A new detection method and application of biosensor chip based on NanoSPR technology

Abstract

The present invention discloses a new detection method and application of a biosensor chip based on NanoSPR technology, which belongs to the field of in vitro qualitative and quantitative detection technology. The biosensor chip provided by the present invention includes a substrate and a protein, antibody or functional group modified on the substrate; the substrate includes a substrate, a titanium film, a silver film and a gold film from bottom to top, and the substrate surface matrix is ​​arranged with nanopores; the thickness of the titanium film is 2-20nm, the thickness of the silver film is 9-90nm, and the thickness of the gold film is 5-50nm; the protein, antibody or functional group specifically refers to a protein, antibody, aptamer or functional group that can specifically bind to the target to be tested, and this application not only involves all animal and human target detections that can be detected by ELISA, but also provides a modification method of the above chip, as well as a detection method, with high detection sensitivity, fast detection speed and high throughput, without labeling, and directly amplifying the antibody to be tested by the secondary antibody sandwich method, which can be used for high-throughput quantification or screening quality control.

Description

Technical Field

[0001] The present invention belongs to the technical field of qualitative and quantitative detection of antibodies in human / animal bodies, in particular to a method for rapid detection of antibodies in human / animal bodies based on pure NanoSPR technology. Background Art

[0002] African swine fever (ASF) is a highly contagious viral disease that affects domestic and wild pigs, with high mortality and rapid spread, causing huge socioeconomic losses. Its pathogen is a large, enveloped DNA virus, the ASF virus (ASFV). Currently, there is no approved vaccine or effective treatment for ASFV. Therefore, disease control mainly relies on early diagnosis, timely culling of infected pigs, and improved biosecurity controls in the pig industry. After infection with ASFV, especially subacute infection, surviving pigs may have detectable levels of ASFV antibodies. Therefore, it is a good marker for detecting ASFV antibodies in endemic areas affected by ASF. Antibody testing is economical, compatible with automated equipment, and suitable for high-throughput screening. Since no commercial vaccine is currently available, antibodies remain a clear indicator of ASFV infection in this context. Therefore, the development of a rapid antibody test for ASFV monitoring is of great significance in this field.

[0003] Nanoplasma chip technology (NanoSPR) is different from the two technical modes of plasma chip technology (SPR) and localized surface plasmon resonance technology (LSPR). Due to its unique three-dimensional structure, it is different from the SPR effect of the planar model and the LSPR effect of metal nanoparticles. It can support both SPR and LSPR modes at the same time.

[0004] The plasma resonance effect of the nanopore array biosensor can be directly incident on the nanopore metal structure, and the surface light field is immediately excited, so there is no need for complex optical paths and large optical instruments. NanoSPR technology retains many advantages of traditional SPR sensors, such as real-time, no labeling, no background interference, and high resolution, and retains the advantages of LSPR sensors in performance modification; by adjusting the aperture, depth, shape, period of the nanopore array, and the type and thickness of the surface metal, high-quality chips that can capture the strongest LSPR signal are selected, and stronger signals can be obtained without a large spectrometer.

[0005] At present, lateral flow immunochromatographic strips based on gold nanoparticles, latex microspheres or fluorescent dye microspheres are designed for on-site detection of ASFV antibodies in serum. However, their sensitivity and stability quantification still need to be further improved, and they cannot accurately quantify the antibody content of ASFV. TaqMan-based real-time quantitative PCR (RT-PCR) and ELISA are the most widely used laboratory immunology-based diagnostic methods in EU reference laboratories and OIE because of their high sensitivity. Unfortunately, the above methods cannot be widely used in the field of rapid detection in pig farms. In recent years, some advanced technologies, such as isothermal amplification, lateral flow chromatography and microfluidic chips, have been used to detect ASFV. However, none of the diagnostic technology products can meet the needs of early, sensitive and quantitative on-site diagnosis.

[0006] So we developed a new biosensor chip based on NanoSPR technology to detect ASFV African swine fever antibodies. Summary of the invention

[0007] In order to meet the requirements of the above-mentioned African swine fever ASFV antibody diagnostic technology, the present invention provides a biosensor chip based on pure NanoSPR technology for ultra-sensitive quantitative detection of ASFV antibodies in serum. It has high detection sensitivity. The use of the biosensor chip to detect African swine fever antibodies and other antibodies has the advantages of high throughput, short time consumption, accurate test results, and potential for point-of-care testing (POCT). No labeling is required. The antibody to be tested is directly amplified by the secondary antibody sandwich method, which can be used for high-throughput quantification or screening quality control. It is specifically achieved through the following technologies.

