JPH07509618A - Lipase labeled probe - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Lipase labeled probe The present invention provides lipase-labeled probes or assay substances and the measurement of biological materials. The invention relates to the use of lipases for.

biologically involved groups, certain genes, antigens, antibodies, bacterial surface proteins. For the detection of substances or similar substances, Markers and assay substances may be developed to facilitate detection. was previously known.

In particular, in the field of genetic engineering, genetic probes are radiolabeled and It is known to use gene probes to search for complementary nucleic acids. Labeled with enzyme In the case of calibrated analytes, the enzyme is peroxidase or alkaline phosph atase has traditionally been used. Both enzymes have relatively good sensitivity and relatively It is characterized by easy detectability, which means that it can be used as a marker or test substance. This emphasizes their suitability as a However, these special enzymes are It also has points. This means that, for example, these enzymes can be used to detect certain substances. It must be accompanied by certain general conditions that make it impossible to use it. This is a good point. Additionally, these enzymes have limited shelf life and thermal stability.

Additionally, these enzymes are required to carry out their actions in strictly specific media. This is a major constraint in terms of practical use. Finally, for example The presence of metal ions, which is necessary when using alkaline phosphatase, has been tested in many tests. Substances that are difficult to determine and complex with metal ions, such as EDTA, are Not only does it make it impossible to use sphatase, but it also makes it impossible to use peroxidase. Even in cases where there is a There are also risks.

It is therefore an object of the present invention to have significantly improved stability and a considerably wider range of applicability. This makes the use of enzyme-labeled probes or calibrators completely impossible previously. Enzyme-labeled proteins characterized by allowing analytical measurements of substances that have been manufacturing a lobe or calibrator.

For this purpose, as enzymes lipases of plant origin, their isoenzymes or Using structural analogs with at least 70% amino acid homology and lipase activity This is basically achieved by: proven to be particularly suitable as an origin. , in the literature, Candida cylindracea (Candida Cylindra) Candida rugosa (Candida rugosa), also described as cea rugosa) has a lipase whose structure has been extensively determined. There is. Cloning and analysis of this lipase sequence were performed in Gene″124:l (February 14.1993.pages 45-55) for details It is described in. This enzyme is described in this literature as a 57kD enzyme with 534 amino acids. a protein, five isoenzymes with 80% amino acid homology have already been structured. It is stated that it has been analyzed. Plant-derived, especially fungal-derived It is an enzyme from the past. Lipase derived from Candida Rugosa DSM 2031 is special preferred. All known enzymes, as well as other known lipases, especially animal lipases. The use of this enzyme results in a substantial improvement in the properties of the product compared to can get. In particular, when using the lipase proposed in the present invention, at least It was also found that thermal stability up to 60°C is guaranteed. This is alkaline phospha This is something that neither tase nor peroxidase can achieve. especially, hives, where the use of high temperatures is a key requirement to obtain acceptable reaction rates. Regarding lidding tests, the thermal stability thus achieved makes these tests reasonable. This is a necessary condition that enables implementation within a reasonable time.

A further advantage of the lipase proposed by the present invention is that for its activity it This means that there is no need for any additional components. In the case of alkaline phosphatase, the enzyme activity requires magnesium and zinc ions, but these metal ions As it is, it activates, for example, a nuclease, and as a result, the gene protein This prevents the use of alkaline phosphatase for

nuclease activation, especially when using enzyme-labeled gene probes. Complexing substances such as EDTA are often used to ensure prevention. Ru. EDTA complexes with metal ions and therefore the use of alkaline phosphatase Not only does EDTA become impossible to denature, but large amounts of EDTA tend to denature proteins. It also has a direction. The lipase proposed by the present invention is more effective than known enzymes. It is distinguished not only by its qualitatively high thermal stability but also by its complete stability towards EDTA. be marked. In addition to the known enzymes of the lipase proposed by the present invention An effective advantage is that urea and its related derivatives also fully function as lipases. (Do not harm. When using lipase, use E to protect the SH group. DTA can be used. This can be done with conventional enzyme-labeled probes or This was completely impossible with the test substance.

The enzymes used in the present invention are characterized by their improved resistance to organic solvents. It can also be characterized. In particular, the lipase proposed by the present invention is can also be used, thereby reducing the fraction of the probe or analyte of the invention. The range of analytical applications is considerably expanded.

