CN112748242A - Secondary antibody buffer confining liquid for polypeptide chip technology platform detection, kit comprising same and application thereof - Google Patents
Secondary antibody buffer confining liquid for polypeptide chip technology platform detection, kit comprising same and application thereof Download PDFInfo
- Publication number
- CN112748242A CN112748242A CN202011522214.7A CN202011522214A CN112748242A CN 112748242 A CN112748242 A CN 112748242A CN 202011522214 A CN202011522214 A CN 202011522214A CN 112748242 A CN112748242 A CN 112748242A
- Authority
- CN
- China
- Prior art keywords
- secondary antibody
- percent
- products
- chip technology
- technology platform
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
- G01N33/53—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
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a secondary antibody buffer confining liquid for polypeptide chip technology platform detection, a kit comprising the secondary antibody buffer confining liquid and application of the secondary antibody buffer confining liquid. Wherein, the secondary antibody buffer sealing liquid comprises the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 1 percent D-mannitol w/v and 0.1 to 1 percent sodium caseinate w/v. The buffer solution can provide a stable solution environment under the condition of incubation of the secondary antibody, reduces non-specific adsorption of the secondary antibody under the condition of not influencing normal combination of polypeptide-antibody, avoids interference on experimental results, and provides real, effective and stable data for subsequent fluorescence imaging.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a secondary antibody buffer confining liquid for polypeptide chip technology platform detection, a kit comprising the same and application thereof.
Background
The polypeptide chip is a chip based on a substrate material, the chip comprises the characteristics of pre-designed quantity, positions and sequences, one characteristic is a cluster of polypeptides with the same sequence, the polypeptide sequences between the characteristics are different frequently, and the characteristics form a high-density polypeptide array.
The polypeptide chip technology is a detection technology based on a polypeptide chip, and is characterized in that a variety of polypeptides on the polypeptide chip are contacted with a sample, then, an image acquisition technology is used for acquiring each characteristic signal (specifically, a fluorescence image carrying each characteristic signal) on the polypeptide chip, and further, the signal intensity of each characteristic in the polypeptide chip is output, namely, the detection result data of the polypeptide chip. Specifically, in the polypeptide chip technology, after a sample is contacted with a polypeptide chip, components which cannot be bound or have weak binding force with the polypeptide chip in the sample are washed away, and then an antibody (secondary antibody) carrying a fluorescent label is labeled on a specific target antigen (a specific object to be observed or detected in the sample, such as IG G, IG M, and the like), so that a fluorescent signal on the polypeptide chip is obtained by an image acquisition technology. Therefore, the polypeptide chip technology can identify the antibody which is expressed differently between different individuals or the antibody which is expressed differently by the same individual at different time points, and further carry out related research according to the information.
In a specific experiment, in order to ensure that the fluorescence-labeled antibody has higher bioactivity, obtain a better imaging result, reduce non-specific binding of the fluorescence-labeled antibody, reduce non-specific background coloring, and improve the specificity and sensitivity of the experiment, the fluorescence-labeled antibody is mostly required to be properly diluted. The diluted fluorescently labeled antibody should generally be preserved and used at 4 ℃ for no less than 6 months and be reusable. In immunological experiments such as ELISA, high background value often appears, and detection of experimental results is affected. This is because, when the relevant immunological reagent is used, the protein is bound to the non-adsorbed solid carrier, and non-specific binding occurs. Such solid support sites are often blocked in practice by blocking reagents to reduce or eliminate the effects of non-specific binding background. The blocking reagent generally contains BSA, animal serum, and Fab fragment single-chain secondary antibody as active ingredients. The BSA component is relatively single and possibly has bovine IgG, so that the BSA component has strong cross reaction with anti-bovine, goat, sheep, horse and other secondary antibodies, and can cause a certain background; some blocking serums may contain sodium azide, so that the blocking serums are not suitable for an HRP enzyme labeling detection system; the Fab fragment single-chain secondary antibody is complex to prepare, high in price and not suitable for being used in large quantities, so that the Fab fragment single-chain secondary antibody is not an ideal choice for the components of a general blocking reagent (blocking liquid) of a polypeptide chip platform. In addition, because high-density polypeptide sequences are arranged on the polypeptide chip, the confining liquid of the polypeptide sequences puts forward higher requirements, and the problems of high cost and unstable confining effect exist in the experiment process of the existing confining liquid for carrying out the polypeptide chip of the immunity surface acupuncture technology.
