CN112748242B - 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 - Google Patents
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 Download PDFInfo
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Abstract
The invention discloses a secondary antibody buffer sealing solution for polypeptide chip technology platform detection, a kit comprising the same and application thereof. Wherein, the secondary antibody buffer sealing solution comprises the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-1% D-mannitol w/v and 0.1-1% sodium caseinate w/v. The buffer solution can provide a stable solution environment under the condition of secondary antibody incubation, reduces the non-specific adsorption of the secondary antibody under the condition of not affecting the normal combination of polypeptide-antibody, avoids the interference to 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, in particular to a secondary antibody buffer sealing solution for polypeptide chip technical platform detection, a kit comprising the same and application thereof.
Background
A polypeptide chip is a chip based on a substrate material, and the chip comprises features with pre-designed numbers, positions and sequences, wherein one feature is a cluster of polypeptides with identical sequences, the polypeptide sequences between the features are often different, and the features form a high-density polypeptide array.
The polypeptide chip technology is a detection technology based on a polypeptide chip, and utilizes various polypeptides on the polypeptide chip to contact with a sample, then utilizes an image acquisition technology to acquire various characteristic signals (particularly can be represented as fluorescent images carrying the various characteristic signals) on the polypeptide chip, and further outputs the signal intensity of each characteristic in the polypeptide chip, namely the detection result data of the polypeptide chip. Specifically, the polypeptide chip technology usually washes out components in a sample, which cannot be combined with or have weak binding force with the polypeptide chip, after the sample contacts with the polypeptide chip, and then marks the components on a specific target antigen (a specific object to be observed or detected in the sample, such as IG G, IG M, etc.) by using an antibody (secondary antibody) carrying a fluorescent label, thereby obtaining a fluorescent signal on the polypeptide chip through an image acquisition technology. Therefore, the polypeptide chip technology can identify antibodies which are differentially expressed among different individuals or antibodies which are differentially expressed by the same individual at different time points, and further related researches are carried out according to the information.
In a specific experiment, in order to ensure that the fluorescent-labeled antibody has higher bioactivity and obtain a better imaging result, the nonspecific binding of the fluorescent-labeled antibody is reduced, the nonspecific background coloring is reduced, the specificity and the sensitivity of the experiment are improved, and the fluorescent-labeled antibody is mostly required to be properly diluted. The diluted fluorescent-labeled antibody should generally be storable and usable at 4 ℃ for not less than 6 months and reusable for multiple times. In immunological experiments such as ELISA, high background values often appear, which affects the detection of the experimental results. This is because, when using the relevant immunological reagents, the proteins bind to the non-adsorbed solid carrier, resulting in non-specific binding. In practice, such solid support sites are often blocked by blocking reagents to reduce or eliminate the effects of non-specific binding background. The effective components in the general blocking reagent include BSA, animal serum, fab fragment single-chain secondary antibody, etc. The BSA component is relatively single and can possibly contain bovine IgG, so that the BSA component has stronger cross reaction with secondary antibodies such as anti-bovine, goat, sheep, horse and the like, and a certain background can be caused; some of the blocked serum may contain sodium azide and is therefore not suitable for HRP enzyme-labeled detection systems; the Fab fragment single-chain secondary antibodies are complex to prepare, high in price and not suitable for large-scale use, so that the Fab fragment single-chain secondary antibodies are not ideal choices of general blocking reagent (blocking solution) components of a polypeptide chip platform. In addition, because the polypeptide chip is arranged with high-density polypeptide sequences, the sealing liquid has higher requirements, and the problem of high cost and unstable sealing effect exists in the experimental process of the existing sealing liquid for the polypeptide chip of the immune surface needle technology.
Disclosure of Invention
The invention aims to provide a secondary antibody buffer sealing solution for polypeptide chip technology platform detection, a kit comprising the same and application thereof, so as to provide a universal sealing solution suitable for polypeptide chip technology platform detection.
In order to achieve the above object, according to one aspect of the present invention, there is provided a secondary antibody buffer blocking solution for a polypeptide chip technology platform. The secondary antibody buffer sealing solution comprises the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-1% D-mannitol w/v and 0.1-1% sodium caseinate w/v.