Due to its high stability, the probe or analyte of the invention can be used in the pH range from 2.5 to 9. The insensitivity to urea makes it possible to use highly hydrophobic It also becomes possible to detect substances. in probes or analytes labeled with other enzymes. The use of urea, for example, could not have been tolerated in the It is possible to elongate the structure to some extent, thereby increasing the reliability of gene probes. It can also be improved.

Due to the considerably high stability of the lipase proposed by the present invention, reactive substances, There are almost no restrictions in terms of the type of conjugation to biological recognition substances.

Their high stability makes it possible to dry and, if necessary, freeze the lipase complexes. In lyophilized form or simply air-dried, it can be stored indoors without loss of activity. It can be stored for several years at room temperature. Overall, labeled by known enzymes Compared to probes, the conversion rate is similar, but the detection ability of lipase is As a result, it has excellent sensitivity.

For example, in a comparative test with alkaline phosphatase, the detection limit was approximately 250 11,001), whereas experiments have shown that lipase is in the range of approximately 5 pg. and purely qualitative analysis as well as comparative analysis down to the 10 ng range. It has been shown that even relatively simple capacity is possible.

Lipase measurements can be carried out using e.g. phenol esters or their derivatives as substrates. It can be carried out by the usual methods used. expressed in this way Color reaction allows for direct and simple analysis. The appropriate operation method is known by those skilled in the field.

The stability advantage of the lipase of the present invention is particularly important for oligonucleotide probes. This is extremely significant.

According to a preferred embodiment of the invention, the probe or assay substance is Othin, avidin, lecithin, protein A.

protein G, antibody binding protein, antibody, antigen, viral protein antigen, virus bacterial surface protein, digoxigenin, DNA, RNA, oligonucleotide complexed with metal colloids or plastic particles (5 μm). It is characterized as a combination. These complexes are suitable for a wide range of applications. have a specific reactive group, in particular a biological recognition substance, and thus this method provides an extremely wide range of applications for labeled probes or analytes.

The present invention further provides plant-derived, specific Lipases from Candida Rugosa DSM 2031 and their isoenzymes or structural similarity with at least 70% amino acid homology and lipase activity body, and in which lipase forms a complex with biological recognition groups. These assay substances are especially used in bioassays, test strips, and biosensors. , or those suitable as gene probes. related to this In particular, typical oligonucleotide probes are used as gene probes. The favorable thermostability of lipases allows for selection under harsh conditions. Selective hybridization is now possible.

Lipid derived from plants, especially Candida rugosa, proposed in the present invention The advantages of enzymes or their isoenzymes and structural analogs mentioned above are illustrated in the diagram below and This will be explained in further detail through examples.

The figure shows the thermostability of lipase from Candida species. Activity is at a temperature of 50℃ At a pH value of 5, it remains almost constant during the test period, and at a pH value of 7, it remains constant after 1 hour. It can be seen that 75% or more of the initial activity is still retained even after 50% of the initial activity. Temperature 60℃ At a pH value of 5, approximately 80% of the initial activity is retained after 1 hour. .

” indicates the detection limit of the color reaction of lipase and alkaline phosphatase. Al In the case of caliphosphatase, the detection limit was almost reached at approximately 250+)g. On the other hand, in the case of lipase, there is no color reaction even in spg in the original diagram. It can be seen that the spots can be detected. For lipase, the spot diameter is 10 ng range also correlates substantially with the actual amount, but for alkaline phosphatase. If the amount is less than approximately 500 pg, simple quantitative evaluation cannot be easily performed.

Figure 3 shows a flow diagram of the use of lipase for oligonucleotide gene probes. It is schematically drawn. In this process, structural gene 2 It is fixed to the microtiter plate 1 via a Gin coupling. Ori Gonucleotide probe 4 is linked to lipase 3, but the microtiter Analyte that binds to both the gene immobilized on plate l and oligonucleotide 4 5, this lipase is immobilized on the microtiter plate. results and When the specific biological substance 5 to be detected is present, the lipase 3 reacts with the hybrid It is immobilized on the microtiter plate l by a hydrogenation reaction. Therefore, suitable by a suitable method, e.g. by a lipase color reaction using a suitable substrate as a reagent. , qualitative detection is possible.