[0008] The biosensor chip based on NanoSPR technology includes a substrate and a protein, antibody or functional group modified on the substrate; the substrate includes a base, a titanium film, a silver film and / or a gold film from bottom to top, and the surface of the substrate is imprinted with nanopores arranged in a matrix; the diameter of the nanopore is 50-800nm, the height is 100-800nm, and the spacing between two adjacent nanopores is 200-1000nm; the thickness of the titanium film is 2-20nm, the thickness of the silver film is 8-80nm, and the thickness of the gold film is 5-50nm; the protein, antibody or functional group specifically refers to a protein, antibody, aptamer or functional group that specifically binds to a virus antibody. For example, the P30 protein and P54 protein that can bind to African swine fever virus antibodies; sheep anti-swine-IgG (Beijing Biolong), Protein A (Yiqiao Shenzhou), sheep anti-swine-IgG (Anti), and rabbit anti-swine-IgG (Boersi) that can bind to African swine fever virus antibodies.

[0009] Preferably, the thickness of the titanium film is 10 nm, the thickness of the silver film is 80 nm, and the thickness of the gold film is 15 nm.

[0010] The present invention also provides a method for preparing the modified biosensor chip based on NanoSPR technology, comprising the following steps:

[0011] S1, washing the substrate, adding 1-50 μl of 5-50 μg / ml P30 protein and / or P54 protein solution; incubating at 4-37° C. for 1.5-24 h, and then washing the substrate coated with P30 protein and / or P54 protein with PBST solution for multiple times;

[0012] S2, adding a blocking solution to the surface of the substrate obtained in step S1, incubating at 25-37° C. for 1-4 h, and removing excess blocking solution;

[0013] S3, dripping protective liquid on the surface of the substrate obtained in step S2, incubating at 25-37° C. for 0.5-1 h, removing excess protective liquid, and drying to obtain a biosensor chip.

[0014] The blocking solution is a complex solution with a concentration of 10 μg / ml prepared by dissolving bovine serum albumin in a CBS solution; the CBS solution contains 4-6 g / L potassium carbonate, 7.5-10 g / L sodium bicarbonate, and 2.5-5 ml / L of preservative Proclin300;

[0015] The protective solution is a complex solution with a concentration of 10-200 μg / ml prepared by dissolving sugar in PBST solution; the sugar is one of dextran, glucose, sucrose, and trehalose;

[0016] Preferably, the PBST solution contains 2-3 g / L disodium hydrogen phosphate dodecahydrate, 0.2-0.5 g / L sodium dihydrogen phosphate, 0.1-0.3 g / L potassium chloride, 6-8 g / L sodium chloride, 0.5-1 ml / L Tween-20, and 0.5-1 ml / L preservative Proclin300.

[0017] The present invention also provides a chip microplate, which integrates the above-mentioned biosensor chip. The chip microplate can be selected from various types of microplates such as 1-hole, 48-hole, and 96-hole.

[0018] The present invention also provides a gene delivery vector detection kit, which includes the above-mentioned biosensor chip, or includes the above-mentioned chip microplate. The kit can be equipped with other different reagents based on different detection methods (label-free detection, sandwich method).

[0019] Preferably, the above detection kit also includes washing solution, antibody purification reference substance, and African swine fever serum sample. Sheep anti-swine IgG antibody, P30 protein and / or P54 protein can bind to African swine fever antibody at different epitopes.

[0020] A method for using the above chip microplate or detection kit to detect the concentration of the gene delivery vector in the sample to be tested, which method does not require the use of luminescent reagents such as colloidal gold, but directly inserts the chip microplate into a full-spectrum microplate reader for detection, and has very good sensitivity and specificity for antibody detection. The specific method is:

[0021] P1. Dilute the pig serum containing African swine fever antibodies or the plasma sample prepared with anticoagulant (heparin / EDTA / sodium citrate) 10-1000 times, add 30-50 μl to each well of the chip microplate, and shake the plate for reaction for 10-60 minutes;

[0022] P2. Discard the liquid in the wells, add 30-150 μL PBST washing solution, wash the plate, tap the plate, and spin dry, repeat 2 times;

[0023] P3, add 30-50 μl PBST diluent to the well, put it into the microplate reader, and record the initial value;

[0024] P4, discard the liquid in the well of step P3, add 30-50μl of 5-40ug / mL secondary antibody diluent, shake the plate for 10 minutes, put it into the microplate reader, record the end point value, and obtain qualitative and quantitative analysis; the full spectrum of the antibody to be tested and the protein incubated on the chip will produce displacement changes in the same wavelength band before and after the binding reaction, and the difference between the spectrum values ​​before and after the displacement will appear as a waveform diagram of peaks and valleys. There is no need to use horseradish peroxidase (HRP) to label antibodies. Only the spectrum changes generated by the Nano SPR chip can detect whether antibodies are bound to the chip surface.