Further, the pH of the secondary antibody buffer sealing solution is 7.2-7.6.
Further, the pH of the secondary antibody buffer sealing solution is 7.38-7.42.
According to another aspect of the invention, a kit for polypeptide chip technology platform detection is provided. The kit comprises any of the secondary antibody buffer sealing solutions for the polypeptide chip technology platform.
Further, the kit also comprises a cleaning solution, wherein the cleaning solution comprises the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, and pH 7.2-7.6.
Further, the kit also comprises a sample diluent, wherein the sample diluent comprises the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-1% D-mannitol w/v and pH of 7.2-7.6.
Further, the kit also comprises a secondary antibody diluent, and the buffer solution comprises the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-0.75% sodium caseinate w/v and pH of 7.2-7.6.
According to still another aspect of the present invention, there is provided an application of the second antibody buffer blocking solution for a polypeptide chip technology platform in the field of preparation of a polypeptide chip technology platform detection product or biological medicine.
Further, 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 assessment products; the biomedical field includes, but is not limited to, drug screening, drug design, vaccine development, vaccine design, and biomarker screening.
According to still another aspect of the present invention, there is provided an application of the above-mentioned kit for detecting a polypeptide chip technology platform in preparing a polypeptide chip technology platform detection product or in the field of biological medicine.
Further, 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 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 secondary antibody incubation condition, reduces the non-specific adsorption of the secondary antibody under the condition of not affecting the normal combination of the polypeptide-antibody, avoids the interference to an experimental result, and provides real, effective and stable data for subsequent fluorescence imaging; the kit has the advantages of low cost and stable performance, and has the effect of improving the specificity and the sensitivity in immunology, particularly in detection and scientific research by using a polypeptide array chip.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 shows the blocking effect of blocking solutions containing different concentrations of Casein or BSA (bovine serum albumin) in example 1;
FIG. 2 shows the effect of blocking solutions containing different concentrations of Casein on P53-specific signals in example 1;
FIG. 3 shows the different blocking effects of blocking solutions containing different concentrations of Casein (sodium caseinate) in example 1 on specific and non-specific peptide fragments in the P53 test experiment; and
FIG. 4 shows the blocking effect of blocking solutions containing different concentrations of Casein (sodium caseinate) in example 1 on serum-induced non-specific background peptide signals.
Detailed Description
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.
According to an exemplary embodiment of the present invention, there is provided a secondary antibody buffer blocking solution for a polypeptide chip technology platform, including the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-1% D-mannitol w/v and 0.1-1% sodium caseinate w/v. Among them, sodium caseinate is also called casein.
Sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate are taken as salt ions in the buffer solution, so that 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 exerting the performance of the polypeptide array on a polypeptide chip platform; tween-20 is used as a nonionic type antigen, has the function of renaturation antigen, and can improve the specific recognition capability; proclin950 is a broad-spectrum antibacterial preservative, can inhibit the growth of microorganisms such as bacteria and fungi in a broad spectrum for a long time under a certain concentration, generates little drug resistance, and can be used for antibacterial and antiseptic of primary antibodies, secondary antibodies, confining liquid, antibody diluent and the like; casein/sodium caseinate is phosphorus-containing protein in animal milk, and has structural formula of NH 2 RCOOH, odorless white to yellow powder, relative density 1.25 to 1.31 (water 1); sodium caseinate is almost insoluble in water, alcohol and ether, in dilute alkali solution, alkaline carbonate solution and concentrated acid, in weak statePrecipitation in acid, hygroscopicity, stability when dry, rapid hardening when wet; the non-specific adsorption of casein/sodium caseinate can be reduced. 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 secondary antibody incubation, can reduce the nonspecific adsorption of the secondary antibody, especially the nonspecific adsorption of the secondary antibody and solid phase components outside a polypeptide array on a polypeptide chip under the condition of not affecting the normal combination of the polypeptide-antibody, further avoids the interference to experimental results, and provides real, effective and stable data for subsequent fluorescence imaging; the kit has the advantages of low cost and stable performance, and has the effect of improving the specificity and the sensitivity in immunology, particularly in detection and scientific research by using a polypeptide array chip.
