CN112730824B - Sealing stabilizer for mechanized preparation of pre-coated plate - Google Patents
Sealing stabilizer for mechanized preparation of pre-coated plate Download PDFInfo
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- CN112730824B CN112730824B CN202011387068.1A CN202011387068A CN112730824B CN 112730824 B CN112730824 B CN 112730824B CN 202011387068 A CN202011387068 A CN 202011387068A CN 112730824 B CN112730824 B CN 112730824B
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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Abstract
The invention belongs to the technical field of biology, and discloses a sealing stabilizer for a pre-coated plate prepared mechanically. On the premise of maintaining the mechanical efficient washing and sealing efficiency, the pre-coated plate sealing stabilizer can effectively solve the problem of uniformity of the pre-coated plate caused by liquid injection impact force. The pre-coated plate sealing stabilizer has a good sealing effect on an unbound antibody region on an ELISA plate, and can effectively stabilize the activity of a coated antibody on the ELISA plate. The invention is applied to enzyme-linked immunoassay detection, ensures the accuracy and the detection sensitivity of the detection result and can effectively improve the validity period of the kit.
Description
Technical Field
The invention relates to the field of biotechnology, and more particularly relates to a sealing stabilizer for mechanized preparation of a pre-coated plate.
Background
Immunoassay detection techniques have been widely used in clinical testing, scientific research, environmental and health testing, and other fields. The immunodetection technology comprises enzyme-linked immunoassay, chemiluminescence immunoassay, time-resolved immunofluorescence assay and the like.
The solid phase carrier used in these detection techniques is mostly an enzyme label plate. Coating the antigen or the antibody on the plate hole surface of the enzyme label plate through the physical adsorption effect or the chemical bonding effect of the antigen or the antibody and the surface group of the enzyme label plate to prepare the pre-coated plate. The pre-coated plate is prepared by two general processes, namely manual coating, washing and sealing and mechanical coating, washing and sealing. When the manual coating is washed and sealed, in the operations of liquid adding, plate beating, spin-drying and the like, the quality of the pre-coated plate is greatly influenced by the artificial factors such as a liquid adding mode, the soaking time, the plate beating force and the like, and the manual plate making operation is complicated and consumes a large amount of manpower and material resources, so that the manual coating is not suitable for the large-batch preparation of the pre-coated plate. The mechanical coating and washing-sealing mode changes the key operation into the completion of a coating and washing-sealing integrated machine, the mechanical operation condition is constant, the artificial introduction factor is avoided, the working efficiency is greatly improved, but because the antigen or the antibody coated on the pre-coated plate is combined with the solid phase carrier in a non-specific adsorption mode mostly, the physical adsorption of the antigen or the antibody and the surface groups of the solid phase carrier is weaker, the fluid shearing force and the unbalanced liquid injection impact force in the mechanical washing-sealing process can damage the physical adsorption and even the activity of the antigen or the antibody, and the pre-coated plate is caused to generate larger inter-hole difference and even intra-batch and inter-batch difference. In addition, the structure of the antigen or antibody may be changed, even partially denatured and inactivated during transportation and long-term storage of the kit, resulting in gradual loss of the activity of the pre-coated plate. The pre-coated plate is a key component of the enzyme-linked immunoassay kit, and the intra-plate and inter-plate precision and the activity stability of the pre-coated plate are key indexes for evaluating the quality of the pre-coated plate. The quality of the pre-coated plate has important influence on the accuracy and detection sensitivity of the detection result. Therefore, the mechanized preparation of the pre-coated plate with excellent precision and stability becomes a difficult problem to be solved urgently in the research and development process of the enzyme linked immunosorbent assay kit.
In order to mainly solve the problems of the precision in the pre-coated plate in batch and between batches and the activity stability of the pre-coated plate introduced in the mechanized preparation process, the invention researches and analyzes the conditions of a closed stabilizer used in the mechanized preparation process of the pre-coated plate.
Disclosure of Invention
In order to overcome the above-mentioned drawbacks of the prior art, the primary object of the present invention is to provide a blocking stabilizer for the mechanized preparation of pre-coated plates.
