NL2026488B1 - Method for preparing a protein chip plate - Google Patents
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- NL2026488B1 NL2026488B1 NL2026488A NL2026488A NL2026488B1 NL 2026488 B1 NL2026488 B1 NL 2026488B1 NL 2026488 A NL2026488 A NL 2026488A NL 2026488 A NL2026488 A NL 2026488A NL 2026488 B1 NL2026488 B1 NL 2026488B1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
- C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00497—Features relating to the solid phase supports
- B01J2219/00527—Sheets
- B01J2219/00529—DNA chips
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00608—DNA chips
Abstract
The present invention relates to a method for preparing a protein chip plate, comprising: Step 1) preparing a membrane treatment solution, immersing a nanomembrane in the membrane treatment solution, and then rinsing the nanomembrane with purified water followed by blow drying; Step 2) preparing a spotting solution, diluting a protein with the spotting solution and spotting on the nanomembrane to obtain a protein chip, Step 3) drying the protein chip at a temperature of 23°C i 2°C and a humidity of 25% or less, and Step 4) preparing a membrane blocking solution, and blocking the protein chip with the membrane blocking solution to obtain a protein chip plate. The method relates to combined use of the membrane treatment solution, drying conditions, and membrane blocking solution, so that the sample spots have clear edges without any overflow, tailing or halo phenomena, the sample spots are regular, the coefficient of variation of the sample spots’ diameters is small, the matriX is regular, the color of different proteins after the reaction is moderate, the gray value is moderate and the coefficient of variation of the gray value is small. The method provides a better repeatability, and a longer storage period, and can meet the demands of production.
Description
Method for preparing a protein chip plate
[0001] The present invention relates to a method for preparing a protein chip plate, which belongs to the field of biotechnology.
[0002] A protein chip is a high-throughput protein functional analysis technology, which can be used for protein expression analysis and protein-protein interaction study, and has outstanding advantages in disease screening, early diagnosis and screening of protein targets for drug action. Protein is the research object of protein chip technology, whose principle includes carrying out special chemical treatment on a solid-phase carrier to make its surface have certain functionality, and then immobilizing a protein molecule product (such as enzymes, antigens, antibodies, receptors, ligands, cytokines, etc.) on it; according to the characteristics of these biomolecules, it can capture a protein to be tested (existing in serum, plasma, lymph, interstitial fluid, urine, exudate, cell lysate, secretion, etc.) that can specifically bind to it, which is then confirmed and biochemically analyzed after being washed and purified; such technology provides strong technical support for obtaining important life information (such as unknown protein components, sequences, in-vivo expression levels, biological functions, mutual regulation relationship with other molecules (such as enzymes, antigens, antibodies, receptors, ligands, cytokines, etc.), drug screening, and drug target selection, etc.).
[0003] A protein microarray is one type of protein chips, whose principle includes designing spotting on the substrate through a spotting mechanical device, sucking the purified protein solution with spotting needles, moving them to the top of the substrate, immobilizing the protein solution on the surface of the substrate through contact or non-contact spraying, and forming a protein chip after subsequent processing.
[0004] However, in the kit preparing process, a protein chip plate is the main reason for intra- 1 batch and inter-batch differentiation. The treatment solution involved in the preparation of protein chip plates affects the morphology and overflow state of the sample spots, and it is extremely easy for the matrix to be irregular, and the blocking solution affects the subsequent reaction results of the protein. Even though there are many research results on spotters, spotting pins or substrates of protein chips in this field, there are few in-depth studies on the treatment solution and the blocking solution. Therefore, there is an urgent need to establish a preparation method that can reduce the differentiation of protein chips in order to meet the demand for mass production.
[0005] In order to solve the shortcomings of the prior art, the aim of the present invention is to optimize a method for preparing protein chip plates, so that, through optimization, protein sample spots have clear edges and regular matrix (or array), without any overflow, tailing, or halo phenomenon; the coefficient of variation of the sample spots’ diameters is small, the color of different proteins after the reaction is moderate, the gray value is moderate and the coefficient of variation of the gray value is small. The method provides a better repeatability, and a longer storage period of the protein chip plates.
[0006] To this end, the present invention provides a method for preparing a protein chip plate, comprising:
[0007] Step 1) preparing a membrane treatment solution, immersing a nanomembrane in the membrane treatment solution, and then rinsing the nanomembrane with purified water followed by blow drying;
[0008] Step 2) preparing a spotting solution, diluting a protein with the spotting solution and spotting on the nanomembrane to obtain a protein chip;
[0009] Step 3) drying the protein chip at a temperature of 23°C + 2°C and a humidity of 25% 2 or less; and
[0010] Step 4) preparing a membrane blocking solution and blocking the protein chip with the membrane blocking solution to obtain a protein chip plate.