Disclosure of Invention
The invention aims to provide a secondary antibody buffer confining liquid for polypeptide chip technology platform detection, a kit comprising the secondary antibody buffer confining liquid and application of the secondary antibody buffer confining liquid, so as to provide a general confining liquid suitable for polypeptide chip technology platform detection.
In order to achieve the above objects, according to one aspect of the present invention, there is provided a secondary antibody buffer blocking solution for use in a polypeptide chip technology platform. The secondary antibody buffer sealing liquid comprises the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 1 percent D-mannitol w/v and 0.1 to 1 percent sodium caseinate w/v.
Further, the pH value of the secondary antibody buffer sealing liquid is 7.2-7.6.
Further, the pH value of the secondary antibody buffer sealing liquid is 7.38-7.42.
According to another aspect of the present invention, a kit for polypeptide chip technology platform detection is provided. The kit comprises any secondary antibody buffer sealing solution used for the polypeptide chip technology platform.
Further, the kit also comprises a cleaning solution, wherein the cleaning solution comprises the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v and pH 7.2 to 7.6.
Further, the kit also comprises a sample diluent, wherein the sample diluent comprises the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 1 percent D-mannitol w/v, and the pH value is 7.2 to 7.6.
Further, the kit also comprises a secondary antibody diluent, and the buffer comprises the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 0.75 percent sodium caseinate w/v, and the pH value is 7.2 to 7.6.
According to another aspect of the invention, the invention provides an application of the secondary antibody buffer sealing solution for the polypeptide chip technology platform in the field of preparation of polypeptide chip technology platform detection products or biological medicines.
Further, the polypeptide chip technology platform detection products include, but are not limited to, sample classification identification products, vaccine effect evaluation products, biomarker detection products, disease diagnosis products, and health condition assessment products; the biomedical field includes, but is not limited to, drug screening, drug design, vaccine development, vaccine design, and biomarker screening.
According to another aspect of the invention, the application of the kit for polypeptide chip technology platform detection in the field of preparation of polypeptide chip technology platform detection products or biomedicine is provided.
Further, the polypeptide chip technology platform detection products include, but are not limited to, sample classification identification products, vaccine effect evaluation products, biomarker detection products, disease diagnosis products, and health condition assessment products; the biomedical field includes, but is not limited to, drug screening, drug design, vaccine development, vaccine design, and biomarker screening.
The buffer solution can provide a stable solution environment under the condition of incubation of the secondary antibody, reduces non-specific adsorption of the secondary antibody under the condition of not influencing normal combination of polypeptide-antibody, avoids interference on experimental results, and provides real, effective and stable data for subsequent fluorescence imaging; and the method has low cost and stable performance, and has the effect of improving specificity and sensitivity in immunology, particularly in detection and scientific research by using a polypeptide array chip.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows the blocking effect of blocking solutions containing different concentrations of Casein (sodium caseinate) or BSA (bovine serum albumin) in example 1;
FIG. 2 shows the effect of different concentrations of Casein (sodium caseinate) containing blocking solutions on the P53-specific signal in example 1;
FIG. 3 shows the different blocking effects of different concentrations of Casein (sodium caseinate) in the blocking solution of example 1 on specific and non-specific peptides in the P53 test experiment; and
FIG. 4 shows the blocking effect of different concentrations of Casein (sodium caseinate) containing blocking solutions on non-specific background peptide signals from serum in example 1.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
According to an exemplary embodiment of the present invention, a secondary antibody buffer blocking solution for a polypeptide chip technology platform is provided, which comprises the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 1 percent D-mannitol w/v and 0.1 to 1 percent sodium caseinate w/v. Among them, sodium caseinate is also known as casein.
