chambers) instead of the unsuitable TaqMan® reaction vessels, which are sealed according to the invention with a suitable, heat-resistant, transparent film after the immune reaction has been carried out and the PCR reaction mix has been dispensed into the cavities. Furthermore, a suitable perforated mask was constructed according to the invention, which allows both optimal excitation by high-energy rays in the reaction cavities and maximum heat transfer from the conventional cover counterheating of selected PCR devices to the processed strips or microtiter plates. In a particularly preferred variant of the design, this perforated mask is made of heat-stable silicone rubber. This closure device according to the invention can be used universally and can also be used to seal reaction chambers such as those used in conventional qualitative or quantitative RT-PCRs.

The essence of the invention lies in a combination of known elements and new solutions, which influence each other and, in their new overall effect, result in a practical advantage and the desired success, which lies in the fact that now for the first time qualitative or quantitative RT-PCR or RT-iPCR analyses can be carried out directly in heat-stable,

■ · a

PCR can be carried out using suitable microtiter plates or strips with high protein binding capacity. In addition to eliminating the above-mentioned disadvantages of the prior art, the method according to the invention thus has a number of advantages:

• The RT-iPCR can now be used in the same way as a conventional RT-PCR in terms of detection, making it significantly easier, faster and much more accurate to use.

• The restriction to specific reaction spaces (optical tubes, optical plates) is no longer applicable, a much wider range of reaction spaces can therefore be used.

• For the first time, reaction chambers can be used that have been developed primarily for protein detection, thus having optimal protein binding properties, but can also be used at high temperatures of up to 11O ⁰ C, as is required for PCR, for example (e.g. TopYield™ Strips, Nunc, Roskilde, Denmark).

• Since a much wider range of reaction spaces is accessible for enzymatic "real-time" amplifications, the costs for consumable plastic can be significantly reduced.

• The perforated mask can be reused as often as required, which significantly reduces the costs for disposable closure material.

The application of the device according to the invention lies in test kits for determining selected nucleic acids or proteins of biological origin by means of RT-PCR or RT-iPCR. The test kits consist of at least one transparent, heat-stable sealing film, a flexible, heat-stable perforated mask, a heat-stable multiple reaction chamber and the associated standard proteins, antibodies and reagents.

In summary, the universally applicable, two-part closure device for multiple reaction chambers is characterized by the fact that it

a) the sealing material for the reaction chambers is a transparent, radiolucent, gas-tight, heat-stable sealing material, through which

b) a congruent, flexible aperture mask is positioned, which consists of a flexible and thermostable material and has at least as many windows of suitable diameter as there are multiple reaction chambers, which are arranged in such a way that when placed on the multiple reaction chamber they are each located exactly above the middle of the individual cavities.

Preferably, the materials a) and b) are arranged in such a way that both excitation of associated chromophores by energy sources of different wavelengths and detection can take place simultaneously directly in the reaction chamber without hindrance by the two-part closure device, whereby at the same time an optimal heat transfer to the reaction chambers, if required, and volume constancy in the detection approach are possible by suitable counter-heating devices.

Self-adhesive films or adhesive strips are preferably used as sealing material a), while the thermostable material b) is preferably silicone rubber.

The device is used to seal reaction chambers that have both high protein binding and thermal stability, which are preferably made of polycarbonate or materials with comparable binding and thermal properties, and which are suitable for subsequent inclusion in suitable measuring devices. Furthermore, the device is suitable for sealing reaction chambers that are used for qualitative or quantitative conventional or real-time PCR, as well as for sealing reaction chambers that can be used for qualitative or quantitative real-time immuno-PCR and which are suitable for subsequent inclusion in corresponding measuring devices, in test kits for determining selected nucleic acids or proteins, wherein the test kits preferably consist of at least one perforated mask, a transparent sealing film, a multiple reaction chamber optionally coated with at least one nucleic acid or protein, as well as necessary, possibly DNA-conjugated antibodies and reagents for carrying out immune reactions, enzymatic amplification reactions or other signal-enhancing detection reactions.

