CN110716057A - Kit for determining HBP (hepatitis B protein) based on latex enhanced immunoturbidimetry and preparation and use methods thereof - Google Patents
Kit for determining HBP (hepatitis B protein) based on latex enhanced immunoturbidimetry and preparation and use methods thereof Download PDFInfo
- Publication number
- CN110716057A CN110716057A CN201911069850.6A CN201911069850A CN110716057A CN 110716057 A CN110716057 A CN 110716057A CN 201911069850 A CN201911069850 A CN 201911069850A CN 110716057 A CN110716057 A CN 110716057A
- Authority
- CN
- China
- Prior art keywords
- latex
- hbp
- buffer solution
- kit
- reagent
- 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.)
- Pending
Links
- 239000004816 latex Substances 0.000 title claims abstract description 189
- 229920000126 latex Polymers 0.000 title claims abstract description 189
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 102000004169 proteins and genes Human genes 0.000 title abstract description 5
- 108090000623 proteins and genes Proteins 0.000 title abstract description 5
- 208000002672 hepatitis B Diseases 0.000 title abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 69
- 239000004005 microsphere Substances 0.000 claims abstract description 63
- 239000000243 solution Substances 0.000 claims abstract description 51
- 239000007853 buffer solution Substances 0.000 claims abstract description 36
- 230000008878 coupling Effects 0.000 claims abstract description 32
- 238000010168 coupling process Methods 0.000 claims abstract description 32
- 238000005859 coupling reaction Methods 0.000 claims abstract description 32
- 239000003755 preservative agent Substances 0.000 claims abstract description 31
- 230000002335 preservative effect Effects 0.000 claims abstract description 31
- 239000000203 mixture Substances 0.000 claims abstract description 25
- 239000011535 reaction buffer Substances 0.000 claims abstract description 25
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 14
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 9
- 239000003381 stabilizer Substances 0.000 claims abstract description 9
- 239000004094 surface-active agent Substances 0.000 claims abstract description 9
- 230000003213 activating effect Effects 0.000 claims description 21
- 230000000903 blocking effect Effects 0.000 claims description 15
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 12
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 12
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 11
- 239000008213 purified water Substances 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 238000005259 measurement Methods 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 10
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 9
- 239000011534 wash buffer Substances 0.000 claims description 8
- 238000002835 absorbance Methods 0.000 claims description 7
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical group CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 239000004471 Glycine Substances 0.000 claims description 6
- 239000002202 Polyethylene glycol Substances 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 6
- 229920001223 polyethylene glycol Polymers 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 235000002639 sodium chloride Nutrition 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 6
- 229940033663 thimerosal Drugs 0.000 claims description 6
- 239000011592 zinc chloride Substances 0.000 claims description 6
- 235000005074 zinc chloride Nutrition 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 5
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- 239000013504 Triton X-100 Substances 0.000 claims description 4
- 229920004890 Triton X-100 Polymers 0.000 claims description 4
- 235000011148 calcium chloride Nutrition 0.000 claims description 4
- 235000011147 magnesium chloride Nutrition 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 4
- 229920000053 polysorbate 80 Polymers 0.000 claims description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 239000003761 preservation solution Substances 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 239000004353 Polyethylene glycol 8000 Substances 0.000 claims description 2
- 239000003623 enhancer Substances 0.000 claims description 2
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 229940113116 polyethylene glycol 1000 Drugs 0.000 claims description 2
- 229940093429 polyethylene glycol 6000 Drugs 0.000 claims description 2
- 229940085678 polyethylene glycol 8000 Drugs 0.000 claims description 2
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 claims description 2
- 239000011550 stock solution Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 25
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 206010040047 Sepsis Diseases 0.000 description 12
- 230000035939 shock Effects 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 238000003908 quality control method Methods 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 208000013223 septicemia Diseases 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 102100030009 Azurocidin Human genes 0.000 description 4
- 101710154607 Azurocidin Proteins 0.000 description 4
