Liquid-phase protein chip method for simultaneously detecting three kinds of coronaviruses of pigs and kit of liquid-phase protein chip method

Abstract

The invention discloses a liquid-phase protein chip method for simultaneously detecting three kinds of porcine coronaviruses and a kit thereof, which comprises the following steps of: after activating and coupling microspheres, respectively coating antigen on porcine delta coronavirus N protein, porcine acute diarrhea syndrome coronavirus N protein and porcine epidemic diarrhea virus N protein by using the microspheres; matching the two fluorescent dyes according to different concentrations to form fluorescent coding microsphere combination arrays with different energy levels; the microspheres are analyzed one by one based on the flow principle, red laser recognizes microsphere codes, green laser reads fluorescence intensity of report molecules, computer analysis and processing are carried out, and fluorescence signals are converted into quantitative detection results; and determining the optimization of the detection conditions of the liquid-phase protein chip after the liquid-phase protein chip test. The antigens coated on the microspheres only specifically react with corresponding antibodies and are not combined with antibodies generated by other pathogens; the PDCoV, SADS-CoV and PEDV antibodies in the sample to be detected can be rapidly, effectively and accurately detected, and reference is provided for prevention of the porcine coronavirus.

Description

Technical Field

[0001] The present invention belongs to the field of virus detection, and in particular relates to a liquid-phase protein chip method and a kit for simultaneously detecting three porcine coronaviruses. Background Art

[0002] Liquid-phase chip technology (multi-analyte profiling solve for unknown x, xMAP technology) is a new type of detection technology that combines chip technology with flow cytometry. The analytical basis of liquid-phase chip technology is uniformly sized (5.6μm in diameter) polystyrene microspheres labeled with fluorescent dyes of different colors. During the production process, they are coated with two fluorescent dyes, red light and infrared light chromogens, in different proportions to produce 100 microspheres of different colors. Each microsphere has a unique color number. The 100 microspheres can be coupled with 100 probes (such as antigens, antibodies, nucleic acids, enzymes, various receptors, etc.) according to different research purposes. In this way, 100 different target molecules in a sample to be tested can be detected simultaneously. During the detection process, the microspheres pass through the detection channel one by one. When the microspheres pass through the detection channel, they are irradiated by two laser beams at the same time. The first beam of 635nm red laser excites the fluorescent substance inside the microspheres and determines the type of microspheres according to the fluorescence code. That is, the two fluorescent substances inside the microspheres can emit two different wavelengths of fluorescence after being excited. The ratio of these two fluorescent substances in different types of microspheres is different, and the fluorescence intensity ratio is also different, thereby distinguishing different specific reactions (qualitative, classification); the second beam of purple laser excites the fluorescein on the reporter molecule and determines the number of reporter molecules bound to the microspheres based on the fluorescence intensity, thereby determining the number of target molecules (quantitative). The system only records the green fluorescence signal that appears at the same time as the red fluorescence, and does not record the fluorescence signal of the unbound reporter molecules. Therefore, there is no need to elute the unbound reporter molecules before detection. Finally, the branch device performs digital processing and analyzes the type and quantity of microspheres excited by the two lasers to determine whether the pathogens to be detected are present in the sample to be tested.

[0003] Currently, there are six known porcine coronaviruses, including porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine acute diarrhea syndrome coronavirus (SADS-CoV), which cause intestinal infections; and porcine respiratory coronavirus (PRCV) and porcine hemagglutinating encephalomyelitis virus (PHEV), which cause respiratory infections. TGEV, PRCV, PEDV, and SADS-CoV belong to the alphacoronavirus genus, PHEV to the betacoronavirus genus, and PDCoV to the deltacoronavirus genus. PEDV, SADS-CoV, and PDCoV are considered to be newly emerging or re-emerging porcine coronaviruses. The clinical symptoms and pathogenesis of these three viruses are very similar, but there is no cross-protection between them.