[0011] As an embodiment of the present invention, the membrane treatment solution in step 1) is a PBS solution containing 4% w/v (g/ml) EDC, 2% w/v (g/ml) NHS, 0.25 %v/v-1%v/v
DMSO, which is added with 0.1%v/v DCC and mixed well before use. The PBS solution is a
PBS solution having a pH of 5.5.
[0012] A content of DMSO can be selected from 0.25%v/v, 0.30%v/v, 0.35%v/v, 0.40%vV/v, 0.45% v/v, 0.50%v/v, 0.55%v/v, 0.60%v/v, 0.65%v/v, 0.70%v/v, 0.75%v/v, 0.80%vV/v, 0.85%v/v, 0.90%v/v, 0.95%v/v, and 1% v/v.
[0013] As an embodiment of the present invention, the membrane treatment solution in the step 1) is a PBS solution containing 4% w/v EDC, 2% w/v NHS, and 0.5% v/v DMSO, which is added with 0.1%v/v DCC and mixed well before use.
[0014] As an embodiment of the present invention, in the step 1), the nanomembrane is immersed in the membrane treatment solution for 30-60 minutes, preferably 50 minutes.
[0015] The membrane treatment solution provided by the present invention is used for the preparation of protein chip plates, which can make the sample spots have clear edges without any overflow, and significantly improve the interference of static electricity on the surface of the nanomembrane, so that the matrix is regular, the coefficient of variation of diameter of the sample spots is small and the coefficient of variation of gray value of the sample spots is small, which indicates excellent uniformity of the nanomembrane treated by the treatment solution.
[0016] As an embodiment of the present invention, the protein in step 2) includes porcine 3 pseudorabies virus gD/gE protein, mycotoxin antigen, avian antibiotic antigen, and rotavirus antibody.
[0017] As an embodiment of the present invention, in the step 3), the protein chip 1s dried at a temperature of 23°C + 2°C and a humidity of 25% or less for 6-8 hours, preferably 6 hours.
[0018] As an embodiment of the present invention, in the step 4), the membrane blocking solution 1s a PBS solution containing BSA, glycerol and Tween-20, and the PBS solution is a
PBS solution having a pH of 7.4; preferably, the membrane blocking solution is a PBS solution containing 1% w/v BSA, 0.1%-0.5% v/v glycerol, and 0.1% v/v Tween-20.
[0019] A content of the glycerol can be selected from 0.10%v/v, 0.11%v/v, 0.12%v/v, 0.13%v/v, 0.14%v/v, 0.15%v/v, 0.16%v/v, 0.17%v/v, 0.18%v/v, 0.19% v/v, 0.20%v/v, 0.21%v/v, 0.22%v/v, 023%v/v, 024%v/v, 0.25%v/v, 0.26%v/v, 0.27% v/v, 028%v/v, 0.29%v/v, 0.30%v/v, 031%v/v, 0.32%v/v, 0.33%v/v, 0.34%v/v, 0.35%v/v, 0.36%v/v, 0.37%v/v, 0.38%v/v, 0.39%v/v, 0.40%v/v, 0.41%v/v, 0.42%v/v, 0.43%v/v, 0.44%v/v, 0.45%v/v, 0.46%v/v, 0.47%v/v, 0.48%v/v, 0.49%v/v, and 0.50% v/v.
[0020] The membrane blocking solution provided by the present invention is used for the preparation of a protein chip plate, which can make sample spots regular, without any phenomenon of tailing or halo, and make the color moderate after reaction with small coefficient of variation, making it easier to determine whether the sample 1s positive or negative.
For accelerated stability testing, the sample can be stored for 6 days, and for real-time stability testing, it can be stored for 15 months.
[0021] The present invention also provides a membrane treatment solution which is a PBS solution containing 4% w/v EDC, 2% w/v NHS, and 0.25% v/v - 1% v/v DMSO, which is added with 0.1%v/v DCC and mixed well before use, and the PBS solution is a PBS solution having a pH of 5.5; preferably, the membrane treatment solution is a PBS solution containing 4
4%w/v EDC, 2%w/v NHS, and 0.5 %v/v DMSO, which is added with 0.1%v/v DCC and mixed well before use.
[0022] The present invention also provides a membrane blocking solution, which is a PBS solution containing BSA, glycerol, Tween-20, and the PBS solution is a PBS solution having a pH value of 7.4; preferably, the membrane blocking solution is a PBS solution containing 1%
W/V BSA, 0.1%-0.5% V/V glycerol, and 0.1% V/V Tween-20.