The optimum pH for lipase, peroxidase and alkaline phosphatase Shows a comparison of values.

Dish] shows pigment precipitation assays for lipase and alkaline phosphatase.

Example General operations Horseradish peroxidase activity assay principle using ABTS: peroxidation in 0.1M citrate/phosphate buffer, pH 4.5 at 23°C. Oxidation rate of ABTS (1,49mM) in the presence of hydrogen (0,045%) Horseradish peroxidase was detected by monitoring at a wavelength of 420 nm. was detected spectrophotometrically.

Method description: A B T S (20 mg/10 ml of water) 0.5 ml, hydrogen peroxide (0. ．． 6%) 1 ml and 0.1 M citrate/phosphate buffer, pH 4,59,5 +++1 substrate mixture was prepared.

Mix 50 μl of the enzyme solution with 500 μl of the above substrate solution and apply the wavelength for 1 minute. The kinetics of the enzymatic reaction was monitored at 420 nm.

The extinction coefficient for ABTS was used to calculate the concentration of oxidized substrate. anti The concentration of the enzyme solution was set to ensure a linear time course of the extinction curve under the reaction conditions. Established.

0.1MTRl5, O, 1M NaCl and 50mM gC12, pH8 , 1mM p-nitrophenyl phosphate at 23°C in Alkaline phosphatase was determined by monitoring the decomposition at a wavelength of 405 nm. spectrophotometrically measured [Lazdunski, C., and Lazdun ski, M. (1966) BBA 113, 551-566].

Method description: Add 5 μl of enzyme solution to O, IMTRIS, 0.1 MNac] and 50 mM g Mixed with 500 μI of CI2 (pH 8,5). 0.6mMp---Trough After adding 100 μl of phenyl phosphate solution, the 4050 At m, the reaction time course was kinetically monitored. of the extinction curve under the reaction conditions. The concentration of the enzyme solution was set to ensure a linear time course. pl(=8. The concentration of hydrolyzed substrate was determined using the p-nitrophenol calibration curve in 5. The degree was measured.

Principle of measuring lipase activity using p-nitrophenylbutyrate: 0.1mM p-nitrate at 23°C in 0.1M phosphate buffer, pH 7.0. Monitoring the hydrolysis of lophenylbutyrate at a wavelength of 405 nm. Lipase was measured spectrophotometrically.

Method description 23 μl of p-nitrophenylbutyrate (pNPB) was added to 1% polyvinyl alcohol in water. A stock solution of pNPB was prepared by mixing with 5 ml of cole with vigorous stirring. undissolved The pNPB-containing phase of the solution was separated by centrifugation (4°C, 1500 g, 7 min). .

The concentration of the remaining aqueous solution of pNPB was adjusted to It was measured using the Nord calibration curve.

Mix 5 μl of enzyme solution with 500 μl of 0.1M phosphate buffer (pH 7.0) did. After adding 100 μl of 0.6 mM p-nitrophenyl butyrate solution, Reaction kinetics were monitored at a wavelength of 405 nm over a period of 1 minute. Reaction conditions The concentration of the enzyme solution was set to ensure a linear time course of the extinction curve under . Using the calibration curve of p-nitrophenol at pH = 7.0, the hydrolyzed The concentration of the substrate was measured.

Lipase pretreatment: According to X-ray crystallography studies of various lipases, their active center is a helical protein part. It was found that it was shaped like a groove that was covered by minutes [Sc hrag, J. D., and Cygler, M., J. Mol.

Biol, 230.575-591 (1993); , U., Brzozowski, A.M.

Lawson, D. M., and Derewenda, Z. S., Bioc. hemistry 31.1532-1541 (1992)]. Various effectors It is possible to elongate and cause steric rearrangement by This allows the active center to contact the substrate more freely.