The sodium chloride, the potassium chloride, the disodium hydrogen phosphate and the potassium dihydrogen phosphate are used as salt ions in a buffer solution, the pH value of the solution environment can be maintained within the range of 7.2-7.6, preferably 7.38-7.42, and the pH value range is particularly suitable for the performance exertion of a polypeptide array on a polypeptide chip platform; tween-20 as a non-ionic type has the function of a renaturation antigen, and can improve the recognition capability of specificity; proclin950 is a broad-spectrum antibacterial preservative which can inhibit the growth of microorganisms such as bacteria and fungi in a long time under a certain concentration and rarely generate drug resistance, and can be used for antibacterial preservation of primary antibody, secondary antibody, confining liquid, antibody diluent and the like; the casein/sodium caseinate is phosphoprotein in animal milk, and has a structural formula of NH2RCOOH, odorless and tasteless white to yellow powder, and has a relative density of 1.25-1.31 (water is 1); the sodium caseinate is almost insoluble in water, alcohol and ether, is dissolved in dilute alkali liquor, alkaline carbonate solution and concentrated acid, is precipitated in weak acid, has hygroscopicity, is stable when being dried, and is quickly hardened when being wet; casein/sodium caseinate can reduce nonspecific adsorption. The pH of the secondary antibody buffer blocking solution is adjusted by 1N sodium hydroxide or 1N hydrochloric acid.
The buffer solution can provide a stable solution environment under the condition of incubation of the secondary antibody, and can reduce nonspecific adsorption of the secondary antibody, particularly nonspecific adsorption of the secondary antibody and solid-phase components outside a polypeptide array on a polypeptide chip under the condition of not influencing normal polypeptide-antibody combination, thereby avoiding interference on experimental results and providing real, effective and stable data for subsequent fluorescence imaging; and the method has low cost and stable performance, and has the effect of improving specificity and sensitivity in immunology, particularly in detection and scientific research by using a polypeptide array chip.
Specifically, in a polypeptide array chip polypeptide screening test of human serum, a P53 specific antibody is doped into the serum, and a polypeptide signal of P53 is used as a specific signal reference. Then, through experiments, the detection results of the serum sample and the serum sample containing P53 are compared, and the detection results of the two samples under the condition of the confining liquid and the condition of the non-confining liquid are compared, so that the confining liquid disclosed by the application has no interference on the specific signal of the P53 antibody, but a large amount of non-specific signals in the serum are obviously reduced. Multiple experiments prove that the result can be repeated, which shows that the effect of the confining liquid is stable.
Specifically, in a preferred embodiment of the present application, the secondary antibody buffer blocking solution is prepared according to the following formula: 135mM sodium chloride, 2.5mM potassium chloride, 3.8mM disodium hydrogen phosphate, 1.2mM potassium dihydrogen phosphate, 0.05% Tween-20v/v, 0.05% Proclin950v/v, 0.1% -1% casein/sodium caseinate w/v, pH adjusted to 7.4 with 1N hydrochloric acid or sodium hydroxide. Then, a polypeptide array chip detection experiment proves that the normal combination of the polypeptide and the antibody can not be influenced, the non-specific adsorption of the second antibody is reduced, and the interference on the experimental result is reduced.
According to an exemplary embodiment of the present invention, a kit for polypeptide chip technology platform detection is provided. The kit comprises any secondary antibody buffer sealing solution used for the polypeptide chip technology platform.
According to a typical embodiment of the present invention, the kit for polypeptide chip technology platform detection further comprises a cleaning solution, wherein the cleaning solution comprises the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, and the pH value is 7.2 to 7.6. The cleaning solution can provide a stable pH environment at room temperature, and can remove free antigen/antibody without influencing normal combination of polypeptide-antibody, thereby avoiding interference on experimental results and providing real, effective and stable data for subsequent fluorescence imaging. Preferably, the cleaning solution comprises the following components in percentage by weight: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium phosphate, 0.05% Tween-20(v/v), 0.1% Proclin950(v/v), and pH 7.38-7.42.
In an embodiment of the present invention, the preparation and storage method of the cleaning solution is as follows: 1) measuring 18.98L of ultrapure water into a 20L solution bottle by using a measuring cylinder; 2) measuring 1L of 20 XPBST solution (PBS containing 0.05% Tween 20 v/v) by using a measuring cylinder, and adding the solution into a 20L solution bottle; 3) transferring 20mL of Proclin950 by using a pipette, and adding the Proclin950 into a 20L solution bottle until the final concentration is 0.1% to obtain a cleaning solution; 4) after shaking, 1N sodium hydroxide or 1N hydrochloric acid was added to the wash solution to adjust the pH to 7.4. + -. 0.02 (about 10mL of sodium hydroxide was added).