[0023] The term "phosphate buffer” refers to a solution containing phosphoric acid or its salt and being adjusted to a desired pH value, which is the most widely used buffer in biochemical research. Generally, phosphate buffers are prepared from phosphoric acid or phosphates (including but not limited to sodium and potassium salts). Some phosphates are known 1n the art, such as sodium dihydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate, sodium phosphate and potassium phosphate. It is known that phosphates exist in the form of salt hydrates. Due to the secondary dissociation effect of the buffer, the buffer has a wide range of pH, for example, pH of about 4-10, preferably about 5-9.
[0024] The protein chip plates prepared in the present invention render the sample spots to have clear edges without any overflow, tailing, or halo phenomena; the sample spots are regular and the coefficient of variation of the sample spots’ diameters is small, the matrix of the sample spots is regular and the color of different proteins after the reaction 1s moderate, the gray value is moderate and the coefficient of variation of the gray value is small. The method provides a better repeatability, and a longer storage period, and can meet the demands of production.
[0025] FIG. 1 shows spotting photos of the nanomembrane after being pretreated by using treatment solution 1, treatment solution 2, treatment solution 3, treatment solution 4 from top to bottom; 5
[0026] FIG. 2 shows the reaction results after the nanomembrane is treated by using condition 1, condition 2, and condition 3 respectively from top to bottom;
[0027] FIG. 3 shows the reaction results after the nanomembrane is treated by using blocking solution 1, blocking solution 2, blocking solution 3, blocking solution 4, blocking solution 5, and blocking solution 6 respectively.
[0028] The description of the present invention is further provided below in connection with the specific embodiments, and features and advantages of the present invention will become more apparent from the following description. However, these embodiments are only exemplary, but not forming any limitation to the scope of the present invention. It should be understood by a person skilled in the art that modifications or alternatives to details and forms of the technical solution of the present invention can be made without deviation from the spirit and scope of the present invention, while those modification and alternatives should all fall within the scope of the present invention.
[0029] The chemical reagents used in the examples of the present disclosure are of analytical grade and are purchased from Sinopharm Group Co. Ltd. The experimental methods described in the present disclosure are conventional methods unless otherwise specified. The biological materials are commercially available unless otherwise specified.
Example 1 Preparation of modified silica gel membrane
[0030] According to US2006/0057180A1, small batches of nanomembranes (also known as modified silica gel membranes) were prepared, and the membranes were directly spotted as protein chips. After the kits were prepared, they could be used to detect antigens or antibodies, but when the same sample was tested, the inter-batch and intra-batch 6 repeatability was poor (CV>20%) and the thermal stability was poor (only can be stored for 2 days) too. There were serious defects in the detection effect of the kits, and the successful production of the kits could not be realized, and the demand of clinical use of the kits could not be met. Based on this, a large number of process optimizations were carried out, and the optimal results were selected for explanation.
Example 2 Preparation and evaluation of membrane treatment solution
[0031] 2.1 Reagents
DCC: Dicyclohexylcarbodiimide;
EDC: (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride),
NHS: N-hydroxysuccinimide;
DMSO: Dimethyl sulfoxide.
[0032] 2.2 Preparation method
[0033] Preparation of 4% EDC solution: 4g of EDC was weighed and added into pure water, stirred to dissolve, diluted to 100ml, and well shaken.
[0034] Preparation of 2% NHS solution: 2g of NHS was weighed and added into pure water, stirred to dissolve, diluted to 100ml, and well shaken.
[0035] Preparation of 1% DMSO solution: DMSO was diluted with pure water in a ratio of 1% (V/V) to form a 1% DMSO solution.
[0036] Preparation of PBS (pH 5.5) solution: 0.2mol/L disodium hydrogen phosphate solution and 0.3mol/L sodium dihydrogen phosphate solution was prepared firstly, and then mixed in a volume ratio of 49:51, of which the pH is adjusted to obtain a phosphate buffer having a pH of 5.5. 7
[0037] 2.3 Preparation of nanomembrane treatment solution
[0038] Membrane treatment solution 1: 4% EDC solution and 2% NHS solution were mixed in aratio of 1:1 (V/V), and well shaken.
[0039] Membrane treatment solution 2: 2g of EDC and lg of NHS were weighed and added into the PBS solution, stirred to dissolve, diluted to 100ml with the PBS solution, and well shaken.
[0040] Membrane treatment solution 3: 2g EDC and 1g NHS were weighed and added into a 1% v/v DMSO solution, stirred to dissolve, diluted to 100ml with 1% DMSO solution, and well shaken.
[0041] Membrane treatment solution 4: 4g EDC and 2g NHS were weighed and added into a 1% v/v DMSO solution, stirred to dissolve, diluted to 50ml, and then mixed with 50ml PBS solution in the same volume and well shaken for use. The solution was added with 100uL of
DCC and well shaken immediately before use.