- 206010040070 Septic Shock Diseases 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 238000012417 linear regression Methods 0.000 description 4
- 230000036303 septic shock Effects 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 208000019206 urinary tract infection Diseases 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 239000007987 MES buffer Substances 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 206010027202 Meningitis bacterial Diseases 0.000 description 1
- 206010021888 Nervous system infections Diseases 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010051379 Systemic Inflammatory Response Syndrome Diseases 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 201000009904 bacterial meningitis Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 102000022382 heparin binding proteins Human genes 0.000 description 1
- 108091012216 heparin binding proteins Proteins 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000008728 vascular permeability Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- 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
- G01N33/557—Immunoassay; Biospecific binding assay; Materials therefor using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction
-
- 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
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Abstract
The invention discloses a kit for measuring HBP (hepatitis B protein) based on a latex enhanced immunoturbidimetry, which comprises a reaction buffer solution R1 and a latex reagent R2; the reaction buffer solution R1 comprises buffer solution, inorganic salt, turbidity-increasing agent, preservative, stabilizer and surfactant; the latex reagent R2 comprises at least two latex reagent composition solutions formed by coupling HBP monoclonal antibody and latex microspheres, and each composition solution comprises latex preservative solution and corresponding HBP monoclonal antibody-labeled latex microspheres preserved in the latex preservative solution; wherein, the mixing ratio of each component liquid is 1: 1. The invention also discloses a preparation and application method of the kit for measuring HBP based on the latex enhanced immunoturbidimetry. The invention can be used on a full-automatic biochemical analyzer, has simple operation, low cost and high automation and saves the detection time; and under the conditions of high stability and high precision, compared with other products, the invention has higher sensitivity and specificity.
Description
Technical Field
The invention relates to the field of preparation of biological detection reagents, in particular to a kit for determining HBP based on a latex enhanced immunoturbidimetry method and a preparation and use method thereof.
Background
Heparin-binding protein (HBP) is a granulocyte-derived granule protein, which has a structure comprising a single-chain protein of 222 amino acids, contains 8 cysteine residues, has a glycosylation site at the 100 th, 114 th or 145 th aspartic acid residue, has a structure similar to that of neutrophil elastin, and has 45% homology thereto.
HBP is mainly released by PMN under external stimulation, so the HBP content in normal human blood is very low and generally does not exceed 10 ng/mL; when infection occurs, part of bacteria invade into blood vessels, and substances such as thalli or toxins released by the bacteria stimulate fine-particle fine packets to release HBP, so that the content of HBP in blood is increased. HBP can reach 20-30ng/mL in normal infection, and severe infection in ICU is higher and may exceed 100 ng/mL. The clinical specific application of HBP is as follows:
1) HBP as a diagnostic indicator of local infection: when the HBP level of cerebrospinal fluid exceeds 20ng/mL, 100% sensitivity, 99.2% specificity, 96.2% positive predictive value and 100% negative predictive value can be achieved for diagnosing acute bacterial meningitis. Thus, HBP levels in the cerebrospinal fluid can improve the accuracy of distinguishing between bacterial and viral nervous system infections.
2) HBP aids in the diagnosis of urinary tract infections: the sensitivity and specificity of U-HBP for judging urinary tract infection are 93.3% and 90.3% respectively, and meanwhile, the negative predictive value also reaches 98.3%, but the positive predictive value of U-HBP is not ideal and only can reach 70%, and the U-HBP is a good biochemical index when combined with nitrite.
3) Prediction of the onset of septic shock by HBP: comparing the serum HBP level of healthy people (control group) and patients without shock (including patients with local infection such as urinary tract infection, pneumonia, gastroenteritis and the like but without shock), the serum HBP level between the groups is not significantly different, but the serum HBP level between patients without septic shock and patients with non-septic shock is significantly different; thus, the level of HBP in the blood of infected patients is not necessarily significantly elevated, but in shock patients the level of HBP must be significantly elevated, a cause for this phenomenon not to be indistinguishable from the altered vascular permeability of HBP.