According to an exemplary embodiment of the present invention, the kit further comprises a sample diluent, the sample diluent comprising: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 1 percent D-mannitol w/v, and the pH value is 7.2 to 7.6. The sample diluent can provide a stable solution environment under the sample incubation condition, avoids interference on an experimental result under the condition of not influencing normal combination of the polypeptide and the antibody, and provides real, effective and stable data for subsequent fluorescence imaging. Preferably, the sample diluent comprises the following components and contents: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium phosphate, 0.05% Tween-20(v/v), 0.1% Proclin950(v/v), 1% D-mannitol (w/v), and pH 7.38-7.42.
In an embodiment of the present invention, the sample diluent is prepared and stored as follows: 1) A1L beaker was prepared, placed on a rotor, and placed on a magnetic stirrer. 2) 900mL of the rinse solution was measured with a measuring cylinder and added to the beaker. 3) And opening a switch of the magnetic stirrer, and adjusting the rotating speed to enable the solution to be in a vortex state. 4) 10g D-mannitol was weighed into the rinse solution using an analytical balance. 5) Stirring for at least 10min to dissolve completely. 6) The pH was adjusted to 7.4. + -. 0.02 by the addition of 1N sodium hydroxide or 1N hydrochloric acid (approximately 10mL NaOH was required). 7) Adding a cleaning solution with the pH value of 7.4 +/-0.02 to a constant volume of 1L. 8) The filtrate was filtered through a 0.22 μm PES (polyethersulfone membrane) filter, and the filtrate was stored at 4 ℃ after being dispensed. It is administered by taking out and standing to room temperature.
According to an exemplary embodiment of the present invention, the kit further comprises a secondary antibody diluent, the buffer comprising the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 0.75 percent sodium caseinate w/v, and the pH value is 7.2 to 7.6. The secondary antibody diluent can provide a stable solution environment under the secondary antibody incubation condition, reduces non-specific adsorption of the secondary antibody under the condition of not influencing normal combination of polypeptide-antibody, avoids interference on experimental results, and provides real, effective and stable data for subsequent fluorescence imaging. Preferably, the secondary antibody diluent comprises the following components and contents: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium phosphate, 0.05% Tween-20(v/v), 0.1% Proclin950(v/v), 0.75% sodium caseinate (w/v), pH 7.38-7.42.
In an embodiment of the present invention, the preparation and storage method of the secondary antibody diluent is as follows: 1) a500 mL beaker was prepared, placed on a rotor, and placed on a magnetic stirrer. 2) 450mL of cleaning solution is measured by a measuring cylinder and added into the beaker. 3) And opening a switch of the magnetic stirrer, adjusting the rotating speed to enable the solution to be in a vortex state, and adjusting the temperature to 100 ℃. 4) 3.75g of sodium caseinate was weighed into the wash on an analytical balance. 5) Stirred for 2 hours to dissolve fully. 6) The pH was adjusted to 7.4. + -. 0.02 by the addition of 1N sodium hydroxide or 1N hydrochloric acid (approximately 500. mu.L NaOH was required). 7) Adding a cleaning solution with the pH value of 7.4 +/-0.02 to a constant volume of 1L. 8) Filtering with 0.22 μm vacuum filter, packaging, and storing at 4 deg.C. It is administered by taking out and standing to room temperature.
The following examples are provided to further illustrate the advantageous effects of the present invention. It should be noted that the polypeptide chip technology used in the following specific examples is a polypeptide chip technology platform of HealthTell corporation, the used polypeptide chip is a HealthTell V13 chip with a model of P/N:600001V13 Slides, 131712 polypeptides are provided on the polypeptide chip, and 131712 polypeptides are polypeptides formed by combining 5 to 13 random amino acids without deviation, and the diverse coverage rate on tetramer (4 peptide) can reach 99.9%, and the diverse coverage rate on pentamer (pentapeptide) is 48.3%.
Example 1
Purpose of the experiment
In order to explore the blocking effect of the secondary antibody blocking solution, Casein (sodium caseinate) and BSA (bovine serum albumin) are selected as main components of the secondary antibody buffer blocking solution, a p53 antibody is selected as a sample, and the fluorescence signal intensity of the sample is read and analyzed. Among them, the P53 gene is a gene which is widely and deeply studied, and the P53 antibody can be used for early diagnosis of various tumors and screening tests of tumors. In patients with positive P53 antibody in tumor serum, the antibody appears 10-20% earlier than the clinical symptoms. It is commonly seen in breast cancer, lung cancer, colon cancer, bladder cancer, prostate cancer, brain cancer, etc.