[0042] 2.4 Comparison test
[0043] EDC is often used as an activating reagent for carboxyl groups in amide synthesis, and is also used for activation of phosphate groups, cross-linking of proteins and nucleic acids, and preparation of immunoconjugates. NHS is often used with EDC to increase the coupling probability. However, when EDC and NHS are used as the treatment solution for nanomembrane, the purpose is to activate the carboxyl groups on the surface of the nanomembrane to the greatest extent, which then can be quickly coupled with amino groups on the surface of protein when spotting. Therefore, EDC is often combined with NHS as the activating solution for carboxyl groups. But the coupling efficiency is low, and only 20%-50% 8 can be achieved.
[0044] In order to improve the coupling efficiency of the protein and the nanomembrane, making the protein more firmly immobilized on the surface of the nanomembrane and the chip have a better stability, we have developed several nanomembrane treatment solutions and screened out the optimal formula through a variety of protein chips. For ease of illustration, the present invention only takes protein chips prepared by mycotoxin antigen as an example for illustration.
[0045] The nanomembrane was placed in a glass container of appropriate size and placed flat.
The membrane treatment solutions were added to the container containing the nanomembrane to completely cover the surface of the nanomembrane. The nanomembrane was left to stand for 50 minutes, removed from the treatment solutions, rinsed repeatedly with purified water 10 times followed by blow drying and was ready for spotting.
[0046] The uniformity of the nanomembrane after activation was verified by experiments: spotting sample: Ing mycotoxin antigen solution (at a concentration of 0.05 ng/nl), spot matrix: 4x4 array (see Table 1), spotting volume: 20nl; measurement was conducted after color reaction with enzyme-labeled mycotoxin antibody and substrate. Measurement method: after spotting the sample, the chip was photographed by using an industrial camera hole by hole, and diameters of sample spots were measured and analyzed through the software; after reaction of the chip, the gray value was read, and variation range and coefficient of variation of the gray value after the reaction was calculated. The results are shown in Table 2, Figure 1. The uniformity of the nanomembrane after activation is comprehensively determined.
Table 1 Corresponding antigens in the spot matrix
Goat anti-mouse | spotting solution | spotting solution | spotting solution
EEE antigen 9
Mycotoxin Mycotoxin antigen spotting solution | spotting solution antigen (blank control) blank control)
Goat anti-mouse | spotting solution | spotting solution | Goat anti-mouse secondary (blank control) (blank control) secondary antibody antibody
Table 2 Summary of evaluation results of the uniformity of treatment membranes with different treatment solutions
Treatment | Observation with naked | Reaction result of | Spot diameter solution eyes (see drawings for | sample spots
No. details) Gray Coefficient | Diameter Coefficient value of range of | of variation sample variation spots (pm)
Membrane | The sample spots have treatment | serious overflow and the | 7688- 0; 424.8- 0 solution 1 | matrix is irregular (the | 14006 15.86% 708.6 16.83% first row in Figure 1)
Membrane | The sample spots have treatment | slight overflow and the 9828- o/ 489.1- 0 solution 2 | matrix is irregular (the 14616 13.08% 722.9 11.45% second row in Figure 1)
Membrane | The sample spots have treatment | clear edges with no solution 3 | overflow, and the matrix I5871- | g 0304 210.9- 1g 3304
Co . 19261 649.3 is still irregular (the third row in Figure 1)
Membrane | The sample spots have treatment clear edges with no 0400. _ 5219. solution 4 | overflow, and the matrix 3.87% 3.47% . 11845 574.8 is regular (the fourth row in Figure 1)
[0047] The first row of FIG. 1 shows the photos of spotting after the nanomembrane is pre- treated with the treatment solution 1, which is repeated 4 times. Therefore, four photos are obtained, each of which takes a 4x4 array of color development. By observing the first row of photos in Figure 1 with naked eyes, we can see that the sample spots have serious overflow, serious tailing and clustering of some sample spots, and the matrix is irregular. Taking the mycotoxin sample spots in the first photo of the first row as an example, spot diameters measured with an industrial camera are 480.2um, 666.4um, 571.2um, 708.64m, 414.8um, and 552.0um, of which the coefficient of variation is 19.51%; the gray values of the spots are 7688, 10
15006, 13651, 12253, 12049, and 12198, of which the coefficient of variation is 20.29%. Thus each coefficient of variation is relatively large, the uniformity is poor, and it is far from meeting production needs.
[0048] The second row of FIG. 1 shows the photos of spotting after the nanomembrane 1s pre- treated with the treatment solution 2, which 1s repeated 4 times. By observing the second row of photos in Figure 1 with naked eyes, we can see that there is a slightly overflow of the sample spots which is improved somewhat, the matrix is slightly improved but still irregular, there is still a tailing phenomenon of the sample spots and the reaction coloration is relatively serious.