4) Prediction of circulating failure in sepsis by HBP: the study on HBP, PCT, IL-6, CRP levels and WBC in 5 groups of blood infected but not infected with shock severe septicemia, sepsis, severe septicemia not accompanied with shock, systemic inflammatory response syndrome but not infected with shock revealed that HBP levels in patients with severe septicemia accompanied with shock were significantly higher than those in patients with severe septicemia not accompanied with shock, and HBP levels in these two groups of patients were significantly higher than those in the other three groups of patients. At present, HBP is clinically used as a marker of severe septicemia, and the sensitivity, specificity, positive predictive value and negative predictive value of HBP can respectively reach 87.1%, 95.1%, 88.4% and 94.5%.
5) Recent studies have found that heparin-binding proteins can be an emerging early biomarker for diagnosing severe sepsis and septic shock, and can predict the impending shock of severely infected patients. The levels of PCT, HBP and blood lactic acid in blood are closely related to the severity of the disease of a patient suffering from sepsis, the diagnosis efficiency of the sepsis can be improved by jointly detecting the PCT, the HBP and the blood lactic acid, the sepsis caused by infection of different pathogenic bacteria can be identified, and the method has important significance for early diagnosis and disease evaluation of the patient suffering from the sepsis.
The existing detection method of heparin binding protein HBP mainly adopts an immunological method, and comprises an immunofluorescence chromatography method, an enzyme-linked immunosorbent assay and the like. The immunofluorescence chromatography can realize rapid detection of HBP, but can only carry out qualitative or semi-quantitative detection, has low accuracy and cannot provide accurate clinical judgment basis; meanwhile, the method is limited by itself, and the detection precision is poor, so that the repeatability and consistency of the test cannot be guaranteed. Although the ELISA can accurately detect the concentration of HBP in a sample, the detection is relatively complicated and time-consuming, and the requirement of rapid detection in outpatient or emergency is not met.
The latex boosting immune ratio method is a detection method for dynamically measuring antigen-antibody combination: in a specific dilution system, antigen and antibody are combined, and when the combination proportion is proper, particles are formed and are separated out from a liquid phase; before and after the antigen and antibody are combined, turbidity changes occur; the turbidity change is detected by a full-automatic biochemical analyzer, and a linear curve is drawn by using a standard substance, so that the content of the substance to be detected in the corresponding sample can be obtained. The method does not need special instruments, and is simple and convenient to operate. In addition, the latex enhanced immunoturbidimetry can enhance the absorbance of reaction by using a latex carrier, so that the sensitivity of detection is greatly improved, the detection is realized automatically by a full-automatic biochemical analyzer, the detection is more convenient and rapid, the time is saved, and the requirement of clinical large sample detection can be met.
Disclosure of Invention
The invention mainly aims to provide a kit for measuring HBP based on a latex enhanced immunoturbidimetry and a preparation and use method thereof, and aims to solve the problems of complex operation, long detection time, poor precision, poor repeatability and consistency of the existing HBP detection method.
In order to achieve the purpose, the invention provides a kit for measuring HBP based on latex enhanced immunoturbidimetry, which comprises a reaction buffer solution R1 and a latex reagent R2;
the reaction buffer R1 comprises the following components in parts by weight: 0.5-5g/L of buffer solution, 10-25g/L of inorganic salt, 15-40g/L of turbidity enhancer, 0.5-2mL/L of preservative, 25-50g/L of stabilizer, 0.5-4mL/L of surfactant and purified water as solvent;
the latex reagent R2 comprises at least two latex reagent constitutional solutions formed by coupling HBP monoclonal antibody and latex microspheres, and each latex reagent constitutional solution comprises a latex preservative solution and corresponding HBP monoclonal antibody-labeled latex microspheres which are preserved in the latex preservative solution and have a final concentration of 0.8-1.0m g/mL; the latex preservation solution comprises the following components in percentage by weight: 5-20g/L of buffer solution, 4-15g/L of inorganic salt, 20-90g/L of stabilizing agent, 0.2-1.0mL/L of preservative, 1-5mL/L of surfactant and purified water as solvent; in the latex reagent R2, the mixing ratio of each latex reagent composition liquid formed by coupling HBP monoclonal antibody and latex microspheres is 1: 1.