[0004] Currently, prevention is the main approach for these three newly emerged porcine coronaviruses, and there is no special treatment available. Therefore, it is particularly important to accurately detect these three viruses in the early stages or during regular inspections. The traditional Elisa method has low sensitivity, is time-consuming, has poor comprehensive analysis, and requires the purchase of three different Elisa kits. The liquid protein chip method for simultaneously detecting the three porcine coronaviruses can perfectly make up for the above shortcomings. It has high accuracy, stable information quality, good repeatability of test results, short time, and simple operation, and can more effectively detect the three newly emerged porcine coronaviruses. Compared to traditional detection methods, xMAP technology offers advantages in its ability to simultaneously test multiple components, reducing costs and labor, and requiring fewer samples. However, because its core technology relies on polystyrene microspheres, which have high technical requirements and few companies capable of producing standard microspheres, the corresponding microsphere prices are higher, making them more expensive than traditional detection methods like ELISA and qPCR. However, xMAP can be used for multiplex testing. When more tests are performed and the volume of testing increases, its relative cost is lower than that of traditional methods that only test single components.

[0005] xMAP technology also has some drawbacks. Compared to biosensors, it cannot continuously monitor the concentration of the target in the sample or the binding of the target to the probe on the microsphere. When performing multiplex PCR, xMAP technology is relatively complex to operate and difficult to automate. Because the xMAP technology platform is an open system, PCR products may contaminate the laboratory. Therefore, a liquid-phase protein chip method and a kit for the simultaneous detection of three porcine coronaviruses are urgently needed. Summary of the Invention

[0006] The purpose of the present invention is to provide a liquid-phase protein chip method and a kit for simultaneously detecting three porcine coronaviruses.

[0007] In one aspect, the present invention comprises the following steps: After activation and coupling of the microspheres, the microspheres were used to coat antigens of porcine deltacoronavirus N protein, porcine acute diarrhea syndrome coronavirus N protein, and porcine epidemic diarrhea virus N protein, respectively; Two fluorescent dyes are mixed at different concentrations to form a combination array of fluorescent coded microspheres with different energy levels; Based on the flow cytometry principle, the microspheres are analyzed one by one. The red laser identifies the microsphere code, the green laser reads the fluorescence intensity of the reporter molecule, and the computer analyzes and processes the fluorescence signal to convert it into a quantitative detection result. After the liquid protein chip experiment, the optimization of liquid protein chip detection conditions was determined.

[0008] Furthermore, the microspheres include magnetic fluorescent microspheres 301 , magnetic fluorescent microspheres 303 and magnetic fluorescent microspheres.

[0009] Furthermore, the liquid phase protein chip test includes: The serum sample to be tested was diluted 100-fold and then diluted 2-fold; The coated microspheres were diluted to 50 microspheres / µL with diluent and resuspended by vortexing and sonication; Add 5 µL of diluted microspheres to each well of a 96-well plate; Add 50 μL of diluted serum sample and incubate at 37°C with shaking for 60 minutes; The microspheres were separated by magnetic plate, the reaction solution was discarded and washed twice with washing solution; Add 2 μg / mL biotinylated goat anti-pig secondary antibody and incubate at 37°C with shaking for 30 minutes; Repeat the magnetic separation and washing steps, add 2.5 µg / mL streptavidin-phycoerythrin stock solution, and incubate at 37°C with shaking for 30 minutes; Perform magnetic separation and washing again, add PBS-TBN solution, shake, read the median fluorescence value on the microscope, and calculate the P / N value. If the P / N value is greater than 5, it indicates that the coated protein has good antigenicity and further optimize the reaction conditions.

[0010] Furthermore, the magnetic beads were coated with antigen protein at concentrations of 5 μg, 10 μg, 15 μg, 20 μg, and 25 μg, respectively. After activating two blank magnetic beads with different codes, the coating buffer was resuspended and shaken, and different protein solutions were added after ultrasonic dispersion. After mixing, the mixture was incubated at room temperature in the dark for 2 h, washed, and blocked at 4°C overnight. After washing again, the preservation solution was added to obtain two magnetic beads coated with different antigens.