[0025] The secondary antibody diluent includes 10mM Tris / NaCl, pH 6-9, 0.01-1wt.% TW20, 0.05-5%wt.BSA / serum + 0.1-10wt.% trehalose / glucose / sucrose + 0.01-1wt.% PEG2w / PEG6k; this diluent can inhibit the nonspecific reaction caused by the impurity protein in the serum, and at the same time, protect the stability of African swine fever antibodies in the sample, and promote the binding of antibodies to the antibodies on the chip surface through PEG6000 or PEG20000 and other aggregation promoters.

[0026] P5. Take another new chip microplate after the operation in step P1, repeat the operation method in steps P2-P4 to add 30-200 μl of the diluted sample to be tested into each well of the chip microplate, record the endpoint value again, and obtain the qualitative and quantitative analysis of the sample to be tested based on the cutoff value specified for the Yin and Yang ginseng.

[0027] In the above detection method, the sample to be tested can be collected from human or animal serum to detect the concentration of virus antibodies. Therefore, whether for the purpose of diagnosis or treatment, or for non-diagnosis or treatment, the above method can be used to perform qualitative and quantitative detection of the virus, and has very good sensitivity and specificity.

[0028] The sample diluent includes 10 mM Tris / NaCl, pH 6-9, 0.01-0.1 wt.% TW20, 0.05-5% wt.% BSA / serum + 0.01-1 wt.% PEG2w / PEG6k.

[0029] Compared with the prior art, the present invention is beneficial in that: based on NanoSPR technology, the present invention provides a biosensor chip that can be used to detect gene delivery vectors, a chip microplate integrating the above biosensor chip, and a corresponding detection kit. The biosensor chip, chip microplate and detection kit provided by the present invention can complete the detection of antibodies in just one step, with high detection sensitivity, low detection limit, fast detection speed and high throughput, and can be used for high-throughput quantification or screening quality control in the field of gene delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Example 1 uses chips with four parameters to detect four samples;

[0031] Figure 2 Full spectra of 11 samples in Example 2 at 560-640nm;

[0032] Figure 3 The absorbance at 590 nm of the 11 samples in Example 2 was subtracted from the absorbance at 570 nm;

[0033] Figure 4 Full spectra of 11 samples in Example 3 at 560-640nm;

[0034] Figure 5 The absorbance at 590 nm of the 11 samples in Example 3 was subtracted from the absorbance at 570 nm;

[0035] Figure 6 Full spectra of three samples of five secondary antibodies in Example 4 at 560-640 nm;

[0036] Figure 7 The absorbance at 590 nm of the three samples of the five secondary antibodies in Example 4 was subtracted from the absorbance at 570 nm;

[0037] Figure 8 The full spectrum of Example 5 at different dilution gradients at 560-640 nm;

[0038] Fig. 9 Example 5 Dilution gradient: the horizontal axis represents Yin ginseng, Yang ginseng and 776 positive African swine fever samples with different gradient dilutions, and the vertical axis represents OD value (absorbance);

[0039] Fig.10 Example 5 Standard curve: the horizontal axis represents samples of different concentrations, and the vertical axis represents OD value (absorbance);

[0040] Fig.11 Example 6 ROC curve;

[0041] Fig.12 Full spectrum of 11 samples of Comparative Example 1 at 560-640nm;

[0042] Fig.13 The absorbance at 590 nm of the 11 samples of Comparative Example 1 was subtracted from the absorbance at 570 nm;

[0043] in: Figure 2 , 4 , 6, 8, 12: The horizontal axis represents the wavelength of 560-640nm, and the vertical axis represents the OD value (absorbance); Figure 3 , 5 , 7, 13: The horizontal axis represents Yin ginseng, Yang ginseng, and sample, and the vertical axis represents OD value (absorbance). DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] In the following examples and comparative examples, the double QDM recombinant viral proteins 30 and 54 (P30 and P54) used were purchased from Keqian Company, Protein A protein, goat anti-swine IgG antibody, and mouse anti-swine IgG antibody were purchased from Beijing Yiqiao Shenzhou Company; bovine serum albumin was purchased from Wuhan Qianzesui Biotechnology Co., Ltd.; the protective solution was prepared by dissolving sucrose in PBST solution, with a concentration of 100 μg / ml. The P30 protein and P54 protein solutions used were prepared by diluting the above P30 protein and P54 protein with CBS solution to the corresponding concentrations.