2 Experimental procedures
The blocking solution was prepared using the sample diluent, and blocking solutions with BSA concentrations of 1%, 0.5%, and 0.1% and blocking solutions with Casein concentrations of 1%, 0.5%, and 0.1% were prepared, respectively. Firstly, 100mg of BSA powder and 100mg of Casein powder are weighed respectively, dissolved in 10mL of sample diluent respectively, and uniformly mixed by oscillation to form BSA confining liquid with the mass concentration of 1% and Casein confining liquid with the mass concentration of 1%. Then, using the sample diluent, 2-fold and 5-fold gradient dilution were performed on 1% BSA blocking solution and 1% Casein blocking solution, to finally form BSA blocking solution with mass concentration of 0.5% and 0.1% and Casein blocking solution with mass concentration of 0.5% and 0.1%. As shown in table 1 below:
TABLE 1
Wherein, the sample diluent comprises the following components and contents: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium phosphate, 1% Tween-20(v/v), 0.1% Proclin950(v/v), 1% D-mannitol (w/v), and the pH range is between 7.38 and 7.42.
2.1 dilution of the sample
Samples were diluted in a gradient using 1.5mL low adsorption centrifuge tubes and 15mL centrifuge tubes.
1) Mu. L p53 was added to 99. mu.L of the sample dilution to form 100-fold diluted sample of p53, and mixed well.
2) 28 μ L of serum was added to 8722 μ L of the sample dilution to form a 312.5-fold diluted sample of serum, and mixed well.
3) mu.L of 100-fold diluted p53 sample was added to 4000. mu.L of 312.5-fold diluted serum sample to form 100000-fold diluted p53 serum sample, and mixed well.
2.2 typesetting and sample adding
2.2.1 sealing
And (3) respectively subpackaging the newly prepared confining liquid, cleaning liquid and sample diluent into a clean 96-hole shallow pore plate by using a single-gun pipettor for 60 mu L, and checking sample application by two persons. And transferring the sealing liquid and the like in the 96-hole shallow hole plate to hole positions corresponding to the case by using a line gun according to the typeset hole position information, wherein the transfer volume is 45 mu L. And sealing the membrane, and incubating for 1h at 37 ℃ in a metal mixing instrument.
2.2.2 sample application
The diluted samples were separately dispensed into clean 96-well shallow-well plates with 60 μ L using a single-gun pipette, and double-person check for sample application. And then transferring the samples in the 96-hole shallow-hole plate to hole sites corresponding to the Cassette respectively by using a line gun according to the hole site information of the typesetting list, wherein the transfer volume is 45 mu L.
2.3 first incubation
Cassette was blocked, placed in a metal mixer (Thermo Mix C) and incubated for 1 hour at 37 ℃. The mixture was mixed every 6 minutes at 600rpm for 15 seconds.
2.4 first washing of the plate
Placing the Cassette tear film in an automatic plate washer, using the automatic plate washer (
405LS microplate magnetic plate washer).
The cleaning liquid used by the automatic plate cleaning machine comprises the following components in percentage by weight: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium phosphate, 1% Tween-20(v/v), 0.1% Proclin950(v/v), and the pH range is 7.38-7.42.
2.5 Add secondary antibody
The mouse secondary antibody and the human secondary antibody were diluted using a 5mL centrifuge tube under dark conditions:
1) adding 1 mu L of mouse secondary antibody into 3000 mu L of secondary antibody diluent to form 3000 times diluted mouse secondary antibody, and uniformly mixing;
2) mu.L of human secondary antibody is added into 3000 mu.L of secondary antibody diluent to form 1500-fold diluted human secondary antibody, and the mixture is mixed evenly.
The two diluted secondary antibodies are respectively poured into a liquid separating tank, and then are respectively transferred to the hole sites corresponding to the Cassette by using a discharging gun, and the transfer volume is 40 mu L.
2.6 second incubation
Cassette was blocked, placed in a metal mixer (Thermo Mix C) and incubated for 1 hour at 37 ℃. The mixture was mixed every 6 minutes at 600rpm for 15 seconds.
5.2.7 second washing plate
Placing the Cassette tear film in an automatic plate washer, using the automatic plate washer (
405LS microplate magnetic plate washer).
The cleaning liquid used by the automatic plate cleaning machine is the same as the cleaning liquid used in the first plate cleaning.