Lipase (protein concentration 0.09 mg/ml; by Bradford method) measurement) at room temperature in 0.1M acetate buffer, 0.1% BSA, pH 5°0. 20 to 44% (V/V) effector solution (e.g. tetrahydrofuran, 2-methyl-bentanediol, 2-isopropanol...) for 1 to 60 minutes. Incubated. Subsequently, the enzyme solution was diluted with 0, 1M acetate buffer, pH 5,0,0. , 1% BSA, or O, 1M acetate buffer, pH 5, 0, 0, 1% BSA. It was diluted with 0.2% (v/v) Tween to a measurable concentration. trial The samples were stored at 4°C. Activity was measured using the pNPB assay.

0.38mM hydrochloride at 23°C in 0.1M buffer, pH 1,5-9,0. Monitoring the hydrolysis of quinone monobutyrate at a wavelength of 295 nm Lipase was measured spectrophotometrically.

Method description: 10 mg of hydroquinone monobutyrate (HMB) was added to an aqueous 1% polyvinyl alcohol solution. A stock solution of HMB was prepared by mixing the mixture into 6 ml of HMB with vigorous stirring. Hydroquino When the concentration of HMB solution was measured using a standard curve, it was found to be 3.08 mmol/ I only had one.

20 μl of enzyme solution was added to 700 μl of O, 1M buffer that had been set to the required pH value. 1 was mixed. After adding 100μm of 3.08mM HM B solution, enzyme reaction Kinetics were monitored at a wavelength of 295 nm over a period of 2 minutes. reaction conditions The concentration of the enzyme solution was set to ensure a linear time course of the absorption curve.

A hydroquinone calibration curve was used to determine the concentration of hydrolyzed substrate.

Example 1: Using p-nitrophenyl butyrate or p-nitrophenyl phosphate Comparison of specific activities of lipase and alkaline phosphatase 1. Purified horseradish peroxidase 1050μmol/min-mg ( ABTS, pH=4.5.23°C)2, purified alkaline phosphatase 900 μmol/min -mg (p-nitrophenyl phosphate, pH= 8.5.23℃) 3. Crude preparation of lipase derived from Candida rugosa 27 00μmol/min-mg (p-nitrophel butyrate, p) (= =7.0.23°C) The activity was related to the total protein concentration of the crude enzyme preparation.

In the case of purified lipase isozyme, a significant increase in specific activity is expected.

Example 2: Optimal p of lipase, horseradish peroxidase and alkaline phosphatase Measurement and comparison of H Linose activity at different pH values was determined by hydroquinone assay.

The enzyme lipase has an extremely wide range of optimum pH (1) between H3 and 8. and is suitable for linked enzyme assays (eg with peroxidase). alkaliho Sphatase is active at pH values above 7, and peroxidase uses A as a substrate. When using BTS, it has an optimum pH of approximately pH=5 (Figure 4).

Example 3 Lipase, horseradish peroxidase and alkaline phosph for EDTA Stability of atase All enzymes mentioned above were incubated at 4°C in the presence of 50mM DTA. Ta. All measured enzyme concentrations were set at a protein content of 20 nmol.

- Horseradish peroxidase is O containing 0.05% BSA, 1M phosphoric acid Measurements were made using salt buffer, pH 7.5.

-Alkaline phosphatase is 1mM MgCI2 and 0.1mM ZnCl2. Dilute in 50mM RIS buffer, pH 7.6, containing 0.05% BSA. Ta.

・Lipase was prepared in 0, 1mM acetate buffer containing 0.05% BSA, pH 5, Diluted with 0.

Example 4: By incubating for 120 minutes at 50°C, the protein concentration was reduced to 0. The thermostability of the above enzymes at 2 mg/ml was compared.

- Horseradish peroxidase was incubated in 0.1M phosphate buffer, pH 7.5. Cubated.

・Alkaline phosphatase is 50mMT RI S, 1mMM g C1g and 0.1 mM ZnCL, pH 7.6.

・Lipase was diluted in 0.1M acetate buffer containing 0.5% BSA, pH 5.0. I interpreted it.

Example 5: All of the above in the presence of 0.05% sodium azide at 25°C and 4°C. Enzymes were incubated. To compare these enzymes with each other, 20 nmol A molar equivalent protein concentration of /ml was used.

- Horseradish peroxidase (HRP) in 0.1M phosphate buffer, 0.5% Diluted with BSA, pH 7.5.

・Alkaline phosphatase is 50mMT RIS, 1mMM g C1t and 0.1mM ZnC+ 2. Diluted with 0.05% BSA, pH 7.6. Ta.