[0046] The raw materials and preparation method of PBST solution are: 2.5g / L disodium hydrogen phosphate dodecahydrate, 0.3g / L sodium dihydrogen phosphate, 0.2g / L potassium chloride, 7.5g / L sodium chloride, 0.8ml / L Tween-20, 0.8ml / L preservative Proclin300, add double distilled water and mix well.

[0047] The CBS solution is prepared by mixing 5.5 g / L potassium carbonate, 8.5 g / L sodium bicarbonate, 4 ml / L preservative Proclin 300, and purified water.

[0048] PBST solution was used as the washing solution; African swine fever antibody samples were purchased from Keqian Biotechnology Co., Ltd.

[0049] Embodiment 1:

[0050] Four samples (two negative samples + two positive samples) were tested using a chip with four parameters, such as Figure 1 As shown, the horizontal axis represents four chips with different thicknesses:

[0051] 15nm Ti+70nm Ag+20nm Au; 15nm Ti+60nm Ag+20nm Au; 15nm Ti+70nm Ag; 15nmTi+70nm Au;

[0052] The ordinate represents OD value (absorbance).

[0053] The chip of the present invention must be connected with a Ti layer or a TiO2 layer, otherwise the metal on the chip will fall off due to the lack of an adhesive (connection) layer.

[0054] Example 2: One-step label-free detection of African swine fever antibodies using P30 protein-coated microplates

[0055] In this embodiment, the preparation method of the chip microplate (96 holes) of the nanoplasmonic resonance biochip is as follows: (1) photolithography is performed on an oxidized silicon wafer to produce a nanopore mold; (2) the nanostructure on the 12-inch silicon wafer nanomold is transferred to a polymer flexible material substrate (PET thin plate substrate) by a nanoimprint method to form an inverse wafer-level nanodevice structure; (3) a titanium film layer (thickness of 10 nm), a silver film layer (thickness of 80 nm), and a gold film layer (thickness of 15 nm) are sequentially plated on its surface to form a 96-hole chip microplate integrated with a plasma sensor chip substrate;

[0056] 1. Modification and preparation of biosensor chips

[0057] (1) washing the substrate, adding 50 μl of 8 μg / ml P30 protein solution, incubating at 37° C. for 2 h, and then washing the P30 protein-coated substrate several times with PBST solution;

[0058] (2) Add blocking solution at a volume of 150 μl / well onto the surface of the substrate, incubate in a 37°C oven for 1 h, and wash with PBST solution to remove excess blocking solution;

[0059] (3) Add protective liquid to the surface of the substrate in step (2) at a volume of 150 μl / well, incubate in a 37°C oven for 30 min, wash with PBST solution to remove excess protective liquid, and continue to place in a 37°C oven for 10 min to ensure that the chip surface is dry, thereby obtaining a biosensor chip.

[0060] 2. Label-free method for testing serum samples to determine the strength of antibodies in samples

[0061] (1) Use a pipette to dilute the African swine fever pig serum 50 times, add 50 μl to each well of the chip microplate, and shake the plate for reaction for 20 minutes;

[0062] The diluent formula is 10 mM Tris / NaCl solution, 0.1 wt.% TWeen20, 5 wt.% fetal bovine serum, 1 wt.% PEG6k, pH = 7;

[0063] (2) Discard the liquid in the wells, add 150 μL of PBST washing solution, wash the plate, tap the plate, and spin dry, repeat twice;

[0064] (3) Add 50 μl of PBST diluent to the well, place it in the microplate reader, and record the initial value;

[0065] (4) Discard the liquid in the wells from step (3), add 50 μl of 5 ug / mL secondary antibody diluent, shake the plate for 10 min, then place in an ELISA reader and record the endpoint value. Based on the cutoff values ​​specified for the Yin and Yang ginseng, the qualitative and quantitative analysis of the sample to be tested can be obtained.