Legends to the figures

Figure 1 shows the scheme for the procedure of a quantitative RT-iPCR for the determination of gliadin for the embodiment example.

Fig. 2 shows the top view of the silicon rubber perforated mask produced according to the invention (scale 1:1) for congruent placement on a multiple reaction chamber previously sealed with a sealing film.

Figure 3 shows two standard reference curves for gliadin recorded independently on different experimental days. A) 100, 200, 400, 2000, 10000, 50000 pg gliadin per well;

B) 500, 1000, 2000, 10000, 50000 pg gliadin per well. All measurements were performed in triplicate.

Example: Measurement of gliadins using quantitative iPCR

The following example serves to illustrate the invention without limiting it to this example.

Materials:

1. Highly purified gliadin (Osman et al, Clin Chim Acta 1996, 255; 145-152)

2. Primary antibodies: These were obtained by immunization of rabbits with gliadin (Osman et al., FEBS Lett. 1998, 433: 103-107).

3. DNA-conjugated secondary antibodies: Anti-rabbit immunoglobulin (Dako, Hamburg, Germany) was used for conjugation with a DNA fragment. This fragment comprised 172 bp from the kanamycin gene (pET-29a-c (+) Vector) and was coupled to the antibody using conventional methods (Niemeyer CM. et al, Nucl. Acids Res. 1994,22: 5530-5539)

4. Spacer plate made of silicone rubber, dimensions: 115 x 80 x 3 mm, autoclavable and UV-stable (Nunc, Roskilde, Denmark). This was produced according to the invention as follows: fix the spacer plate on a paper base and place a conventional 96-well carrier plate for 0.2 ml PCR tubes on top, use a permanent marker to fix the well centers on the silicone plate and then use a hand cork drill and a hammer or, if necessary, a hole punch to punch out discs with a diameter of 4.0 mm and discard them. Repeat the process for all positions (i.e. 96 times in total). The holes (windows) are arranged in such a way that when placed on a 96-well microtiter plate they are located exactly over the center of the "wells" (Fig. 2).

5. Scotch 3M 550 Transparent Tape (3M France, Cergy-Pontoise, France), 12 mm &khgr; 30 m or Adhesive Sealing Film (ThinSeal™, Excel Scientific, Wrightwood, USA)

6. Top Yield™ Strips (Nunc, Roskilde, Denmark)

7. Wash buffer: 10 mM Tris-HCl; pH 7.3, 150 niM NaCl, 0.05% Tween 20

8. Blocking buffer: Wash buffer with 3% bovine serum albumin

9. TaqMan® primer and probe (BioTez GmbH, Berlin, Germany) (GenBank Accession Code: AJ 008006)

OSSI-fol (7430-7451) S^-TCA.GGT.GCG.ACA.ATC.TAT.CGA.T^

OSSI-re2 (7475-7498) 5'-TTT.GCC.ATG.TTT.CAG.AAA.CAA.CTC-3'

OSSI-Taql-3 (7455-7473) 5 ^λ -FAM-ATG.GGA.AGCCCG.ATG.CGC.C-TAMRA

10. Reagent premix for a 50^1 PCR approach: PCR grade H ₂ O (ad 50 μέ); 1Ox ROX buffer (PE Biosystems; 5 μέ); 50 mM MgCl ₂ (4 μέ); nucleotide mix (10 niM dATP, 10 raM dCTP, 10 mM dGTP, 20 mM dUTP) (4 μέ); 50 ng λ-DNA (AGS, Heidelberg, Germany; 5 μέ); OSSI-fol (50 ng/μέ) (2 μέ); OSSI-re2 (50 ng/µλ) (2 µλ); OSSI-Taql-3 (1.64 pmol, 5 μ?); AmpliTaq Gold (1.25 U)

Measurement procedure

Scheme see Fig.l, where 1 to 6 have the following meaning

Real-time PCR

dsDNA-Kanamycin fragment

Anti-rabbit immunoglobulin conjugated to DNA

Rabbit anti-gliadin

Gliadin

Plastic surfaces

Coating the plate with gliadin and incubating with antibodies

1. One milligram of gliadin is dissolved in 1 ml of 60% ethanol. This solution is diluted in carbonate buffer (50 mmol/l, pH 9.6) to produce a gliadin dilution series in the concentration range from 50 ng/ml to 200 pg/ml.