Taking the mycotoxin sample spots in the first photo of the second row as an example, the spot diameters, which are measured with an industrial camera, are 630.9um, 532. 4um, 585.7um, 727.6um, 809.3um, 594.3um, of which the coefficient of variation is 15.88%; the gray values of the spots are 13171, 19261, 17869, 15871, 16486, 16370, of which the coefficient of variation 1s 12.41%. Although the result of preparation 1s slightly better than that of treatment solution 1, the uniformity is still poor, and it still cannot meet the production requirements.
[0049] The third row of Fig. 1 shows the photos of spotting after the nanomembrane is pre- treated with the treatment solution 3, which is repeated 4 times. By observing the third row of photos in Figure 1 with naked eyes, it can be seen that the overflow of the sample spots has completely disappeared, and the edges of the sample spots are clear while there is a tailing phenomenon for several sample spots, and the matrix is still slightly affected by static electricity and is irregular. Taking the mycotoxin sample spots in the first photo of the third row as an example, the spot diameters measured with an industrial camera are 567.7um, 545.9um, 568.3um, 592. 1um, 489. 1pm and 558.6um, of which the coefficient of variation is 6.33%; the gray values of the sample spots are 11002, 13041, 13860, 13023, 13188 and 11217, of which the coefficient of variation is 9.26%. Although the result of preparation is slightly better than that of treatment solution 1 and treatment solution 2, it still cannot meet the production requirements. tl
[0050] The fourth row of Fig. 1 shows the photos of spotting of the nanomembrane after the nanomembrane is pre-treated with the treatment solution 4, which is repeated 4 times. By observing the fourth row of photos in Figure 1 with naked eyes, it can be seen that the overflow of sample spot has disappeared completely, the edges of the sample spots are clear, and the matrix 1s regular. Measured with an industrial camera, taking the mycotoxin sample spots in the first photo as an example, the spot diameters are 498 9m, 521.9um, 526.8um, 528.8um, 527. 3pm and 548.5um, of which the coefficient of variation is 3.03%; the gray values of the sample spots are 10490, 11845, 11151, 11053, 11393 and 11322, of which the coefficient of variation is 3.98%. The result is better than the results of preparation with treatment solution 1, treatment solution 2, and treatment solution 3, and can meet production requirements.
[0051] In order to further explore the final volume ratio of DMSO contained in the treatment solution, with the other components unchanged, treatment solutions 5, 6, 7 and 8 were prepared in sequence with the content of DMSO set to 0.05%V/V, 0.25%V/V, 1%V/V and 1.5%V/V respectively. The result is as follows: when the content of DMSO is 0.05%V/V, the coefficient of variation of the diameters of the sample spots is 10.1%, and the coefficient of vanation of the gray value is 9.8%; when the content of DMSO is 1.5%V/V, the coefficient of variation of the diameters of the sample spots is 9.7%, and the coefficient of variation of the gray value is 9.5%; when the content of DMSO is 0.25%V/V or 1%V/V, the coefficient of variation of the diameters of the sample spots as well as the coefficient of variation of the gray value are both equal to or less than 3.90. Therefore, the considered content of DMSO in the membrane treatment solution is 0.25% V/V-1%V/V.
[0052] The above results show that the membrane treatment solution containing 0.25%V/V- 1%V/V DMSO can completely infiltrate the surface of the nanomembrane, reduce the surface tension of water, and increase the coupling efficiency of the carboxyl groups on the surface of nanomembrane with EDC; in the research process, it was also found that DMSO can also significantly improve the interference of static electricity on the surface of the nanomembrane, so that it can prevent the spot matrix from being irregular due to static electricity, DCC can 12 significantly improve the tailing phenomenon of sample spots.
[0053] In order to facilitate subsequent research, the treatment solution 4 is used as the pretreatment solution of the nanomembrane, and spotting 1s performed after the treatment.
[0054] 2.5 Porcine pseudorabies virus gD/gE protein chip
[0055] The porcine pseudorabies virus gD protein was spotted after treating the nanomembrane with the treatment solution 4. Observed by naked eyes, there was no overflow of the sample spots and the edges of the spots were clear, and the matrix was regular. The measured spot diameters of the PRV gD sample spots were 536.5um, 522.8um, 549.4um, 568.6um, 549. Oum, and 576.4um, of which the coefficient of variation is 3.60%; the gray values measured after the reaction are 23096, 21608, 22481, 23059, 23469 and 23292, of which the coefficient of variation is 3.01%; observed by naked eyes, the sample spots have clear edges with no overflow and the matrix is regular.