[0066] Secondary antibody diluent: 10mM Tris / NaCl (PH7.3) + 0.1wt.% TW20 + 0.1wt.% BSA + 0.2wt.% sucrose + 0.05% PEG2W

[0067] The sample contains 11 samples: 6 negative (including 3 specific) + 3 weak positive + 2 positive Figure 2-3 As shown, the test results for 11 samples were: 6 negative and 5 positive. Both strong positive and weak positive samples were uniformly detected as positive samples. The compliance rate of this test result was as high as about 100%.

[0068] Example 3: One-step label-free detection of African swine fever antibodies using P54 protein-coated microplates

[0069] The differences between this embodiment and embodiment 2 are: (1) 8 μg / ml P54 protein (Keqian Company); (2) chip thickness 15+70+20.

[0070] The sample contains 11 samples: 6 negative (including 3 specific) + 3 weak positive + 2 positive

[0071] like Figure 4-5 As shown, the test results were for 11 samples: 7 negative (1 weakly positive sample was not tested accurately) + 4 positive, and the compliance rate of this test result was about 91%.

[0072] Example 4: One-step label-free detection of African swine fever antibodies using five different detection antibodies

[0073] Example 1

[0074] Use different porcine secondary antibodies or proteins:

[0075] (1) Beijing Biolon sheep anti-swine IgG, 10ug / mL;

[0076] (2) Protein A from Sino Biological Shenzhou, 10ug / mL;

[0077] (3) Anti-rat anti-swine IgG, 10ug / mL;

[0078] (4) Anti-goat anti-swine IgG, 10ug / mL;

[0079] (5) Borsi rabbit anti-swine IgG, 10ug / mL;

[0080] like Figure 6-7 As shown, three samples (negative + positive + specific) were selected for the detection of African swine fever antibodies, and the antibody or protein test results numbered (1, 2, 4, 5) were consistent with the actual results. Among them, the pig secondary antibody (4), namely Antigen's sheep anti-swine-IgG, had the best detection result.

[0081] Example 5: One-step label-free detection of African swine fever antibody quantitative experiment using P30 protein coated chip and goat anti-swine IgG antibody as secondary antibody

[0082] Under the optimal experimental conditions, the chip was coated with P30 protein (Keqian Company) and goat anti-pig IgG antibody (Beijing Biolong) was used to measure the standard positive serum with different dilutions from 1:256 to 1:16384 using NanoSPR sensor. Fig. 9 The OD value is Figure 8The difference between the peak and the trough of the curve for each concentration; Fig.10 The standard curve equation obtained for the detection of African swine fever antibodies is, Y = (AD) / [1 + (X / C) B ]+D; A=0.15545, B=-0.68142, C=2.46710, D=-0.04771, r 2 =0.9977.

[0083] Example 6: Using P30 protein coated chip, goat anti-swine IgG antibody as secondary antibody to perform a one-step label-free method to detect African swine fever antibody quantitative experiment using 164 samples (146 negative or specific samples + 18 positive samples) to make ROC curves, such as Fig.11 As shown. The ROC curve is a curve that reflects the relationship between sensitivity and specificity. The horizontal axis X-axis is 1-specificity, also known as the false positive rate (false alarm rate). The closer the X-axis is to zero, the higher the accuracy; the vertical axis Y-axis is called sensitivity, also known as the true positive rate (sensitivity). The larger the Y-axis is, the better the accuracy.

[0084] Comparative Example 1:

[0085] Different from Example 3, the sample diluent and secondary antibody diluent formulas in this comparative example are changed to use 10mM Nacl solution, 0.1wt.% TWeen20, 3wt.% fetal bovine serum, 2wt.% PEG2w, pH value = 7; the full spectrum of 11 samples in comparative example 1 is tested at 560-640nm and the absorbance at 590nm minus the absorbance at 570nm.

[0086] like Figure 12-13 As shown in the figure, the use of different sample diluents and secondary antibody diluents reduced the sensitivity of the reaction. Even though the strong positive samples, specific samples, and negative samples all met the experimental results, the three weak positive samples were not detected, and the expected detection effect was not achieved.