In a preferred embodiment of the present invention, in the reaction buffer R1:
the buffer solution is one of a tris buffer solution, a glycine buffer solution, a2- (N-morpholine) ethanesulfonic acid buffer solution (MES buffer solution) and a 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution (MOPSO buffer solution);
the inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride, zinc chloride and calcium chloride;
the turbidity increasing agent is one or more of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000 and polyethylene glycol 8000;
the preservative is one or more of Proclin300 and thimerosal;
the stabilizer is one of BSA and sucrose;
the surfactant is one or more of Tween-20, Tween-80 and Triton X-100.
In a preferred embodiment of the present invention, the reaction buffer R1 has a pH of 7.3 to 7.7.
In a preferred embodiment of the present invention, in the latex preservative solution:
the buffer solution is 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution;
the inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride, zinc chloride and calcium chloride;
the stabilizer is one or more of BSA, sucrose and mannitol;
the preservative is one or more of Proclin300 and thimerosal;
the surfactant is one or more of Tween-20, Tween-80 and Triton X-100.
In a preferred embodiment of the present invention, the latex preservative solution has a pH of 7.4 to 7.7.
In a preferred embodiment of the present invention, the latex microspheres used in the HBP mab-labeled latex microspheres have a diameter of 100-300 nm.
In a preferred embodiment of the present invention, the latex reagent R2 is prepared by a method comprising:
① adding latex microsphere with solid content of 10% into washing buffer solution, centrifuging, removing supernatant, ultrasonic resuspending the latex microsphere precipitate with activating buffer solution, activating with activating agent, centrifuging again, and ultrasonic resuspending the latex microsphere precipitate with coupling buffer solution to obtain activated latex microsphere;
②, at least two HBP monoclonal antibodies are taken and respectively mixed with the prepared activated latex microspheres according to a specific proportion, coupling is carried out for a certain time, and the coupled latex microspheres are prepared, wherein the v/w ratio of the activated latex microspheres to each HBP monoclonal antibody is respectively 1 (5-6), and the coupling time is 2 h;
③ adding blocking buffer solution into the coupled latex microspheres for blocking, centrifuging again, removing supernatant, adding latex preservative solution, resuspending and ultrasonically treating to obtain latex reagent composition liquid, and mixing the latex reagent composition liquids to obtain the required latex reagent R2.
The preparation method of the kit for measuring HBP based on latex enhanced immunoturbidimetry comprises the following steps:
(1) preparing a reaction buffer R1:
according to the component content of the reaction buffer solution R1, mixing all the components in the same container, and uniformly mixing to obtain a reaction buffer solution R1;
(2) formulation of latex reagent R2:
① adding latex microsphere with solid content of 10% into washing buffer solution, centrifuging, removing supernatant, ultrasonic resuspending the latex microsphere precipitate with activating buffer solution, activating with activating agent, centrifuging again, and ultrasonic resuspending the latex microsphere precipitate with coupling buffer solution to obtain activated latex microsphere;
②, at least two HBP monoclonal antibodies are taken and respectively mixed with the prepared activated latex microspheres according to a specific proportion, coupling is carried out for a certain time, and the coupled latex microspheres are prepared, wherein the v/w ratio of the activated latex microspheres to each HBP monoclonal antibody is respectively 1 (5-6), and the coupling time is 2 h;
③ adding blocking buffer solution into the coupled latex microspheres for blocking, centrifuging again, removing supernatant, adding latex preservative solution, resuspending and ultrasonically treating to obtain latex reagent composition liquid, and mixing the latex reagent composition liquids to obtain the required latex reagent R2.
In a preferred embodiment of the present invention, in step ①, the washing buffer, the activating buffer and the coupling buffer are phosphate buffers, the activating agent is EDAC/NHS, the ratio of the corresponding latex particles to the activating agent is 1 (10-20) (weight ratio), the blocking buffer is one or more of BSA and glycine, and the blocking time is 1 h.