The purpose of the invention is realized by the following technical scheme:
the sealing stabilizer for the mechanized preparation of the pre-coated plate is characterized by comprising the following substances in parts by mass:
the balance is phosphate buffer.
Preferably, the thickener is one or more of gelatin, organic macromolecular polymer PEG1500, PEG2000, PEG4000, PEG20000, PVP K30 and PVP K40.
Preferably, the stabilizer is one or more of sucrose, trehalose and glycerol.
Preferably, the blocking agent is one or more of bovine serum albumin, donkey serum albumin, gelatin and casein.
Preferably, the biological preservative is ProClin 300.
As a specific embodiment, the blocking stabilizer for the mechanized preparation of the pre-coated plate comprises the following substances in percentage by mass:
the rest is phosphate buffer solution.
Bovine serum albumin can better block the unbound antibody region on the surface of the solid phase carrier; PEG20000 is a polyhydroxy high molecular polymer, gelatin is a complex protein macromolecule composed of multiple amino acids, and the two are used together to effectively adjust the solution viscosity and effectively weaken the injection impact force in the mechanized washing and sealing operation, thereby eliminating the difference between the inner batch and the inter-batch of the pre-coated plate introduced by the mechanized washing and sealing operation.
The invention also provides application of the sealing stabilizer in improving the quality stability of the pre-coated board.
Compared with the prior art, the invention has the following beneficial effects:
because the mechanized preparation pre-coated plate has high working efficiency, the quality of the pre-coated plate is prevented from being influenced by manual operation, so that the coating, washing and sealing integrated machine is widely applied, but in the efficient mechanized washing and sealing operation, under the working mode of unbalanced liquid injection impact force, system deviation is introduced rigidly, and the precision of the pre-coated plate is poor. In addition, since the antibody itself is unstable in properties, the antibody coated on the solid phase carrier by physical adsorption is easily inactivated depending on the storage time and storage conditions, resulting in poor stability of the pre-coated plate. The quality problem of the pre-coated plate directly causes the quality problems of the kit comprising the pre-coated plate, such as short storage period, poor detection accuracy, low sensitivity and the like. On the premise of maintaining the mechanical efficient washing and sealing efficiency, the pre-coated plate sealing stabilizer can effectively solve the problem of uniformity of the pre-coated plate caused by liquid injection impact force. The pre-coated plate sealing stabilizer has a good sealing effect on the area of the ELISA plate not combined with the antibody, and can effectively stabilize the activity of the coated antibody on the ELISA plate. The invention is applied to enzyme-linked immunoassay detection, ensures the accuracy and the detection sensitivity of a detection result, and can effectively improve the validity period of the kit.
Drawings
FIG. 1 is a comparison of in-batch differences for pre-coated plates obtained using modified and conventional blocking stabilizers in a mechanized manufacturing process;
FIG. 2 is a comparison of the batch-to-batch differences of pre-coated plates obtained using modified and ordinary blocking stabilizers in a mechanized manufacturing process.
Detailed Description
The following further describes embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Experimental example 1
1. In-batch difference comparison
Two performance indices of pre-coated plates were evaluated using modified blocking stabilizers (2% bovine serum albumin, 4% gelatin, 0.5% PEG20000, 2% sucrose, 0.1% Proclin300, phosphate buffer) and general blocking stabilizers (2% BSA,2% sucrose, 0.1% Proclin300, phosphate buffer) during mechanized preparation. The two pre-coated plates are randomly sampled and tested for uniformity in the plates and batches, wherein the two pre-coated plates are prepared in a mechanized batch by 200 blocks respectively. The detection result shows that the precision difference in batches of the pre-coated plate treated by the improved sealing stabilizer in the mechanized preparation process is less than 5%, and the precision difference in batches of the pre-coated plate treated by the common sealing stabilizer in the mechanized preparation process is close to 10%, which indicates that the pre-coated plate treated by the improved sealing stabilizer in the mechanized batch production has excellent uniformity in plates and batches. The results are shown in FIG. 1.