・Lipase was diluted in O.1M acetate buffer, 0.05% BSA, pH 5.0 .

Azides can be used for the protection of all the above enzymes. alkaliho For sphatase, a slight decrease in enzyme activity (10-20% per day) was observed. Furthermore, in the presence of hydrogen peroxide (during the assay), azide This results in a decrease in activity due to inactivation of oxidase (HRP).

Example 6: All the above enzymes were incubated with 7M urea at 25°C and 4°C. I bet. To compare these enzymes, 20 nmol, 7 ml of protein concentration was used.

・Peroxidase (HRP) is 0.1M phosphate buffer (pH 7.5), 0 ．． Diluted with 0.05% BSA.

・Alkaline phosphatase is 50mMT RIS (pH 7,6), 1mN igc12 and 0.1mM ZnCl 2. Diluted with 0.05% BSA .

・Lipase is diluted with 0.1M acetate buffer (pH 5.0) and 0.05% BSA. I interpreted it.

At 4°C, 7M urea solution results in only slight denaturation of all the above enzymes. (10% activity reduction per day). At 25°C, the activity of these enzymes decreases. sex is reduced by 50%.

Example 7: As the final protein concentration is 3 mg/ml, O, IME D T A, O, 1 M A 10-fold excess of 2-iminothiolein was used in phosphate buffer, pH 7.5. Therefore, we introduced a thiol group into lipase.

After 20 minutes of incubation at room temperature, Centricon-3 Excess 2-iminothio is removed from the modified enzyme by ultracentrifugation using a 0 tube. Rain was separated. Elman's reagent [5,5'-dithio-bis-(2-nito The degree of modification was determined using (benzoic acid). Proteins modified with SH groups texture in 0.1 MEDTA, 0.1 M acetate buffer, pH 5 at 4°C. saved.

Example 8: (Sulfo)succinimidyl-4-(N-maleimidomethylcyclohexane); (PIERCE 22322X) m-7 Reimidobenzoyl-N-hydroxy Cisuccinimide ester; (PIERCE 22312X) (sulfo)sc Cinimidyl-4-(p-maleimidophenyl)butyrate; (PIERCE2 2317X) (sulfo)succinimidyl = (4-iodoacetyl) aminobe (PIERCE 22327X) At least two different anti- The commercially available heterobifunctional cross-linking agents with reactive groups described above allow for sequential coupling. and minimize undesirable polymerization and self-complexation. these racks The crosslinking agent was tested as follows: 50mM borate buffer, 0. 50 molar excess of hete in IMEDTAS pH=7 The lipase was modified with the crosslinker described above using a bifunctional crosslinker. lipase painting The protein concentration was determined by the Bradford test and was found to be 3 mg/min. It was ml. This lipase did not show any significant decrease in activity.

Example 9: The carboxyl group of lipase was replaced with polyethylene glycol, -dimethyl-aminopropyl)carbodiimide (EDC) and N-hydroxy Modified in the presence of succinimide (NH3).

Method description: Modified lipase 0.0゛-bis-(2-a Minopropyl) polyethylene glycol 1900 (M, W: 2000) ( A stock solution of diamino PEG) was prepared at a concentration of 0.59 g/ml in water and diluted with dilute hydrochloric acid. The pH was set to 4.6.

It was 5mg/ml. EDC the reaction with 500mM acetate buffer, pH 5. and a solution of NH8 in dimethylformamide (DMF) in a molar ratio of 15: 1 to prepare a stock solution of EDC/NH8. This stock solution was used immediately.

Lipase (384ug; 6.7nmol) and diamino PEG solution (12, 2 mg, 6.4 μmol) were mixed. Then EDC/NH3 mixture (E DC: 0.15mg, 780nmol; NHS: 0.006mg , 52 nmol) and the mixture was incubated for 16 h at 4°C in the dark. (pH of reaction mixture was 6.8). The final protein concentration is 5 It was mg/ml. The reaction was stopped with 500mM acetate buffer, pH 5. Excess diamide was removed by electrodialysis against , 50mM acetate buffer, pH 5.0. PEG was separated. Reaction products were analyzed on untreated polyacrylamide gels. , indolyl acetate/nitroblue tetrazolium salt (NBT) was used. Detected by staining.