2. Coating of TopYield™ strips with gliadin: 50 μl of the sample per well are incubated overnight at 4 ⁰ C.

3. The wells are emptied, fixed with 10% formaldehyde and then washed three times with washing buffer.

4. The free binding sites are saturated with blocking buffer (1h at 37 ⁰ C) and the wells are washed again.

5. The primary antibody is diluted 1:400 (wash buffer with 1% bovine serum albumin), applied to the wells and incubated for 1 hour at room temperature. Then washed four times.

6. The DNA-conjugated secondary antibody is diluted 1:20,000 (wash buffer with 1% bovine serum albumin), applied to the wells and incubated for 2 hours at room temperature. The plate is then washed thirty times

Carrying out quantitative RT-iPCR

7. After dispensing 50 μl of the PCR reagent premix (see above) into the designated cavities, individual strips are sealed gas-tight with Scotch 3M 550 Transparent Tape or entire plates are sealed gas-tight with Adhesive Sealing Film. The sealed strips or microtiter plates are then inserted into the thermoblock of an ABI PRISM™ 7700 Sequence Detection System and the aperture mask is centered.

• · • ·

• * ♦

&idgr;**; ^&Lgr;

positioned over the cavities. Then carefully close the lid of the ABI PRISM 7700 without moving the aperture mask!

8. Start PCR program (10 min at 95 ⁰ C, then 40 PCR cycles of a 2-step PCR: 30 cycles at 95°C, 2 min at 59°C)

9. After the program has ended, the amplification curves are automatically evaluated and the reference curves are displayed (Fig. 3).

Results

From Fig. 3 it can be seen that the linearity of the reference curve is guaranteed over a gliadin concentration range of approximately 200 pg to 50 ng/ml. If gliadin concentrations of 100 pg/ml are included in the calculation of the calibration curve (Fig. 3A), the linearity decreases significantly, as can be seen from the fact that the slope of the reference curve is < 3 (optimal: 3.3-3.4, corresponding to approximately 100% amplification efficiency). Thus, a lower detection limit of about 200 pg gliadin/ml can be assumed for the exemplary embodiment, which corresponds to at least 15 times higher sensitivity compared to competing ELISAs (lower detection limits to date 3 ng/ml or 4 ng/ml or 156 ng/ml in [Sorell L. et al 1998, FEBS Lett. 439: 46-50, Ellis HJ. et al 1998, Gut 43: 190-195, Skerritt J.H. and Hill A.S. 1991, J. Assoc. Off. Anal. Chem. 74: 257-264]).

Claims (4)

1. Universally applicable, two-part closure device for multiple reaction chambers, characterized in that it a) the sealing material for the reaction chambers is a transparent, radiolucent, gas-tight, heat-stable sealing material, through which b) a congruent, flexible aperture mask is positioned, which consists of a flexible and thermostable material and has at least as many windows of suitable diameter as there are multiple reaction chambers, which are arranged in such a way that when placed on the multiple reaction chamber they are each located exactly above the middle of the individual cavities. 2. Closure device according to claim 1, characterized in that the materials a) and b) are arranged in such a way that both an excitation of associated chromophores by energy sources of different wavelengths and a detection can take place simultaneously directly in the reaction chamber unhindered by the two-part closure device, wherein at the same time an optionally required optimal heat transfer to the reaction chambers and volume constancy in the detection approach are possible by suitable counter-heating devices. 3. Closure device according to claim 1 or 2, characterized in that the sealing material a) is transparent, self-adhesive film or adhesive strip. 4. Closure device according to one of claims 1 to 3, characterized in that the thermostable material b) is silicone rubber.