2. Comparison of inter-batch differences
The performance indexes of two pre-coated plates obtained by using the improved sealing stabilizer and the common sealing stabilizer in the mechanized preparation process are evaluated. And (3) mechanically preparing 200 pieces of each of the two pre-coated plates in batch, randomly sampling the two pre-coated plates, and detecting the precision difference between the two pre-coated plates in batch. The detection results show that the precision difference between batches of the pre-coated plate using the improved blocking stabilizer in the mechanized preparation process is 1.95%, and the precision difference between batches of the pre-coated plate using the improved blocking stabilizer in the mechanized preparation process is 9.12%. Indicating that pre-coated plates treated with the improved post-blocking stabilizer in mechanized batch production have excellent batch-to-batch precision. The results are shown in FIG. 2.
3. Performance index
The performance index of pre-coated plates treated with the improved post-blocking stabilizer in mechanized mass production was evaluated. 200 pieces of pre-coated plate were prepared in batches mechanically, and the batches were randomly sampled to check for within-batch and between-batch uniformity. The detection result shows that the precision difference between the pre-coated plate in batch and between the pre-coated plate in batch is less than 5 percent, which indicates that the pre-coated plate treated by the improved sealing stabilizer in mechanized batch production has excellent uniformity between batches. Through an accelerated experiment, the stability of the mechanically prepared pre-coated plate is evaluated, namely the pre-coated plate is accelerated and aged for 4, 7 and 10 days in a constant temperature incubator at 37 ℃, and the quality change of the pre-coated plate before and after acceleration is compared. The detection result shows that after the acceleration for 7 days, the activity of the pre-coated plate is almost not lost, the standard curve R2 is more than 0.99, and the blank value is less than 0.05. Indicating that the pre-coated plate treated with the improved post-blocking stabilizer in the mechanized batch production has excellent stability. The results are shown in tables 1, 2 and 3.
TABLE 1 blank values of different batches of pre-coated plates as a function of accelerated aging time
TABLE 2 calibration curve of different batches of pre-coated plates as a function of accelerated aging time
TABLE 3 Activity of different batches of Pre-coated plates as a function of accelerated aging time
Claims (2)
1. The application of the sealing stabilizer in improving the precision of an in-batch and in-batch process of a pre-coated plate prepared by a coating, washing and sealing integrated machine in a mechanized manner so as to solve the problem of uniformity of the pre-coated plate caused by liquid injection impact force of the coating, washing and sealing integrated machine is characterized in that the sealing stabilizer is composed of the following substances in percentage by mass:
2% of bovine serum albumin;
2% of sucrose;
ProClin 300 0.1%；
4% of gelatin;
PEG20000 0.5%；
the rest is phosphate buffer solution.
2. The use according to claim 1, wherein the blocking stabilizer is also used to improve the quality stability of the pre-coated board.
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| CN113267621B (en) * | 2021-05-14 | 2021-12-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
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| CN102455360A (en) * | 2010-10-20 | 2012-05-16 | 新疆农业大学 | Neosporosis recombinant enzyme-linked immunosorbent assay (rELISA) antibody assay kit |
| CN102331494A (en) * | 2011-06-16 | 2012-01-25 | 广州艺佳生物科技有限公司 | Sealing and stabilizing agent for microporous board |
| CN102565410A (en) * | 2012-01-12 | 2012-07-11 | 吉林大学 | Immunologic detection method for sika deer prion protein |
| WO2016052690A1 (en) * | 2014-10-02 | 2016-04-07 | コニカミノルタ株式会社 | Blocking method for immunoassay, and immunoassay instrument |
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Denomination of invention: Sealing stabilizer for mechanized preparation of pre coated plates Effective date of registration: 20230505 Granted publication date: 20221115 Pledgee: Bank of China Limited Guangzhou Development Zone Branch Pledgor: GUANGZHOU DEAOU BIOTECHNOLOGY Co.,Ltd. Registration number: Y2023980039825 |