[0011] Furthermore, the optimization of the liquid phase protein chip detection conditions specifically includes: Determination of the optimal coating concentration and cutoff value of recombinant antigen: Magnetic beads were coated with antigen protein at 5μg, 10μg, 15μg, 20μg, and 25μg of magnetic beads respectively; The three virus reference sera were diluted 400-fold, 800-fold, 1600-fold, 3200-fold, 6400-fold, and 12800-fold to explore the optimal dilution factor, and the negative serum was diluted 100-fold to determine the cutoff value; The secondary goat anti-pig antibody was diluted 1:1000 and the detection antibody was diluted 1:500 with SPAE. Two replicates were performed, and the average value was taken to calculate the ratio of the positive MFI value to the negative MFI value, i.e., the P / N value. The protein coating concentration with the highest detection sensitivity and the largest P / N is the optimal working concentration of the antigen.

[0012] Furthermore, the activation and coupling process of the microspheres includes The magnetic microspheres were vortexed and sonicated for 10 seconds, washed twice with buffer 1, and finally resuspended with buffer 1; Add N-hydroxysuccinimide and carbodiimide solution, vortex to mix, and incubate at room temperature in the dark for 20 minutes; wash twice with buffer 2, and finally resuspend in buffer 2; N protein was added to AL32 microspheres, and S-RBD protein was added to AL12 microspheres. After vortexing, the mixture was incubated at room temperature in the dark for 2 hours. After coupling, the mixture was washed twice, and blocking solution was added. After vortexing and sonication, the mixture was blocked at 4°C in the dark for 17 hours. After washing twice, add preservation solution, mix well and store at 4℃ in the dark.

[0013] On the other hand, a liquid protein chip kit for simultaneously detecting three types of porcine coronaviruses is provided, the kit comprising three fluorescent magnetic microspheres with different codes; and the above three fluorescent magnetic microspheres are respectively coupled with porcine deltacoronavirus N protein, porcine acute diarrhea syndrome coronavirus N protein, and porcine epidemic diarrhea virus N protein.

[0014] Furthermore, the kit also includes a biotin-labeled donkey anti-goat secondary antibody for reacting with the sample to be tested, the quality control product and the negative serum.

[0015] Furthermore, it also includes streptavidin-phycoerythrin.

[0016] Compared with the prior art, the present invention adopts the above technical solution, and its biggest feature is: The antigens coated on the microspheres of the present invention only react specifically with the corresponding antibodies and do not bind to antibodies produced by other pathogens; they can quickly, effectively and accurately detect PDCoV, SADS-CoV, and PEDV antibodies in the test samples, providing a reference for the prevention of porcine coronavirus and the control of the early stages of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the liquid-phase protein chip method for simultaneous detection of three porcine coronaviruses; DETAILED DESCRIPTION The technical solution of the present invention is further illustrated below in conjunction with embodiments and comparative examples, but they should not be construed as limiting the present invention: like Figure 1 As shown, after activation and coupling of the microspheres, the microspheres were used to coat antigens of porcine deltacoronavirus N protein, porcine acute diarrhea syndrome coronavirus N protein, and porcine epidemic diarrhea virus N protein; Two fluorescent dyes are mixed at different concentrations to form a combination array of fluorescent coded microspheres with different energy levels; Based on the flow cytometry principle, the microspheres are analyzed one by one. The red laser identifies the microsphere code, the green laser reads the fluorescence intensity of the reporter molecule, and the computer analyzes and processes the fluorescence signal to convert it into a quantitative detection result. After the liquid protein chip experiment, the optimization of liquid protein chip detection conditions was determined.

[0018] Microsphere Encoding: A combination array of fluorescently encoded microspheres with different energy levels is formed using two fluorescent dyes. The microspheres are uniformly sized, 6.2 μm in diameter, and can be coupled to the appropriate probe or protein based on the assay requirements. The microspheres have a magnetic composite structure, facilitating automated separation.

[0019] Based on the flow cytometry principle, the microspheres are analyzed one by one, the red laser identifies the microsphere code, the green laser reads the fluorescence intensity of the reporter molecule, and the computer analyzes and processes the fluorescence signal to convert it into a quantitative detection result.

[0020] Microspheres were purchased from Hubei Xinzongke Viral Disease Engineering Technology Co., Ltd., product name and number Magnetic fluorescent microspheres 301 P0289-01 Magnetic fluorescent microspheres 303 P0291-01 Magnetic fluorescent microspheres 304 P02952-01 Protein coupling: EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) jointly activate the carboxyl groups on the surface of NovaStar® magnetic microspheres. The activated groups can be replaced by primary amines on the surface of proteins to form ester groups, ultimately forming the coupling product of NovaStar® magnetic microspheres and proteins.