Claims

1. Biosensor chip based on pure NanoSPR technology, characterized in that: It comprises a substrate and a protein, antibody or functional group modified on the substrate; the substrate comprises a base, a titanium film, a silver film and / or a gold film from bottom to top, and the surface of the substrate is imprinted with nanopores arranged in a matrix; the diameter of the nanopores is 50-800nm, the height is 100-800nm, and the distance between two adjacent nanopores is 200-1000nm; the thickness of the titanium film is 10-20nm, the thickness of the silver film is 80-90nm, and the thickness of the gold film is 15-50nm; the protein, antibody or functional group specifically refers to a protein, antibody, aptamer or functional group that specifically binds to the antibody of the object to be detected; The modification and preparation method of the biosensor chip based on pure NanoSPR technology comprises the following steps: S1, washing the substrate, adding 1-50 μl of 5-50 μg / ml P30 protein and / or P54 protein solution; incubating at 4-37° C. for 1.5-24 h, and then washing the substrate coated with P30 protein and / or P54 protein with PBST solution for multiple times; S2, adding a blocking solution to the surface of the substrate obtained in step S1, incubating at 25-37° C. for 1-4 h, and removing excess blocking solution; S3, dripping protective liquid on the surface of the substrate obtained in step S2, incubating at 25-37° C. for 0.5-1 h, removing excess protective liquid, and drying to obtain a biosensor chip.

2. The biosensor chip based on pure NanoSPR technology according to claim 1, characterized in that: The thickness of the titanium film is 10 nm, the thickness of the silver film is 80 nm, and the thickness of the gold film is 15 nm.

3. The biosensor chip based on pure NanoSPR technology according to claim 1, characterized in that: The blocking solution is a composite solution with a concentration of 10 μg / ml prepared by dissolving bovine serum albumin in a CBS solution; the CBS solution contains 4-6 g / L potassium carbonate, 7.5-10 g / L sodium bicarbonate, and 2.5-5 ml / L preservative Proclin300.

4. The biosensor chip based on pure NanoSPR technology according to claim 1, characterized in that: The protective solution is a complex solution with a concentration of 10-200 μg / ml prepared by dissolving sugar in PBST solution; the sugar is one of dextran, glucose, sucrose and trehalose.

5. The biosensor chip based on pure NanoSPR technology according to claim 1, characterized in that: The PBST solution contains 2-3 g / L of disodium hydrogen phosphate dodecahydrate, 0.2-0.5 g / L of sodium dihydrogen phosphate, 0.1-0.3 g / L of potassium chloride, 6-8 g / L of sodium chloride, 0.5-1 ml / L of Tween-20, and 0.5-1 ml / L of preservative Proclin300.

6. A chip microplate integrated with the biosensor chip of claim 1.

7. A pure NanoSPR antibody detection kit, characterized in that: The biosensor chip comprises the biosensor chip according to claim 1 or 2, or the chip microplate according to claim 6.

8. The pure NanoSPR antibody detection kit according to claim 7, characterized in that: It also includes washing solution, African swine fever antibody purification reference substances, and African swine fever serum samples.

9. A method for detecting African swine fever antibodies not for the purpose of diagnosis and treatment, characterized in that: Using the chip microplate according to claim 6, the specific steps are: P1. Dilute the pig serum or plasma sample prepared with anticoagulant containing African swine fever antibodies 10 to 1000 times, add 30 to 50 μl to each well of the chip microplate, and vibrate the plate for reaction for 10 to 60 minutes; The sample diluent includes 10 mM Tris / NaCl, pH 6-9, 0.01-0.1 wt.% TW20, 0.05-5% wt.% BSA / serum + 0.01-1 wt.% PEG2w / PEG6k; P2. Discard the liquid in the wells, add 30-150 μL PBST washing solution, wash the plate, tap the plate, and spin dry, repeat 2 times; P3, add 30-50 μl PBST diluent to the well, put it into the microplate reader, and record the initial value; P4, discard the liquid in the wells of step P3, add 30-50 μl of 5-40 ug / mL secondary antibody diluent, shake the plate for 10 minutes, then place in the microplate reader, record the endpoint value, and obtain qualitative and quantitative analysis; The secondary antibody diluent includes 10 mM Tris / NaCl at pH 6-9, 0.01-1 wt.% TW20, 0.05-5% wt.% BSA / serum + 0.1-10 wt.% trehalose / glucose / sucrose + 0.01-0.1 wt.% PEG2w / PEG6k; P5. Take another new chip microplate after the operation in step P1, repeat the operation method in steps P2-P4 to add 30-200 μl of the diluted sample to be tested into each well of the chip microplate, record the endpoint value again, and obtain the qualitative and quantitative analysis of the sample to be tested based on the cutoff value specified for the Yin and Yang ginseng.

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