2.8 imaging and data acquisition
The chip in the Cassette is disassembled, cleaned, dried and assembled into an Imaging Cassette, and the Imaging Cassette is placed into an ImageXpress micro4 imager of Molecular Device company for scanning Imaging. And finally, obtaining a TIFF picture file as the original data by each detection sample. And converting and analyzing the original data.
3 results of the experiment
The whole confining liquid reduces the peptide fragment signal of the polypeptide chip, which means that the confining liquid has a confining effect.
FIG. 1 shows the blocking effect of blocking solutions containing different concentrations of Casein (sodium caseinate) or BSA (bovine serum albumin). As shown in FIG. 1, Casein (sodium caseinate) has a better blocking effect than BSA (bovine serum albumin), and the concentration is selected to be 0.1%. The box plot shows that 0.1% casein blocking solution significantly reduced the overall signal, above the highest concentration of BSA seen in the drop; and a concentration of 0.1% is sufficient, no more significant signal reduction is observed with more sodium caseinate.
FIG. 2 shows the effect of different concentrations of Casein (sodium caseinate) blocking solution on P53-specific signal, as shown in FIG. 2, P53-specific signal peptide signal is stable and not affected by the blocking solution, and FIG. 2 shows the signal intensity of 1 peptide in various specific signal peptides detected by P53 on the polypeptide chip technology platform as a graph of the concentration of sodium caseinate in the blocking solution.
FIG. 3 shows that the blocking solutions of Casein (sodium caseinate) at different concentrations had no effect on the signal intensity of the specific signal peptide of P53 (as shown by A, B in FIG. 3, A, B in FIG. 3 is a graph of the signal intensity of 2 (as representative) peptides of P53 detected on the polypeptide chip technology platform versus the concentration of sodium caseinate in the blocking solution), and had a significant reduction effect on the signal intensity of the non-specific signal peptide of P53 (as shown by C, D in FIG. 3, and C, D in FIG. 3 is a graph of the signal intensity of 2 (as representative) peptides of P53 detected on the polypeptide chip technology platform versus the concentration of sodium caseinate in the blocking solution). FIG. 3 illustrates that the signal intensity of the P53-specific signal peptide is not affected by the blocking solution, i.e., it is first ensured that the blocking solution does not negatively affect the detection of the specific signal; meanwhile, the sealing liquid of Casein (sodium caseinate) with different concentrations can have a sealing effect on non-specific binding peptide fragments, and a better effect can be achieved by 0.1% concentration.
FIG. 4 shows the blocking effect of different concentrations of Casein (sodium caseinate) blocking solutions on non-specific background peptide signals from serum; as shown in fig. 4, A, B in fig. 4 represents 2 of the non-specific background peptide signals (as representative) found when detecting the secondary antibody sample and the serum sample by using the polypeptide chip technology platform, and as shown in fig. 4, the graph of the signal intensity of the peptide in 2 and the sodium caseinate concentration in the blocking solution shows that the blocking solutions of different concentrations of Casein can have the effect of blocking the non-specific background peptide brought by the serum, and the better effect can be achieved by 0.1% concentration.
In addition, the invention verifies through specific experiments that the components of the secondary antibody buffer confining liquid, the cleaning liquid, the sample diluent and the secondary antibody diluent can achieve the technical effects (specific experimental data are shown) similar to those shown in the example 1 within the range defined by the invention.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects:
the polypeptide chip-based confining liquid is suitable for polypeptide array chips, has low cost and stable performance, and has the effects of improving specificity and sensitivity in detection experiments and scientific researches based on the polypeptide chips.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A secondary antibody buffer sealing liquid for a polypeptide chip technology platform is characterized by comprising the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 1 percent D-mannitol w/v and 0.1 to 1 percent sodium caseinate w/v.
2. The secondary antibody buffer blocking solution according to claim 1, wherein the pH of the secondary antibody buffer blocking solution is 7.2-7.6; preferably, the pH value of the secondary antibody buffer sealing liquid is 7.38-7.42.
3. A kit for detecting a polypeptide chip technology platform, which comprises the secondary antibody buffer sealing solution for the polypeptide chip technology platform as claimed in claim 1 or 2.
4. The kit of claim 3, further comprising a wash solution comprising the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, and the pH value is 7.2 to 7.6.
5. The kit of claim 3, further comprising a sample diluent comprising the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 1 percent D-mannitol w/v, and the pH value is 7.2 to 7.6.