Modified lipase 0.0"-bis-(2-aminopropylene) with current point pl=7 to pI=9 Pill) Polyethylene glycol 1900 (M, W: 2000) (Diamino A stock solution of PEG) was prepared in water at a concentration of 0.59 g/ml, and diluted with dilute hydrochloric acid to pH = 4. ．． It was set to 6.

An aqueous solution of EDC and a solution of NH8 in dimethylformamide (DMF) were prepared in a molar ratio of 1. A stock solution of EDC/NHS was prepared by mixing 5:1. This stock solution was used immediately. .

Lipase (384ug; 6.7nmol) and diamino PEG solution (12, 2 mg, 6.4 μmol) were mixed. Then EDC/NH3 mixture (E DC: 0.61 mg, 3.2 μmol; NHS + 0.024 mg , 0.21 μmol) and the mixture was incubated at 4°C in the dark for 16 hours. (pH of the reaction mixture was 6.8). Final protein concentration The degree is Example 1O: Stop and remove excess by electrodialysis against 50mM acetate buffer, pH 5.0. diamino PEG was separated. Reaction products were transferred onto untreated polyacrylamide gels. and indolyl acetate/nitroblue tetrazolium salt (NBT). Modified lipase 0.0% was detected by the staining used and the type point pI=7 to pl=9. 0°-bis-(2-aminopropyl) polyethylene glycol 1900 (M, W: 2000) (diamino PEG) at a concentration of 0.59 g/ml in water. and set the pH to 4.6 with dilute hydrochloric acid.

The molar ratio of an aqueous solution of EDC and a solution of NH8 in dimethylformamide (DMF) A stock solution of EDC/NH8 was prepared by mixing at 15:l. Use this stock solution immediately Ta.

Lipase (384ug; 6.7nmol) and diamino PEG solution (12,2 mg, 6.4 μmol) were mixed. Then EDC/NH3 mixture (ED C: 0.61 mg, 3.2 μmol; NH3: 0.024 mg, 0 ．． 21 μmol) was added and the mixture was inked for 16 hours at 4°C in the dark. (pH of the reaction mixture was 6.8). Final protein concentration is 5 m g/ml. The reaction was stopped by 500mM acetate buffer, pH 5; Excess diamine was removed by electrodialysis against 50mM acetate buffer, pH 5.0. PEG was separated. Reaction products were analyzed on untreated polyacrylamide gels, Dyeing using indolyl acetate/nitroblue tetrazolium salt (NET) Detected by color.

Coupling of lipase to deoxyoligonucleotides Lipase using SMCC ( Modification of 8) modified with polyethylene glycol: 20mM phosphate buffer, 150mM NαC], 1mM EDTA pH7 , PEG lipase at a final protein concentration of 10 mg/ml in 4 (5 amino acids). Modification was performed using a 20-fold molar excess of SMCC (assuming that the groups were modified).

After 30 min incubation at room temperature in the dark, 211 mM phosphate buffer + Sephadex (Sephadex) equilibrated with 2mM EDTA (pH 6,5) Excess crosslinker was separated on a PHADEX-10 column.

Both portions containing the desired lipase activity were placed in Centricon at 4°C. con)-30 tubes for concentration followed by lyophilization. qualified link The pase was stored at -20°C and used directly for conjugation to oligonucleotides. .

Production of SH-oligonucleotide: Oligonucleotides were synthesized using the phosphoramidite method. Final synthesis stage In step 1, a C6-thiol modifier is used to introduce a terminal sulfhydryl group at the 5' end. I entered. Reversed phase HPLC (O, IM triethylammonium acetate as mobile phase) , pH 7 and methanol) and then open the trityl group using silver nitrate. The cells were cleaved and stored at -20°C in DTT (operating instructions provided by C1ontech).

SM CC lipase and deoxyoligonucleotide binding: in 50 μl volume 5 μg of lythyl-free oligonucleotide was placed on a Sephadex G-25 column. and directly mixed with the lyophilized enzyme.

After dissolving the enzyme, it was incubated first for 1 hour at room temperature and then for 1 hour at 4°C to complete the reaction. The reaction was carried out for 6 hours.