Specifically, in the polypeptide array chip polypeptide screening test of human serum, a P53 specific antibody is doped into the serum, and a P53 polypeptide signal is used as a specific signal reference. Then comparing the detection results of the serum sample and the serum sample containing P53 and the detection results of the two samples under the condition of sealing liquid and under the condition of no sealing liquid through experiments, the sealing liquid has no interference on the specific signal of the P53 antibody, but a large amount of non-specific signals in serum are obviously reduced. Multiple experiments prove that the result can be repeated, and the effect of the sealing liquid is stable.
Specifically, in a preferred embodiment of the present application, the secondary antibody buffer seal is formulated as follows: 135mM sodium chloride, 2.5mM potassium chloride, 3.8mM disodium hydrogen phosphate, 1.2mM potassium dihydrogen phosphate, 0.05% Tween-20v/v,0.05%Proclin950 v/v,0.1% -1% casein/sodium caseinate w/v, pH adjusted to 7.4 with 1N hydrochloric acid or sodium hydroxide. Then, the detection experiment of the polypeptide array chip proves that the polypeptide-antibody normal combination is not affected, the non-specific adsorption of the secondary antibody is reduced, and the interference to the experimental result is reduced.
According to an exemplary embodiment of the present invention, a kit for detection of a polypeptide chip technology platform is provided. The kit comprises any of the secondary antibody buffer sealing solutions for the polypeptide chip technology platform.
According to an exemplary embodiment of the present invention, the kit for detecting a polypeptide chip technology platform further comprises a cleaning solution, wherein the cleaning solution comprises the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, and pH of 7.2-7.6. The cleaning liquid can provide a stable pH environment at room temperature, and free antigen/antibody is removed under the condition that normal combination of polypeptide-antibody is not affected, so that interference to experimental results is avoided, and real, effective and stable data are provided 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 potassium dihydrogen phosphate, 0.05% Tween-20 (v/v), 0.1% Proclin950 (v/v), pH 7.38-7.42.
In one embodiment of the invention, the cleaning solution is prepared and stored as follows: 1) Measuring 18.98L of ultrapure water into a 20L solution bottle by using a measuring cylinder; 2) 1L of 20 XPBST solution (PBS containing 0.05% Tween 20 v/v) was measured with a measuring cylinder and added to a 20L solution bottle; 3) Transferring 20mL of Proclin950 by a pipette, adding into a 20L solution bottle to a final concentration of 0.1% to obtain a cleaning solution; 4) Shaking, adding 1N sodium hydroxide or 1N hydrochloric acid to the washing solution, and adjusting pH to 7.4+ -0.02 (about 10mL sodium hydroxide is required).
According to an exemplary embodiment of the invention, the kit further comprises a sample diluent comprising the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-1% D-mannitol w/v and pH of 7.2-7.6. The sample diluent can provide a stable solution environment under the condition of sample incubation, avoid interference to experimental results under the condition of not affecting normal combination of polypeptide-antibody, and provide 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 potassium dihydrogen phosphate, 0.05% Tween-20 (v/v), 0.1% Proclin950 (v/v), 1% D-mannitol (w/v), pH 7.38-7.42.
In one embodiment of the present invention, the preparation and preservation method of the sample diluent is as follows: 1) A 1L beaker was prepared, placed in a rotor and placed on a magnetic stirrer. 2) The cartridge was used to measure 900mL of cleaning solution into the beaker. 3) The magnetic stirrer switch is turned on, and the rotation speed is adjusted to enable the solution to be in a vortex state. 4) 10g D-mannitol was weighed using an analytical balance and added to the wash solution. 5) Stirring for at least 10min to dissolve thoroughly. 6) 1N sodium hydroxide or 1N hydrochloric acid was added to adjust the pH to 7.4.+ -. 0.02 (about 10mL NaOH was added). 7) The washing liquid with pH 7.4+ -0.02 was added to a constant volume of 1L. 8) The mixture was filtered by a vacuum filtration apparatus using a 0.22 μm PES (polyether sulfone) membrane, and the mixture was stored at 4℃after packaging. When in use, the product needs to be taken out in advance and placed to room temperature.
According to an exemplary embodiment of the invention, the kit further comprises a secondary antibody diluent, the buffer comprising the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-0.75% sodium caseinate w/v and pH of 7.2-7.6. The secondary antibody diluent can provide a stable solution environment under the secondary antibody incubation condition, reduce the non-specific adsorption of the secondary antibody under the condition of not affecting the normal combination of the polypeptide-antibody, avoid the interference to the experimental result and provide real, effective and stable data for the subsequent fluorescence imaging. Preferably, the secondary anti-dilution liquid comprises the following components and contents: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate, 0.05% Tween-20 (v/v), 0.1% Proclin950 (v/v), 0.75% casein sodium salt (w/v), pH 7.38-7.42.
In one embodiment of the invention, the preparation and preservation methods of the secondary antibody diluent are as follows: 1) Prepare 500mL beaker, place into rotor and place on magnetic stirrer. 2) The cartridge measures 450mL of cleaning solution into the beaker. 3) The magnetic stirrer switch is turned on, the rotation speed is adjusted to enable the solution to be in a vortex state, and the temperature is adjusted to be 100 ℃. 4) 3.75g casein sodium salt was weighed into the wash solution using an analytical balance. 5) Stirring for 2 hours to dissolve thoroughly. 6) 1N sodium hydroxide or 1N hydrochloric acid was added to adjust the pH to 7.4.+ -. 0.02 (about 500. Mu.L NaOH was added). 7) The washing liquid of pH 7.4.+ -. 0.02 was added to a constant volume of 1L. 8) Filtering with 0.22 μm vacuum filter, packaging, and storing at 4deg.C. When in use, the product needs to be taken out in advance and placed to room temperature.
The advantageous effects of the present invention will be further described below with reference to examples. The polypeptide chip technology used in the following specific examples is a polypeptide chip technology platform of HealthTell company, the used polypeptide chip is a HealthTell V13 chip, the model is P/N:600001V13 Slides, 131712 polypeptides are arranged on the polypeptide chip, the 131712 polypeptides are respectively formed by combining 5-13 unbiased random amino acids, the diversity coverage rate of the tetramer (4 peptide) can reach 99.9%, and the diversity coverage rate of the pentamer (pentapeptide) is 48.3%.
Example 1
1 purpose of 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 fluorescent 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 positive for P53 antibodies in tumor serum, the occurrence of the antibodies is 10% -20% earlier than that of clinical symptoms. Is commonly found in breast cancer, lung cancer, colon cancer, bladder cancer, prostate cancer, brain cancer, and the like.
2 experimental procedure
A blocking solution was prepared using a sample diluent, and blocking solutions were prepared at BSA concentrations of 1%, 0.5% and 0.1% and Casein concentrations of 1%, 0.5% and 0.1%, respectively. Firstly, 100mg of BSA powder and 100mg of Casein powder are respectively weighed and dissolved in 10mL of sample diluent, and the BSA confining liquid and the Casein confining liquid with the mass concentration of 1% and 1% are formed by shaking and mixing uniformly. Then, the sample dilution was used to sequentially perform 2-fold and 5-fold gradient dilutions of 1% BSA blocking solution and 1% Casein blocking solution, to finally form 0.5% and 0.1% BSA blocking solution, and 0.5% and 0.1% Casein blocking solution. As shown in table 1 below:
TABLE 1
The sample diluent comprises the following components in percentage by weight: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate, 1% Tween-20 (v/v), 0.1% Proclin950 (v/v), 1% D-mannitol (w/v), and pH in the range of 7.38-7.42.
2.1 dilution of samples
Samples were gradient diluted using a 1.5mL low adsorption centrifuge tube and a 15mL centrifuge tube.
1) Mu. L p53 was added to 99. Mu.L of the sample dilution to form a 100-fold p53 diluted sample, which was mixed well.
2) 28. Mu.L of serum was added to 8722. Mu.L of the sample dilution to form a 312.5-fold serum diluted sample, which was mixed well.
3) 4 mu L of 100-fold p53 diluted sample is added into 4000 mu L of 312.5-fold serum diluted sample to form a 100000-fold p 53-containing serum sample, and the serum sample is uniformly mixed.
2.2 typesetting and sample addition
2.2.1 closure
And (3) respectively subpackaging the newly prepared sealing liquid, cleaning liquid and sample diluent into 60 mu L to a clean 96-hole shallow hole plate by using a single-gun pipette, and checking and sample adding by two persons. And according to typeset hole site information, using a gun to transfer the sealing liquid and the like in the 96-hole shallow hole plate into the hole site corresponding to the Cassette, wherein the transfer volume is 45 mu L. Sealing the membrane, and incubating for 1h at 37 ℃ in a metal mixing instrument.
2.2.2 sample addition
And (3) respectively subpackaging the diluted samples into 60 mu L to clean 96-hole shallow hole plates by using a single-gun pipette, and checking and loading the samples by two persons. And then according to the hole site information of the typesetting table, using a gun to transfer samples in the 96-hole shallow hole plate into holes corresponding to the Cassette respectively, wherein the transfer volume is 45 mu L.
2.3 first incubation
The Cassette was covered and placed in a metal mixer (Thermo Mix C) and incubated at 37 ℃ for 1 hour. Mixing was carried out every 6 minutes at 600rpm for 15s.
2.4 first washing plate
The Cassette dyestripping is placed in an automatic plate washing machine, and the automatic plate washing machine is used for washing plates [ ]405LS microplate magnetic washer).
The cleaning liquid used for the automatic plate washer comprises the following components in percentage by weight: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate, 1% Tween-20 (v/v), 0.1% Proclin950 (v/v), pH in the range of 7.38-7.42.
2.5 adding secondary antibody
The murine and human secondary antibodies were diluted in a 5mL centrifuge tube under light-protected conditions:
1) Adding 1 mu L of the mouse secondary antibody into 3000 mu L of secondary antibody diluent to form 3000 times diluted mouse secondary antibody, and uniformly mixing;
2) 2 mu L of human secondary antibody is added into 3000 mu L of secondary antibody diluent to form 1500 times diluted human secondary antibody, and the mixture is uniformly mixed.
The two diluted secondary antibodies are respectively poured into a liquid separating groove, and then are respectively transferred into corresponding hole sites of the Cassette by using a gun, wherein the transfer volume is 40 mu L.
2.6 second incubation
The Cassette was covered and placed in a metal mixer (Thermo Mix C) and incubated at 37 ℃ for 1 hour. Mixing was carried out every 6 minutes at 600rpm for 15s.
5.2.7 second washing plate
The Cassette dyestripping is placed in an automatic plate washing machine, and the automatic plate washing machine is used for washing plates [ ]405LS microplate magnetic washer).
The cleaning liquid used by the automatic plate washer is the same as the cleaning liquid used in the first plate washer.
2.8 imaging and data acquisition
The chip in the Cassette is disassembled, cleaned and dried, then assembled into Imaging Cassette, and then placed into a ImageXpress micro imager of Molecular Device company for scanning Imaging. And finally, obtaining a TIFF picture file which is the original data by each detection sample. And converting and analyzing the original data.
3 results of experiments
The blocking liquid reduces the peptide signal of the polypeptide chip as a whole, which means that the blocking liquid has a blocking 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 can significantly reduce overall signal, higher than the highest concentration BSA in terms of amplitude reduction; and a concentration of 0.1% is sufficient, no more significant signal reduction is observed for more sodium caseinate.
Fig. 2 shows the effect of blocking solutions of different concentrations of Casein (sodium caseinate) on P53 specific signals, as shown in fig. 2, the P53 specific signal peptide signals are stable and not affected by the blocking solution, and fig. 2 shows a graph of the signal intensity of 1 peptide of the multiple specific signal peptides detected by P53 on a polypeptide chip technology platform and the concentration of sodium caseinate in the blocking solution.