6. The kit of claim 3, further comprising a secondary antibody diluent, wherein the buffer comprises the following components: 130 to 137mM sodium chloride, 2.5 to 2.7mM potassium chloride, 3.8 to 4.3mM disodium hydrogen phosphate, 1.2 to 1.4mM monopotassium phosphate, 0.05 to 1 percent Tween-20v/v, 0.05 to 0.1 percent Proclin950v/v, 0.5 to 0.75 percent sodium caseinate w/v, and the pH value is 7.2 to 7.6.
7. The use of the secondary antibody buffer blocking solution for the polypeptide chip technology platform according to claim 1 or 2 in the preparation of polypeptide chip technology platform detection products or in the field of biomedicine.
8. The use of claim 7, wherein the polypeptide chip technology platform test products comprise sample classification identification products, vaccine effect evaluation products, biomarker test products, disease diagnosis products and health condition assessment products; the biomedical field includes drug screening, drug design, vaccine development, vaccine design, and biomarker screening.
9. Use of the kit for polypeptide chip technology platform assay according to any one of claims 3 to 6 in the preparation of polypeptide chip technology platform assay products or in the field of biomedicine.
10. The use of claim 9, wherein the polypeptide chip technology platform test products comprise sample classification identification products, vaccine effect evaluation products, biomarker test products, disease diagnosis products and health condition assessment products; the biomedical field includes drug screening, drug design, vaccine development, vaccine design, and biomarker screening.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011522214.7A CN112748242B (en) | 2020-12-21 | 2020-12-21 | Secondary antibody buffer sealing solution for polypeptide chip technology platform detection, kit comprising secondary antibody buffer sealing solution and application of secondary antibody buffer sealing solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011522214.7A CN112748242B (en) | 2020-12-21 | 2020-12-21 | Secondary antibody buffer sealing solution for polypeptide chip technology platform detection, kit comprising secondary antibody buffer sealing solution and application of secondary antibody buffer sealing solution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112748242A true CN112748242A (en) | 2021-05-04 |
| CN112748242B CN112748242B (en) | 2023-07-21 |
Family
ID=75648141
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011522214.7A Active CN112748242B (en) | 2020-12-21 | 2020-12-21 | Secondary antibody buffer sealing solution for polypeptide chip technology platform detection, kit comprising secondary antibody buffer sealing solution and application of secondary antibody buffer sealing solution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112748242B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114778846A (en) * | 2022-03-23 | 2022-07-22 | 北京利德曼生化股份有限公司 | Kidney injury molecule (KIM-1) determination kit and detection method thereof |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684145A (en) * | 2008-09-23 | 2010-03-31 | 北京市结核病胸部肿瘤研究所 | Antigen peptide identified by p53 antoantibody, kit and application thereof in preparing tumor detection kit |
| CN202189053U (en) * | 2011-08-12 | 2012-04-11 | 张灿 | Biological chip |
| CN103293145A (en) * | 2013-05-16 | 2013-09-11 | 赫利森（厦门）生物科技有限公司 | Chemiluminescence reagent |
| CN105572353A (en) * | 2014-10-17 | 2016-05-11 | 广州瑞博奥生物科技有限公司 | Antibody chip reagent kit for detecting hepatoma marker |
| CN105925599A (en) * | 2016-04-21 | 2016-09-07 | 深圳华大基因研究院 | Preparation method of high-purity monoclonal antibodies, monoclonal antibodies and applications of monoclonal antibodies |
| CN108375559A (en) * | 2018-02-08 | 2018-08-07 | 南京岚煜生物科技有限公司 | Cardiac troponin kit and preparation based on micro-fluidic chip and detection method |
| CN108398551A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | It is a kind of for the composition of enzyme linked immunological kit and antinuclear antibodies spectrum detection kit and preparation method thereof |
| CN108398554A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | A kind of kit of anti-torch-IgM types antibody spectrum chip and preparation method thereof and TORCH detections |
| CN108398550A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | A kind of composition, chip and preparation method thereof and include the chip detection device |
| CN109725141A (en) * | 2018-12-21 | 2019-05-07 | 东莞东阳光科研发有限公司 | The preparation method of working solution, SA- magnetic bead dried frozen aquatic products and dried frozen aquatic products is lyophilized in SA- magnetic bead |