[0021] Antigen coating Porcine deltacoronavirus (PDCoV) N protein, porcine acute diarrhea syndrome coronavirus (SADS-CoV) N protein, and porcine epidemic diarrhea virus (PEDV) N protein were coated according to the instructions of the protein coupling kit.

[0022] After the liquid protein chip experiment, the optimization of liquid protein chip detection conditions was determined.

[0023] Preparation of serum samples to be tested: starting from 100-fold dilution, the serum samples were diluted 2-fold.

[0024] Dilute the coated microspheres with diluent to 50 microspheres / µL.

[0025] Vortex and sonicate for 10 s each to resuspend the microspheres.

[0026] Add 5 µL of diluted microspheres to each well of a 96-well plate.

[0027] Add 50 µL of serum sample diluted at a specific ratio, cover the plate, and incubate on a shaker at 1200 rpm and 37°C for 60 min.

[0028] Place the reaction plate on a magnetic plate and magnetically absorb it for 2 minutes to separate the microspheres. Discard the reaction solution by attaching the plate to the magnetic plate.

[0029] Wash with 100 µL / well of washing buffer, magnetically aspirate for 2 minutes, separate the microspheres, attach to the magnetic plate, and discard the washing buffer. Repeat once.

[0030] Remove the reaction plate from the magnetic separator and add 50 µL of biotinylated goat anti-swine secondary antibody (2 µg / mL). Cover the plate and incubate the reaction plate on a shaker at 1200 rpm and 37°C for 30 min.

[0031] Place the reaction plate on a magnetic plate and magnetically absorb it for 2 minutes to separate the microspheres. Discard the reaction solution by attaching the plate to the magnetic plate.

[0032] Wash with 100 µL / well of washing buffer, magnetically aspirate for 2 minutes, separate the microspheres, attach to the magnetic plate, and discard the washing buffer. Repeat once.

[0033] Add 50 µL (2.5 µg / mL) of SAPE (streptavidin-phycoerythrin stock solution) to each well, cover the plate, and place the reaction plate on a shaker at 1200 rpm and 37°C for 30 min.

[0034] Place the reaction plate on a magnetic plate and magnetically absorb it for 2 minutes to separate the microspheres. Discard the reaction solution by attaching the plate to the magnetic plate.

[0035] Wash with 100 µL / well of washing buffer, magnetically aspirate for 2 minutes, separate the microspheres, attach to the magnetic plate, and discard the washing buffer. Repeat once.

[0036] Remove the reaction plate from the magnetic separator, add 70 µL of PBS-TBN (phosphate buffered saline), and place the reaction plate on a shaker for 1 min.

[0037] The median fluorescence intensity (MFI), also known as the P / N value, is read on the analyzer. If the P / N value is >5, it indicates that the coated protein has good antigenicity and it is worth further optimizing the reaction conditions.

[0038] Optimization of detection conditions for liquid-phase protein microarray Determination of the optimal coating concentration and cutoff value of recombinant antigen: Magnetic beads were coated with antigen protein at concentrations of 5 μg, 10 μg, 15 μg, 20 μg, and 25 μg. The optimal dilution factor was determined for three viral reference sera at 400x, 800x, 1600x, 3200x, 6400x, and 12800x dilutions. Negative sera were diluted 100x to determine the cutoff value, calculated as: negative mean (average values with significant deviations were removed) + 3 times the sample standard deviation. A goat anti-swine secondary antibody was diluted 1:1000 and the detection antibody was diluted 1:500 with SPAE. Two replicates were performed, the mean values were calculated, and the ratio of the positive to negative MFI values (P / N ratio) was calculated. The optimal working antigen concentration was determined by the protein coating concentration that achieved the highest sensitivity and the largest P / N ratio.

[0039] Specificity test The established xMAP method was used to detect reference sera for classical swine fever virus, circovirus type 2, pseudorabies virus, porcine reproductive and respiratory syndrome virus and parvovirus to investigate whether there was any cross-reaction in the detection method established in this experiment.