Example 11: Synthesis of immobilized lipase and antigen colloidal gold on metal colloids by Frens, G. , et al, , [Nature Lond, Phys, Sci.

241, 20 (1973)]. In 0.1M phosphate buffer lipase (10 μm, protein concentration 1 mg/ml) and virus Mix 6 μl of antigen (0.8 mg/ml) and 1 ml of colloidal gold solution and incubate at room temperature. Incubated for 30 minutes.

Unbound protein was removed by centrifugation at 1600 g for 15 min. The texture was removed. Remove the supernatant with a pipette and transfer the precipitate to 10% polyethylene glycol. Le20000 (PEG20000) (Polyscience)100 Dissolved in 0 μl. Use 10% 5DS 50 μm and ultrasound for several minutes for each lysis. (Bandelin 5ONOREX 5per RK5101 1). This operation was repeated three times.

After the final centrifugation, the precipitate was diluted with 50 μl of 1% PE and PBST- It was dissolved in 200 μl of 3% B5A. For lipase activity and antigen activity, supernatant and the dissolved precipitate were tested. Only the first supernatant showed lipase activity.

This indicates the presence of unbound protein. subsequent centrifugation After separation, no enzyme activity was observed in the supernatant. In contrast, the dissolved precipitate showed lipase activity as expected.

Example 12: Lipase sedimentation assay: Substrate stock solution 2100% Indolylacetic acid in 1 ml dimethylformamide (DMF) Tate 50mg; source of nitro blue tetrazolium chloride (NBT) Solution = 50 mg of NBT dissolved in 1 ml of 70% dimethylformamide, substrate The solution was 66 μm of NET stock solution in 510 ml of O, 1 M phosphate buffer, pH 6. Prepared by mixing. The mixture was used immediately.

Alkaline phosphatase sedimentation assay; Substrate stock solution + 5-butane dissolved in 1 ml of 100% dimethylformamide (DMF) Lomo-4-chloro-3-indolylphosphate (BCI P) 50mg; NBT 50 dissolved in 1ul of NBT stock solution Nia 0% dimethylformamide mg0 substrate solution was O.1M substrate buffer (0.1MT RIS, 0.1M Na C1, 50mM g C12, pH 8,5) Substrate stock solution 33μ in 10ml 1 and 66 μl of NBT stock solution. Use the mixture immediately Ta.

In both cases, higher concentrations are better due to the low solubility of the lipase reaction products. The results are obtained (Figure 5).

The enzyme used in this case had not been optimally purified. Therefore, the reserve Better detection limits can be achieved using pre-treated and pre-treated lipases. .

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Fig, 2 Fig, 5 alkali Lipase Phosphatase Continuation of front page (72) Inventor Eker, Bernhard Austria A-2551 Enz Essfeld Hirtenbergerstrasse 130 Ei (72) Inventor: Kinklov, Austria, A-1140 Wie hmm.

Linzerstrasse 83/11 (72) Inventor: Wuakolbinger, Werner Austria A-4061 Persinquim Backerfeld 1st address

Claims (6)

[Claims] 1. The enzyme is a lipase of plant origin, the corresponding isoenzyme or at least 70% amino Enzyme-labeled probes or assays that are structural derivatives with acid homology and lipase activity. fixed substance. 2. An enzyme with lipase activity was obtained from Candida rugosa DSM2031. The enzyme-labeled probe or assay substance according to claim 1, which is an enzyme-labeled probe or assay substance. 3. Lipase is piotin, avidin, lecithin, protein A, protein G, Antibody binding proteins, antibodies, antigens, viral protein antigens, bacterial surface proteins Protein, digoxigenin, DNA, RNA, oligonucleotide or synthetic Composite with analogs, metal colloids or plastic particles (<5μm) The enzyme-labeled probe or test substance according to claim 1 or 2. 4. Lipases of plant origin, in the form of complexes with biological recognition groups; enzyme or having at least 70% amino acid homology and lipase activity. Use of these structural analogs for the production of analytical probes or assay substances. 5. Lipase is obtained from Candida Rugosa DSM2031 , the use according to claim 4. 6. Manufacture of assay materials for bioassays, test strips, biosensors or gene probes The method according to claim 4 or 5 for.