[0056] The porcine pseudorabies virus gE protein was spotted after treating the nanomembrane with the treatment solution 4. Observed by naked eyes, there was no overflow of the sample spots and the edges of the spots were clear, and the matrix was regular. The measured spot diameters of the PRV gE sample spots were 526.4um, 538.5m, 555.5um, 537. 7um, 576.5um and 554.5um, of which the coefficient of variation is 3.24%; the gray values measured after the reaction are 24593, 22664, 22155, 23541, 23957 and 23272, of which the coefficient of variation is 3.76%; observed by naked eyes, the sample spots have clear edges with no overflow and the matrix is regular.
[0057] 2.6 Avian antibiotic antigen
[0058] The avian antibiotic antigen was spotted after treating the nanomembrane with the 13 treatment solution 4. Observed by naked eyes, there was no overflow of the sample spots and the edges of the spots were clear, and the matrix was regular. The measured spot diameters of the avian antibiotic antigen sample spots were 563.3 um, 529.3 um , 568.6um, 559.5um, 576.4um and 528.5um, of which the coefficient of variation is 3.69%; the gray values measured after the reaction are 16096, 14608, 15481, 16059, 16466, and 15856, of which the coefficient of variation is 4.12%; observed by naked eyes, the sample spots have clear edges with no overflow and the matrix is regular.
[0059] 2.7 Rotavirus antibody
[0060] The rotavirus antibody was spotted after treating the nanomembrane with the treatment solution 4. Observed by naked eyes, there was no overflow of the sample spots and the edges of the spots were clear, and the matrix was regular. The measured spot diameters of the rotavirus antibody sample spots were 542.1um, 537.3um, 534.7um, 553.5um, 563.9um and 585.7um, of which the coefficient of variation is 3.52%; the gray values measured after the reaction are 24152, 22664, 23537, 24115, 24525 and 25346, of which the coefficient of variation is 3.77%; observed by naked eyes, the sample spots have clear edges with no overflow and the matrix is regular.
[0061] For protein chips of mycotoxin antigen, porcine pseudorabies virus gD/gE protein, avian antibiotic antigen, and rotavirus antibody, the coefficients of variation of the spot diameters and gray values of sample spots between different batches are less than 5%, indicating that the protein chips after treatment with the treatment solution 4 has good uniformity and can be mass- produced.
Example 3 Preparation and inspection of a protein chip plate
[0062] 3.1 Preparation of a spotting solution 14
[0063] Preparation of 5% glycerol solution: 5.00g glycerol was accurately weighed and placed in a 100ml volumetric flask, added with a small amount of purified water and gently rotated to dissolve fully, avoiding too many bubbles, then added with purified water to the scale mark, and shaken by turning upside down 10 times for use;
[0064] Preparation of 5% sorbitol solution: 5.00g sorbitol was accurately weighed and placed in a 250ml beaker, added with an appropriate amount of purified water and stirred to completely dissolve, and then completely transferred to a 100ml volumetric flask, added with purified water to the scale mark, and shaken by turning upside down 10 times for use;
[0065] Preparation of 0.05% Triton solution: 50ul of Triton was measured by using a pipette and placed in a 100ml volumetric flask, added with an appropriate amount of purified water to completely dissolve, then added with purified water to the scale mark, and shaken by turning upside down 10 times for use;
[0066] DMSO solution: DMSO reagent can be used directly;
[0067] Preparation of PBS (pH 6.8) solution: 0.2mol/L disodium hydrogen phosphate solution and 0.3mol/L sodium dihydrogen phosphate solution was prepared firstly, and then mixed in a volume ratio of 49:51, of which the pH is adjusted to obtain a phosphate buffer having a pH of 6.8;
[0068] The above solutions were mixed uniformly in a volume ratio of 10:15:0.1:50:100 as a spotting solution.
[0069] 3.2 Preparation of a protein chip plate
[0070] The preparation method of the protein chip plate includes:
Step 1) preparing a membrane treatment solution, immersing a nanomembrane in the membrane 15 treatment solution for 50 minutes, and then rinsing the nanomembrane with purified water followed by blow drying;
Step 2) preparing a spotting solution, diluting a protein with the spotting solution and spotting the diluted protein on the nanomembrane to obtain a protein chip;
Step 3) drying the protein chip at a temperature of 23°C + 2°C and a humidity of 25% or less for a different period; and
Step 4) preparing a blocking solution and blocking the protein chip with the blocking solution to obtain a protein chip plate.
Example 4 Drying conditions of the protein chip
[0071] Drying conditions 1 to 3 for the protein chip were set as follows:
Condition 1: drying for 4 hours a temperature of 23°C + 2°C and a humidity of 25% or less;
Condition 2: drying for 6 hours at a temperature of 23°C+2°C and a humidity of 25% or less;
Condition 3: drying for 16 hours at a temperature of 23°C + 2°C and a humidity of 25% or less.