| CN111487413A (en) * | 2019-01-29 | 2020-08-04 | 艾维可生物科技有限公司 | Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method |
-
2020
- 2020-12-21 CN CN202011522214.7A patent/CN112748242B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684145A (en) * | 2008-09-23 | 2010-03-31 | 北京市结核病胸部肿瘤研究所 | Antigen peptide identified by p53 antoantibody, kit and application thereof in preparing tumor detection kit |
| CN202189053U (en) * | 2011-08-12 | 2012-04-11 | 张灿 | Biological chip |
| CN103293145A (en) * | 2013-05-16 | 2013-09-11 | 赫利森（厦门）生物科技有限公司 | Chemiluminescence reagent |
| CN105572353A (en) * | 2014-10-17 | 2016-05-11 | 广州瑞博奥生物科技有限公司 | Antibody chip reagent kit for detecting hepatoma marker |
| CN105925599A (en) * | 2016-04-21 | 2016-09-07 | 深圳华大基因研究院 | Preparation method of high-purity monoclonal antibodies, monoclonal antibodies and applications of monoclonal antibodies |
| CN108375559A (en) * | 2018-02-08 | 2018-08-07 | 南京岚煜生物科技有限公司 | Cardiac troponin kit and preparation based on micro-fluidic chip and detection method |
| CN108398551A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | It is a kind of for the composition of enzyme linked immunological kit and antinuclear antibodies spectrum detection kit and preparation method thereof |
| CN108398554A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | A kind of kit of anti-torch-IgM types antibody spectrum chip and preparation method thereof and TORCH detections |
| CN108398550A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | A kind of composition, chip and preparation method thereof and include the chip detection device |
| CN109725141A (en) * | 2018-12-21 | 2019-05-07 | 东莞东阳光科研发有限公司 | The preparation method of working solution, SA- magnetic bead dried frozen aquatic products and dried frozen aquatic products is lyophilized in SA- magnetic bead |
| CN111487413A (en) * | 2019-01-29 | 2020-08-04 | 艾维可生物科技有限公司 | Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114778846A (en) * | 2022-03-23 | 2022-07-22 | 北京利德曼生化股份有限公司 | Kidney injury molecule (KIM-1) determination kit and detection method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112748242B (en) | 2023-07-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7027330B2 (en) | Antigen array | |
| CN107817232B (en) | Automated immunoassay system for performing diagnostic assays for allergies and autoimmune diseases | |
| US20230213510A1 (en) | Methods relating to improving accuracy of capture object-based assays | |
| EP3270160A1 (en) | Method for detecting test substance and reagent kit used in said method | |
| WO2006128362A1 (en) | Method and its kit for quantitatively detecting specific analyte with single capturing agent | |
| EP3903103B1 (en) | Direct detection of single molecules on microparticles | |
| US11639938B2 (en) | Multiplexed bead-based analytical assays | |
| CN104169710A (en) | Method and apparatus for time-resolved fluorescence immunoassay testing | |
| CN114994304B (en) | cTnI dry-type homogeneous phase chemiluminescence detection kit | |
| CN111175494A (en) | Thyroglobulin antibody detection kit and use method thereof | |
| CN108603880A (en) | The detection method of tested substance and the detection kit of tested substance | |
| CN107533052B (en) | Method for reusing test probes and reagents in immunoassays | |
| JP2008268043A (en) | Method for forming detection part of examination device, and examination device for measuring lateral flow immunity | |
| CN112748242A (en) | Secondary antibody buffer confining liquid for polypeptide chip technology platform detection, kit comprising same and application thereof | |
| Li et al. | Simultaneous detection of various contaminants in milk based on visualized microarray | |
| CN113049835A (en) | Combined detection kit, detection method and immunoassay system | |
| KR101628221B1 (en) | Test reagent, and method for measuring analyte in test sample using same | |
| CN117030991A (en) | Extracellular vesicle detection device and detection method | |
| CN101910843A (en) | Reagent for detecting HIV-1 antigen and detection method | |
| CN111363789B (en) | Kit and method for simultaneously detecting protein and RNA | |
| JP2010101703A (en) | Immunoassay and measuring reagent kit | |
| JP2012225726A (en) | Sandwich immunoassay method and biosensor using the same | |
| US20060223194A1 (en) | Methods of screening for post-translationally modified proteins | |
| Matson | Well-based antibody arrays | |
| JP2009030997A (en) | Blood group determining kit for type and screening and blood group determining device using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |