CN117603941A - Specific LP-PLA2 epitope peptide, antigen, antibody, application and kit - Google Patents
Specific LP-PLA2 epitope peptide, antigen, antibody, application and kit Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
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- G—PHYSICS
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- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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Abstract
The invention discloses a specific LP-PLA2 epitope peptide, an antigen, an antibody, application and a kit. The specific LP-PLA2 epitope peptide in the invention is as follows: as set forth in SEQ ID NO: 1-3, and a polypeptide shown in any one of amino acid sequences. The specific LP-PLA2 epitope peptide provided by the invention has good antigenicity, and the antigen (immunogen) prepared by the specific LP-PLA2 epitope peptide can be used for immunizing animals to generate monoclonal antibodies and polyclonal antibodies with high specificity. The antibody latex reagent prepared by the antibody has no cross reaction with other proteins in blood, has the same affinity to the Lp-PLA2 protein in a lipoprotein binding state and a non-lipoprotein binding state, and improves the detection specificity and accuracy; the stability is good. The kit provided by the invention is simple and convenient to operate, has short time consumption and avoids errors of manual operation by adopting a method for detecting the LP-PLA2 by using a full-automatic specific protein analyzer or a biochemical analyzer.
Description
Technical Field
The invention relates to the technical field of polypeptides and immunology, in particular to a specific LP-PLA2 epitope peptide, an antigen, an antibody, application and a kit.
Background
The principle of the method for clinically detecting the content of LP-PLA2 (lipoprotein-related phospholipase A2 in human body) is mainly immunological determination and enzymatic biochemical method. Enzymatic biochemistry is mainly to catalyze an additional substrate analogue by using lipoprotein-related phospholipase A2 with activity to generate a substance which can be detected, and calculate the activity of the lipoprotein-related phospholipase A2 by detecting the change amount of the substance. The method is convenient to operate, is less influenced by environmental factors, has high requirement on the solubility of the substrate, has short reagent storage time and is greatly interfered by other substances in the sample. The immunoturbidimetry is that the LP-PLA2 antibody (or monoclonal antibody) is coupled with latex particles, and the content of the LP-PLA2 in the sample is determined by adopting latex enhanced turbidimetry, so that the method has the advantages of no need of processing the sample, simple operation, good repeatability and capability of being determined on a full-automatic protein analyzer, a biochemical analyzer and a spectrophotometer.
The antibodies used in the current LP-PLA2 immunoturbidimetry kit are almost monoclonal antibodies (poly) generated by recombinant antigens, and the LP-PLA2 is combined with lipoproteins in the blood in a great part. However, epitope peptides recognized by monoclonal (poly) antibodies produced based on recombinant antigens may be covered, making these monoclonal (poly) antibodies unable to accurately recognize the native LP-PLA2 protein in blood, and environmental impact during testing may lead to inactivation of enzymes in the sample, resulting in unstable accuracy.
Accordingly, there is still a need in the art for further improvements and enhancements.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention provides a specific LP-PLA2 epitope peptide, an LP-PLA2 specific antigen prepared by using the epitope peptide, a corresponding monoclonal antibody or polyclonal antibody, application of the antigen in preparing an LP-PLA2 in-vitro diagnosis kit and a kit for detecting the content of LP-PLA2 in human serum.
Specifically, the invention provides the following technical scheme:
in a first aspect, a specific LP-PLA2 epitope peptide, wherein the specific LP-PLA2 epitope peptide is: as set forth in SEQ ID NO: 1-3, and a polypeptide shown in any one of amino acid sequences.
In a second aspect, a specific LP-PLA2 epitope peptide, wherein the specific LP-PLA2 epitope peptide is: any two polypeptides shown in SEQ ID NO. 1-3 amino acid sequences are combined.
In the invention, the conservation of human LP-PLA2 in human beings, rabbits and mice is analyzed according to Blast software on NCBI by using a biological information method. And then comprehensively predicting and analyzing the epitope of the human LP-PLA2 by using software such as Kolaskar & Tongaonkar prediction algorithm tools, IEDB Antibody Epitope Prediction, ABCpred Prediction Server, bcePred Prediction Server, bepippred 1.0b Server, DNASTAR and the like, removing signal peptide, glycosylation site, lipoprotein site and polymorphism site of the LP-PLA2, and then comprehensively predicting and analyzing the polypeptide segment with high specificity, which is verified by experiments. Illustratively, in the present invention, the selected polypeptide fragments are as follows:
Peptide 1：79-91(13aa)
the sequence is as follows: 79TFLRLYYPSQDND 91
Peptide 2：133-145(13aa)
The sequence is as follows: 133NWNSPLRPGEKYP 145
Peptide 3：330-342(13aa)
The sequence is as follows: 330KMKKCYSPDKERK 342.
In a third aspect, a LP-PLA2 antigen, wherein the LP-PLA2 antigen comprises the specific LP-PLA2 epitope peptide of the first aspect.
In a fourth aspect, an LP-PLA2 antibody, wherein the LP-PLA2 antibody is a monoclonal or polyclonal antibody made from the LP-PLA2 antigen of the third aspect.
In a fifth aspect, the use of an antibody to LP-PLA2 in the preparation of an LP-PLA2 in vitro diagnostic kit.
In a sixth aspect, a kit for detecting the level of LP-PLA2 in human serum, comprising: sample diluent, buffer solution, antibody latex reagent and calibrator; the antibody in the antibody latex reagent is the antibody of the fifth aspect.
According to the application of the LP-PLA2 immunoturbidimetry detection kit provided by the invention, particularly, the kit is adopted, and the method for detecting the LP-PLA2 by using the full-automatic specific protein analyzer or the biochemical analyzer is simple and convenient to operate, short in time consumption and capable of avoiding errors of manual operation.
As a preferred embodiment, the kit, wherein the sample diluent comprises a sample dissociating agent, a preservative, and an antifoaming agent; the sample dissociating agent is selected from one or more of Tris, EDTA and sodium chloride.
As a preferred technical scheme, the kit is characterized in that the preservative is one or more selected from sodium benzoate, nitrate, nitrite and sulfur dioxide; the defoaming agent is one or more selected from organosilicon defoaming agents, surfactant defoaming agents and mineral oil defoaming agents.
As a preferred technical scheme, the kit, wherein the buffer solution is a buffer solution with a PH of 6.5-7.5, mainly comprises: inorganic salt, accelerator, preservative and defoamer, wherein the inorganic salt can be one or more of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride; the accelerator comprises one or more of triton-X100 and tween-20; the preservative comprises one or more of sodium sorbate, sodium benzoate, sodium azide, sodium nitrite and Proclin 300; the defoaming agent comprises one or more of organic silicon defoaming agents, surfactant defoaming agents and mineral oil defoaming agents.
As a preferred embodiment, the kit, wherein the antibody latex reagent comprises: one or more of lipoprotein-resistant anti-human LP-PLA2 monoclonal antibody latex particles, stabilizers (irrelevant proteins, inorganic salts, surfactants, suspending agents, antioxidants), preservatives (sodium sorbate, sodium benzoate, sodium azide, sodium nitrite), defoamers, silicone defoamers, surfactant defoamers and mineral oil defoamers.
As a preferred technical scheme, the kit, wherein the preparation method of the antibody latex reagent comprises the following steps:
adding the carboxyl microsphere into HEPES solution to obtain emulsion solution;
dispersing the LP-PLA2 antibody into the emulsion solution, and carrying out concussion reaction to obtain a precursor solution;
dispersing carbodiimide hydrochloride solution into the precursor liquid, and carrying out concussion reaction to obtain a reaction product;
and (3) purifying and resuspending the reaction product to obtain the antibody emulsion reagent.
As a preferable technical scheme, the kit is characterized in that the reaction temperature of the concussion reaction is 35-40 ℃ and the reaction time is 30-60min.
The beneficial effects are that: compared with the prior art, the specific LP-PLA2 epitope peptide provided by the invention has good antigenicity, and the antigen (immunogen) prepared by the specific LP-PLA2 epitope peptide can be used for immunizing animals to generate monoclonal antibodies and polyclonal antibodies with high specificity. The antibody latex reagent prepared by the antibody has no cross reaction with other proteins in blood, has the same affinity to the Lp-PLA2 protein in a lipoprotein binding state and a non-lipoprotein binding state, and improves the detection specificity and accuracy; the stability is good. The kit provided by the invention is simple and convenient to operate, has short time consumption and avoids errors of manual operation by adopting a method for detecting the LP-PLA2 by using a full-automatic specific protein analyzer or a biochemical analyzer.
Drawings
FIG. 1 is a graph showing the correlation between the detection results of the kit and the ELISA kit according to the embodiment of the invention.
Detailed Description
The invention provides specific LP-PLA2 epitope peptides, antigens, antibodies, applications and kits, and the invention is further described in detail below for the purpose, technical scheme and effect of the invention to be clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The reagent and the material adopted by the invention are all common commercial products and can be purchased in the market.
The invention will be further illustrated by the following examples
Example 1
The kit mainly comprises the following components: sample diluent R1, latex reaction buffer R2, latex reaction solution R3 (i.e., antibody latex reagent). The solution of R1 contains sodium chloride, nitrite and surfactant defoamer. The specific configuration process of R1 is as follows:
adding quantitative purified water into a beaker, accurately weighing 9g of sodium chloride, and weighing 0.05ml of Tween-20 by using a pipette; the pH was adjusted to 7.5 with stirring and finally the volume was set to 1000ml with a volumetric flask.
The solution of R2 contains disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, bovine serum albumin, preservative proclin300 and purified water
The specific configuration process of R3 is as follows:
a fixed amount of purified water was added to a beaker, 5.6g of disodium hydrogen phosphate dodecahydrate, 0.572g of sodium dihydrogen phosphate dihydrate, 8.46g of sodium chloride, 19.302g of bovine serum albumin were accurately weighed into the beaker, 0.965ml proclin300 g of bovine serum albumin was added to the beaker, pH was adjusted to 7.5 with stirring, and finally the volume was set to 1000ml with a volumetric flask.
The preparation process of the latex reaction particle solution R3 comprises the following steps:
9.8ml of 50mM HEPES solution (pH 6.0) was added to the clean centrifuge tube, and 0.25ml of the above solution was added thereto, according to the instructions for use of the latex microspheres; the JSR P0113 carboxyl microsphere is uniformly mixed. 2.0mg of the Lp-PLA2 monoclonal antibodies of the invention are respectively taken and added into the latex solution under the shaking condition after being uniformly mixed, and the latex solution is subjected to shaking reaction for 30min at 37 ℃. 125ul of 10mg/ml carbodiimide hydrochloride solution is added into the reaction system, and the shaking reaction is continued for 60min at 37 ℃. After the reaction was completed, unbound antibody was removed from the supernatant by high-speed centrifugation at 15000rpm for 30min, the latex was resuspended with 1% bovine serum albumin solution, and the shaking blocking reaction was continued at 37℃for 60min. After the reaction, the mixture was centrifuged at 15000rpm for 30min, and the latex precipitate was resuspended in 25ml of a latex preservation solution (preservation solution formulation: 100mM Tris, 1% NaCl, 0.5% BSA, 3% trehalose, 0.1% proclin 300), and the resuspension was sonicated for 5min to obtain a latex particle solution R3.
Example 2 comparison of clinical examples of Lp-PLA2 detection kit
This example was used to evaluate the performance of the recombinant single chain antibody-based LP-PLA2 latex enhanced turbidimetry assay kit of the above examples.
1. Determination of sensitivity
The kit provided by the invention is used for repeatedly measuring a blank sample (normal saline) for 20 times, and the minimum detection limit is the mean value concentration of the blank plus 2 standard deviations, so that the minimum detection limit is 4.02ng/mL, and the specific table is shown in Table 1.
TABLE 1 minimum detection limit measurement results table of the kit of the present invention
2. Comparison correlation of clinical examples of Lp-PLA2 detection kit
The correlation between the kit of the present invention and the ELISA kit was verified by measuring 100 samples (59 positive serum samples and 41 negative serum samples), and the detection results are shown in Table 2. The detection result is plotted as a graph. As can be seen from fig. 1, the correlation coefficient r2= 0.9979 between the kit of the present invention and the kit of the enzyme-linked immunosorbent assay method is as follows: y=0.994x+ 0.3272. The Lp-PLA2 latex turbidimetric kit prepared based on the monoclonal antibody can accurately detect the content of the Lp-PLA2, provides a basis for diagnosis of atherosclerosis, and can fully meet the clinical in-vitro diagnosis and detection requirements.
Table 2 comparison table of the test results of the kit of the present invention and the enzyme-linked immunosorbent assay kit
The correlation chart of the detection results of the kit and the ELISA method kit is shown in figure 1.
Example 3
Stability and repeatability analysis of Lp-PLA2 detection kit
Long-term stability: the Lp-PLA2 detection kit is stored under the refrigerating condition of 2-8 ℃, clinical samples frozen at the temperature of-20 ℃ are detected respectively in the examination time of 0, 1, 3, 6, 9, 12 and 14 months, and the result shows that the detection result is relatively changed within +/-10% after the kit is placed for 12 months, which indicates that the long-term stability of the kit is more than 12 months.
Acceleration stability: the Lp-PLA2 detection kit is subjected to an acceleration test at 37 ℃ to simulate long-term stability, clinical samples frozen at-20 ℃ are detected on days 0, 1, 3, 5, 8 and 12, and the result shows that the relative change is within +/-10% after acceleration for 12 days at 37 ℃.
Repeatability: the high and low quality control products are repeatedly tested for 10 times in a single operation, the variation coefficient at the high and low level is calculated, and the result shows that the variation coefficient CV is less than 3%.
Interference resistance: selecting clinical samples near a reference value as basic samples, and dividing the basic samples into 4 parts, wherein the 1 st part of samples are not treated and serve as blank control group samples; adding solvent into the 2 nd sample to prepare a solvent control group sample; storage solutions of potential interfering substances were added to the 3 rd and 4 th samples to prepare test group samples. Wherein C1 is a blank control sample, C2 is a solvent control sample, and T1 and T2 are test group samples. And repeatedly measuring each sample, recording the average value of the test results, and calculating the interference rate of the potential interference substances or the cross substances according to the formulas (1) and (2). The allowable concentration of common interfering substances is analyzed, and the result shows that the kit has better anti-interference capability.
TABLE 3 Long-term stability examination of the kit of the invention
TABLE 4 accelerated stability testing of the kits of the invention
TABLE 5 results of the reproducibility test of the kit of the invention
TABLE 6 influence of interferents on kits
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In conclusion, the kit provided by the invention has higher detection sensitivity, can accurately detect the Lp-PLA2 content, provides a basis for atherosclerosis diagnosis, and can fully meet the clinical in-vitro diagnosis detection requirement. Meanwhile, the kit has the advantages of high stability and good repeatability.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (10)
1. A specific LP-PLA2 epitope peptide, wherein the specific LP-PLA2 epitope peptide is: as set forth in SEQ ID NO: 1-3, and a polypeptide shown in any one of amino acid sequences.
2. A specific LP-PLA2 epitope peptide, wherein the specific LP-PLA2 epitope peptide is: any two polypeptides shown in SEQ ID NO. 1-3 amino acid sequences are combined.
3. An LP-PLA2 antigen, wherein the LP-PLA2 antigen comprises the specific LP-PLA2 epitope peptide of claim 1.
4. An LP-PLA2 antibody, wherein the LP-PLA2 antibody is a monoclonal or polyclonal antibody prepared from the LP-PLA2 antigen of claim 3.
5. The use of the LP-PLA2 antibody of claim 4, in the preparation of an LP-PLA2 in vitro diagnostic kit.
6. A kit for detecting LP-PLA2 content in human serum, comprising: sample diluent, buffer solution, antibody latex reagent and calibrator; the antibody in the antibody emulsion reagent is the antibody of claim 4.
7. The kit of claim 6, wherein the sample diluent comprises a sample dissociating agent, a preservative, and an antifoaming agent; the sample dissociating agent is selected from one or more of Tris, EDTA and sodium chloride.
8. The kit of claim 7, wherein the preservative is selected from one or more of sodium benzoate, nitrate, nitrite and sulfur dioxide; the defoamer is selected from one or more of silicone defoamer, surfactant defoamer and mineral oil defoamer.
9. The kit of claim 6, wherein the method for preparing the antibody latex reagent comprises:
adding the carboxyl microsphere into HEPES solution to obtain emulsion solution;
dispersing the LP-PLA2 antibody into the emulsion solution, and carrying out concussion reaction to obtain a precursor solution;
dispersing carbodiimide hydrochloride solution into the precursor liquid, and carrying out concussion reaction to obtain a reaction product;
and (3) purifying and resuspending the reaction product to obtain the antibody emulsion reagent.
10. The kit according to claim 9, wherein the reaction temperature of the shaking reaction is 35-40 ℃ and the reaction time is 30-60min.
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