[0072] In the present invention, multiple products were prepared under conditions 1 to 3, and the results were the same. For ease of explanation, taking the porcine pseudorabies virus (PRV) gDgE protein dual detection chip (see Table 3 for the spot matrix, 2“3 matrix) as an example, the following samples were evaluated and analyzed.
Table 3 Antigens corresponding to porcine pseudorabies virus gD/gE protein spot matrix antibody control) gD protein gE protein antibody antibody
[0073] PRV gD-positive and PRV gE-negative swine serum (referred to as Pl): the 16 neutralization titer determined by the porcine pseudorabies virus neutralization test was 1:51.3; the test result of the Biochek PRV gB kit was positive, with an S/P value of 2.393; the test result of the IDEXX PRV gE kit is negative, with an S/N value of 0.989.
[0074] PRV gE-positive and PRV gD-positive swine serum (referred to as P2): the neutralization titer determined by the porcine pseudorabies virus neutralization test was 1:11.2; the test result of the Biochek PRV gB kit was positive, with an S/P value of 1.152; the test result of the IDEXX PRV sE kit is negative, with an S/N value of 0.454.
[0075] PRV gD-negative and PRV gE-negative swine serum (referred to as N): the neutralization titer determined by the porcine pseudorabies virus neutralization test is less than 1:2; the test result of the Biochek PRV gB kit was negative, with an S/P value of 0.191; the test result of the IDEXX PRV gE kit is negative, with an S/N value of 0.958.
Table 4 Summary of reaction results under different drying conditions
Drying PRV ¢D antibod PRV gE antibod Observed | Accelerate condition Naverage/P 1 average CV Naverage/P2average CV by naked d stability s (n=10 (n=10 | eves after | test reaction ) )
Condition 3.24 13.50% 4.37 12.73% | The color | Poor, only 1 is light and | 2 days nonunifor m
Condition 3.47 3.46% 5.72 3.69% The color | Better, 4 2 is days moderate and uniform
Condition 2.86 4.48% 3.71 439% | Thecolor | __ 3 is dark and | Slightly uniform better, 4 days
[0076] The above-mentioned serums were used for the porcine pseudorabies virus gD and gE 17 protein dual detection antibodies to evaluate different drying conditions. Because the principle of this method is ELISA blocking test, the N/P values were calculated and the drying condition corresponding to the maximum N/P value was selected as the best condition. The reaction results are shown in Table 4 and Figure 2. The result analysis is as follows: the repeatability of the reaction results after drying under condition 1 is relatively poor (10% <<CV value < 15%), and observed by naked eyes, there is a tailing phenomenon, the spots are irregular and the positive sample spots are darker; for accelerated stability testing, the protein chip can only be stored at 37°C for 2 days. Although the repeatability of the reaction results after drying under condition 3 is relatively good (CV value <5 %), observed by naked eyes, there is a tailing phenomenon, the color of the positive sample spots is dark, and the high gray value of Pl and
P2 leads to low N/P 1 and N/P2 under the same condition, which 15 not favorable to the accurate
Judgment of negative and positive; the result of the accelerated stability test is slightly improved compared with condition 1. In contrast, the reaction results after drying under condition 2 have good repeatability (CV value <4%), observed by naked eyes, the reaction is moderate and the N/PI and N/P2 values are both the highest, which makes it easier to judge negative and positive, and the result of the stability test is better.
Example 5 Preparation and evaluation of the membrane blocking solution
[0077] 5.1 Preparation of the membrane blocking solution
[0078] On the basis of the previous optimization, in order to further study the stability, the blocking solutions 1 to 6 were prepared according to the following table.
Table 5 Formulations of different membrane blocking solutions
Tween-20 18
0.1%V/V Tween-20 0.1%V/V Tween-20 0.1%V/V Tween-20
[0079] 5.2 Evaluation of blocking effects
[0080] In the present invention, multiple products were prepared by using membrane blocking solutions 1 to 6, and the results are the same. For ease of explanation, taking the porcine pseudorabies virus gDgE protein dual detection chip (2x3 matrix) as an example, samples N,
P1, and P2 in Example 4 were evaluated and analyzed.