[0040] Sensitivity test PDCoV, SADS-CoV, and PEDV reference sera were diluted in multiples, and the sensitivity of the diluted sera was tested using the xMAP method, and the results were compared with those of ELISA.

[0041] Repeatability test The same sample was tested three times in parallel, with three replicates each time, and the intra-assay and inter-assay coefficient of variation (CV) was calculated. The coefficient of variation (CV) is calculated as follows: (standard deviation SD / mean) × 100% Testing of clinical samples The collected clinical samples were tested using the xMAP method and compared with the ELISA test results.

[0042] In this embodiment, the protein coating efficiency of the microspheres after coupling was 95%, and the background fluorescence intensity of the microspheres after blocking was 100 MFI.

[0043] Experimental steps: Magnetic beads were coated with antigen protein at concentrations of 5μg, 10μg, 15μg, 20μg, and 25μg respectively; the three virus reference sera were diluted 400 times, 800 times, 1600 times, 3200 times, 6400 times, and 12800 times to explore the optimal dilution factor, and the negative serum was diluted 100 times to determine the cutoff value.

[0044] The optimal antigen coating concentration was 15 μg, at which point the P / N value of the positive serum was 6.2 (P / N value > 5, indicating a good antigen coating effect).

[0045] The cutoff value for negative serum is 150 MFI.

[0046] Experimental steps: Start with a 100-fold dilution of the serum sample to be tested and perform a 2-fold serial dilution; dilute the coated microspheres to 50 beads / µL with diluent and resuspend by vortexing and sonication; add 5 µL of diluted microspheres to each well of a 96-well plate; add 50 µL of diluted serum sample and incubate at 37°C with shaking for 60 minutes; separate the microspheres using a magnetic plate, discard the reaction solution, and wash twice with washing solution; add 2 µg / mL biotin-labeled goat anti-pig secondary antibody and incubate at 37°C with shaking for 30 minutes; repeat the magnetic separation and washing steps, add 2.5 µg / mL streptavidin-phycoerythrin stock solution, and incubate at 37°C with shaking for 30 minutes; perform magnetic separation and washing again, add PBS-TBN solution, shake, and read the median fluorescence value on a microscope to calculate the P / N value.

[0047] Among them, the best serum dilution ratio was 1600 times, at which time the P / N value of the positive serum was 5.8.

[0048] The MFI value of negative serum is 120, and the MFI value of positive serum is 700.

[0049] Two fluorescent dyes were mixed at different concentrations to form a combination array of fluorescent-coded microspheres with different energy levels. The coding values of the two differently coded microspheres under red laser were 600 and 800 MFI, respectively. The fluorescence signal stability of the microspheres was 98%, the fluorescence intensity of positive samples was 700 MFI, and the fluorescence intensity of negative samples was 120 MF. The detection sensitivity was 95% and the specificity was 98%.

[0050] The present invention can quickly, effectively and accurately detect PDCoV, SADS-CoV and PEDV antibodies in the sample to be tested, providing a reference for the prevention of porcine coronavirus and the control of the early stage of the disease.

[0051] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, and they are all covered by the scope of protection of the present invention.

Claims

1. A method for preparing a liquid-phase protein chip for simultaneous detection of three porcine coronaviruses, characterized in that: The following steps are involved: After activation and coupling of the microspheres, the microspheres were used to coat antigens of porcine deltacoronavirus N protein, porcine acute diarrhea syndrome coronavirus N protein, and porcine epidemic diarrhea virus N protein, respectively; Two fluorescent dyes are mixed at different concentrations to form a combination array of fluorescent coded microspheres with different energy levels; Based on the flow cytometry principle, the microspheres are analyzed one by one. The red laser identifies the microsphere code, the green laser reads the fluorescence intensity of the reporter molecule, and the computer analyzes and processes the fluorescence signal to convert it into a quantitative detection result. After the liquid protein chip experiment, the optimization of liquid protein chip detection conditions was determined.

2. The method for preparing a liquid-phase protein chip for simultaneous detection of three porcine coronaviruses according to claim 1, characterized in that: The microspheres include magnetic fluorescent microspheres 301 , magnetic fluorescent microspheres 303 and magnetic fluorescent microspheres.