FIG. 3 shows that blocking solutions of different concentrations of Casein (sodium caseinate) have no effect on the signal intensity of specific signal peptides of P53 (as shown in FIG. 3, A, B. A, B in FIG. 3 is a graph of the signal intensity of 2 peptides (as a representative) of the plurality of specific signal peptides detected by P53 on a polypeptide chip technology platform versus the concentration of sodium caseinate in the blocking solution), and that blocking solutions have a significant reduction effect on the signal intensity of non-specific signal peptides of P53 (as shown in FIG. 3, C, D. C, D in FIG. 3 is a graph of the signal intensity of 2 non-specific signal peptides (as a representative) detected by P53 on a 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 fluid, i.e., it is first ensured that the blocking fluid does not negatively affect the specific signal discovery; meanwhile, blocking solutions of Casein (sodium caseinate) with different concentrations can have a blocking effect on non-specific binding peptide fragments, and a better effect can be achieved at a concentration of 0.1%.
FIG. 4 shows the blocking effect of blocking fluid of different concentrations of Casein on serum-derived non-specific background peptide signals; as shown in fig. 4, A, B in fig. 4 represents 2 (representative) non-specific background peptide signals found when a polypeptide chip technology platform is used for detecting a secondary antibody sample and a serum sample, and as shown in fig. 4, the signal intensity of the peptide in fig. 2 and the concentration of sodium caseinate in a blocking solution show that the blocking solution of Casein (sodium caseinate) with different concentrations can have a blocking effect on the non-specific background peptide brought by serum, and the concentration of 0.1% can achieve a better effect.
In addition, the present invention has been verified by specific experiments that the components of the secondary antibody buffer sealing liquid, the cleaning liquid, the sample diluent and the secondary antibody diluent all achieve technical effects similar to those shown in example 1 within the scope defined by the present invention (specific experimental data are shown).
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:
the blocking liquid is suitable for the polypeptide array chip, has low cost and stable performance, and has the effect of improving the specificity and the sensitivity in detection experiments and scientific researches based on the polypeptide chip.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. Application of secondary antibody buffer sealing liquid for P53 antibody in serum in polypeptide chip technology platform in preparation of polypeptide chip technology platform detection product or biological medicine field, the secondary antibody buffer sealing liquid is composed of the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-1% D-mannitol w/v and 0.1% sodium caseinate w/v, wherein the pH of the secondary antibody buffer sealing solution is 7.2-7.6; the biomedical field comprises drug screening, drug design, vaccine development, vaccine design and biomarker screening.
2. The use according to claim 1, wherein the secondary antibody buffer blocking solution has a pH of 7.38-7.42.
3. The use of claim 1, wherein the polypeptide chip technology platform detection products include sample classification identification products, vaccine effect evaluation products, biomarker detection products, disease diagnosis products, and health assessment products.
4. The application of the kit for detecting the P53 antibody in serum by the polypeptide chip technology platform in the preparation of the polypeptide chip technology platform detection product or in the field of biological medicine comprises the steps of a secondary antibody buffer sealing solution for the polypeptide chip technology platform; the secondary antibody buffer sealing solution consists of the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-1% D-mannitol w/v and 0.1% sodium caseinate w/v, wherein the pH of the secondary antibody buffer sealing solution is 7.2-7.6; the biomedical field comprises drug screening, drug design, vaccine development, vaccine design and biomarker screening.
5. The use according to claim 4, wherein the secondary antibody buffer blocking solution has a pH of 7.38-7.42.
6. The use according to claim 4, wherein the kit further comprises a cleaning solution comprising the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, and pH 7.2-7.6.
7. The use of claim 4, wherein the kit further comprises a sample diluent comprising the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-1% D-mannitol w/v, and pH 7.2-7.6.
8. The use of claim 4, wherein the kit further comprises a secondary anti-dilution comprising the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950v/v, 0.5-0.75% sodium caseinate w/v, and pH 7.2-7.6.
9. The use of claim 4, wherein the polypeptide chip technology platform detection products include sample classification identification products, vaccine effect evaluation products, biomarker detection products, disease diagnosis products, and health assessment products.
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| CN101684145B (en) * | 2008-09-23 | 2012-10-17 | 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 |
| CN103293145B (en) * | 2013-05-16 | 2016-08-10 | 赫利森（厦门）生物科技有限公司 | A kind of chemical illuminating reagent |
| CN105572353B (en) * | 2014-10-17 | 2018-10-30 | 广州瑞博奥生物科技有限公司 | A kind of antibody chip kit for detecting liver cancer 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 |
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