[0081] The reaction results after the treatment of membrane blocking solutions 1 to 6 are shown in Table 6 and Figure 3. The analysis of the results are as follows: after the reaction of the blocking solution 1, observed by naked eyes, the sample spots are irregular and there is a tailing phenomenon, and each coefficient of variation is large; the result of the accelerated stability test is poor (the protein chip can only be stored for 2 days). After the reaction of blocking solution 2, observed by naked eyes, the sample spots are irregular and there is a tailing phenomenon, and each coefficient of variation is large; the result of the accelerated stability test is slightly better (the protein chip can be stored for 4 days). After the reaction of blocking solution 3, observed by naked eyes, the sample spots are irregular and there is a tailing phenomenon, and each coefficient of variation is slightly large; the result of the accelerated stability test 1s slightly better (the protein chip can be stored for 4 days). After the reaction of the blocking solution 4, observed by naked eyes, the sample spot has a halo phenomenon, and the color of the positive sample spots after reaction is darker and the high gray value leads to false negatives, and each coefficient of variation is slightly large; the result of accelerated stability test 1s poor (only can be stored for 2 days). After the reaction of blocking solution 5, the color of positive samples is darker and the high gray value leads to false negatives, and each coefficient of variation is slightly large; the result of the accelerated stability test is not good 19 enough (can be stored for 4 days). In contrast, observed by naked eyes after the reaction of the blocking solution 6, the color of the sample spots is moderate, there is no tailing or halo phenomenon; the protein chip can be stored for 6 days in the accelerated stability test.
Table 6 Summary of reaction results after treatment with the different membrane blocking solutions
PRV gD antibod PRV gE antibod Observed by | Accelerated
Blocking | Naverage/P laverage CV Naverage/P2average CV naked eyes stability solutions (n=10) (n=10) after reaction test
The sample | Poor, only
Blocking rea and 2 days solution 2.59 15.50% 4.21 9.47% = 1 there is a tailing phenomenon
The sample /
Block spots are Slightly oc ing 287 13 26% 586 2 TO irregular and better 4
SO ution : 3.26% 5. 79% there is a 2 - tailing days phenomenon
The sample / lock spots are Slightly
B oc ing ss 14 18% 399 _— irregular and better 4
SO ution - 18% 3.9¢ 32% there is a , tailing days phenomenon
The sample | Poor, only a 2 a
Blocking have 2 halo 2 doe solution 2.34 6.35% 3.52 5.29% 4 phenomenon, and the color is darker
Slightly
Blocking The color is solution 2,68 4.39% 3.58 4.01% dark better, 4 5 days . The color is | Better, 6
Blocking moderate days solution 3.45 3.36% 5.70 3.45% Tae, y 6 there is no tailing or ob eee] phenomenon
[0082] In order to further study the content of glycerol in the membrane blocking solution, with the other components unchanged, blocking solution 9, blocking solution 7, blocking solution 8, and blocking solution 10 were prepared in sequence with the content of glycerol set to 0.05%V/V, 0.1%V/V (blocking solution 7), 0.5%V/V (blocking Solution 8), 1%V/V respectively. The result is as follows: when the glycerol contents are 0.05%V/V and 1%V/V, the coefficients of variation are both no less than 15%, the sample spots are irregular and there is a tailing phenomenon after the reaction; for the accelerated stability test, the protein chip can only be stored for 4 days. Only when the glycerol contents in the blocking solution are 0.1% V/V and 0.5%V/V, the sample spots are uniform and there is no tailing or halo phenomenon, and the coefficients of variation are both less than 5%, and for the accelerated stability test, the protein chip can be stored for 6 days. Therefore, the considered content of glycerol in the membrane blocking solution is 0.1% V/V~0.5%V/V.
[0083] The antigen plates prepared with the blocking solution 6, the blocking solution 7, and the blocking solution 8 were subjected to a real-time storage period test at 2 to 8°C. The result is as follows: no significant change was detected when stored for 15 months, which can meet the production requirements.
[0084] In summary, the membrane treatment solution, drying conditions, and membrane blocking solution are used in combination in the preparation process of the protein chip plate of the present invention, so that the sample spots have clear edges without any overflow, tailing, or halo phenomena, the sample spots are regular and the coefficient of variation of the sample spots’ diameters is small, the matrix is regular and the color of different proteins after the reaction is moderate, the gray value is moderate and the coefficient of variation of the gray value is small. The method provides a better repeatability, and a longer storage period, and can meet the demands of production. 21
[0085] The foregoing descriptions are merely preferred examples of the present disclosure and are not intended to limit the present disclosure in any form.
Although the present disclosure has been disclosed by way of preferred examples, it is to be understood that the disclosure is not limited thereto.
A person skilled in the art can make some equivalent variations or modifications to the above-disclosed technical content without departing from the scope of the technical solutions of the present disclosure to obtain equivalent examples.
Simple modifications, equivalent changes and modifications made to the above examples according to the technical essence of the present disclosure all fall within the scope of the technical solutions of the present disclosure without departing from the contents of the technical solutions of the present disclosure. 22
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