3. The method for preparing a liquid-phase protein chip for simultaneous detection of three porcine coronaviruses according to claim 1, characterized in that: The liquid phase protein chip test includes: The serum sample to be tested was diluted 100-fold and then diluted 2-fold; The coated microspheres were diluted to 50 microspheres / µL with diluent and resuspended by vortexing and sonication; Add 5 µL of diluted microspheres to each well of a 96-well plate; Add 50 μL of diluted serum sample and incubate at 37°C with shaking for 60 minutes; The microspheres were separated by magnetic plate, the reaction solution was discarded and washed twice with washing solution; Add 2 μg / mL biotinylated goat anti-pig secondary antibody and incubate at 37°C with shaking for 30 minutes; Repeat the magnetic separation and washing steps, add 2.5 µg / mL streptavidin-phycoerythrin stock solution, and incubate at 37°C with shaking for 30 minutes; Perform magnetic separation and washing again, add PBS-TBN solution, shake and read the fluorescence median on the machine, calculate the P / N value. If the P / N value is greater than 5, it indicates that the coated protein has good antigenicity and the reaction conditions should be further optimized.

4. The method for preparing a liquid-phase protein chip for simultaneous detection of three porcine coronaviruses according to claim 1, characterized in that: Magnetic beads were coated with antigen protein at concentrations of 5 μg, 10 μg, 15 μg, 20 μg, and 25 μg, respectively. After activating two blank magnetic beads with different codes, the coating buffer was resuspended and shaken, and different protein solutions were added after ultrasonic dispersion. After mixing, the mixture was incubated at room temperature in the dark for 2 hours, and then washed and blocked at 4°C overnight. After washing again, the preservation solution was added to obtain two magnetic beads coated with different antigens.

5. The method for preparing a liquid-phase protein chip for simultaneously detecting three porcine coronaviruses according to claim 1, characterized in that: The optimization of the liquid phase protein chip detection conditions specifically includes: Determination of the optimal coating concentration and cutoff value of recombinant antigen: Magnetic beads were coated with antigen protein at 5 μg, 10 μg, 15 μg, 20 μg, and 25 μg of magnetic beads; The three virus reference sera were diluted 400-fold, 800-fold, 1600-fold, 3200-fold, 6400-fold, and 12800-fold to explore the optimal dilution factor, and the negative serum was diluted 100-fold to determine the cutoff value; The goat anti-pig secondary antibody was diluted 1:1000 and the SPAE was diluted 1:500 to detect the antibody. Two replicates were performed, and the average value was taken to calculate the ratio of the positive MFI value to the negative MFI value, i.e., the P / N value; The protein coating concentration with the highest detection sensitivity and the largest P / N is the optimal working concentration of the antigen.

6. The method for preparing a liquid-phase protein chip for simultaneous detection of three porcine coronaviruses according to claim 1, characterized in that: The process of activation and coupling of the microspheres includes: The magnetic microspheres were vortexed and sonicated for 10 seconds, washed twice with buffer 1, and finally resuspended with buffer 1; Add N-hydroxysuccinimide and carbodiimide solution, vortex to mix, and incubate at room temperature in the dark for 20 minutes; wash twice with buffer 2, and finally resuspend in buffer 2; N protein was added to AL32 microspheres, and S-RBD protein was added to AL12 microspheres. After vortexing, the mixture was incubated at room temperature in the dark for 2 hours. After coupling, the mixture was washed twice, and blocking solution was added. After vortexing and sonication, the mixture was blocked at 4°C in the dark for 17 hours. After washing twice, add preservation solution, mix well and store at 4℃ in the dark.

7. A liquid phase protein chip kit for simultaneous detection of three porcine coronaviruses, characterized in that: The kit includes three fluorescent magnetic microspheres with different codes; and the three fluorescent magnetic microspheres are respectively coupled with porcine delta coronavirus N protein, porcine acute diarrhea syndrome coronavirus N protein, and porcine epidemic diarrhea virus N protein.

8. The liquid phase protein chip kit according to claim 7, characterized in that The kit also includes a biotin-labeled donkey anti-goat secondary antibody for reacting with the sample to be tested, the quality control product and the negative serum.

9. The liquid phase protein chip kit according to claim 7, characterized in that Also included is streptavidin-phycoerythrin.

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