The use method of the kit for measuring HBP based on latex enhanced immunoturbidimetry comprises the following specific steps:
(1) pipetting 20. mu.L of the sample, adding 240. mu.L of reagent R1, and incubating at 37 ℃ for 5 min;
(2) adding 60 μ L reagent R2, and incubating at 37 deg.C;
(3) and (3) after incubation for 30s, reading the absorbance A1, incubating for 4.5min, reading the absorbance A2, finally calculating delta A, wherein △ A is A2-A1, and calculating the HBP content in the sample according to the delta A.
As one of the preferred embodiments of the present invention, each of the HBP monoclonal antibodies used in the present invention is derived from a conventional HBP monoclonal antibody which is commercially available in the art.
As one of the preferred embodiments of the present invention, the reaction principle of the kit of the present invention:
the heparin binding protein in the sample and the anti-human heparin binding protein monoclonal antibody latex particles have antigen-antibody reaction, the absorbance of the latex particles is detected under specific wavelength, and the change degree of the absorbance is in direct proportion to the content of HBP in the sample.
Compared with the prior art, the invention has the advantages that:
(1) the invention can be used on a full-automatic biochemical analyzer, has low cost and high automation, can save the detection time and is convenient for clinical application;
(2) compared with other methods, the kit disclosed by the invention utilizes a latex enhanced immunoturbidimetry to measure HBP, and has the advantages of simplicity in operation, high detection speed, high precision, good repeatability and good consistency;
(3) the kit prepared by the method can simultaneously improve the sensitivity and specificity of detection, so that the kit has wider linear range and higher accuracy and better meets the clinical requirements;
(4) in the application of the latex enhanced immunoturbidimetry, if the polyclonal antibody is adopted for marking, the sensitivity of the reagent can be effectively improved, but the specificity is not strong, so that the accuracy of the detection result is reduced; if a monoclonal antibody is used for marking latex, the specificity can be improved, but the sensitivity is influenced; aiming at the problems, in the kit, at least two HBP monoclonal antibodies with stronger specificity are selected to mark latex respectively and then are mixed together; the method has the advantages that firstly, the situation that the specificity is not strong and the accuracy of a detection result is reduced due to multi-resistance is avoided, the accuracy of the reagent prepared by the method is high when the performance is evaluated, and the relative deviation can be controlled within 5% and is far lower than the requirements of 10-15% of other detection methods; secondly, the requirement of low sensitivity caused by a monoclonal antibody is avoided, the reagent prepared by the method has the linear high end reaching 300ng/mL and the low end detection reaching 2ng/mL in performance evaluation, and the linear correlation coefficient R2 is more than 0.99 in the performance evaluation of the reagent.
Drawings
FIG. 1 is a linear-range linear regression plot of the kit for the latex-enhanced immunoturbidimetry-based determination of HBP in example 6.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
This example is a kit for measuring HBP based on latex enhanced immunoturbidimetry, comprising reaction buffer R1 and latex reagent R2.
The reaction buffer R1 comprises the following components in parts by weight: 0.5g/L of tris buffer solution, 10g/L of sodium chloride, 100015 g/L of polyethylene glycol, 3000.5 mL/L of Proclin, 25g/L of BSA, 200.5 mL/L of Tween, and purified water as a solvent, wherein the pH value is 7.3.
The latex reagent R2 comprises two latex reagent compositions formed by coupling HBP monoclonal antibody and latex microspheres, and each latex reagent composition comprises a latex preservative solution and corresponding HBP monoclonal antibody-labeled latex microspheres which are preserved in the latex preservative solution and have a final concentration of 0.8m g/mL. Wherein, the latex preservative fluid comprises the following components in percentage by weight: 5g/L MOPSO buffer solution, 4g/L sodium chloride, 20g/L BSA, 3000.2mL/L proclin, 201 mL/L Tween-5, purified water as solvent, and pH 7.4.
Further, in the latex reagent R2, the mixing ratio of each latex reagent composition liquid obtained by coupling the HBP mab and the latex microspheres was 1: 1.
Further, in the latex reagent R2, the latex microspheres used were 100nm in diameter.
Example 2
This example is a kit for measuring HBP based on latex enhanced immunoturbidimetry, comprising reaction buffer R1 and latex reagent R2.
The reaction buffer R1 comprises the following components in parts by weight: 5g/L of glycine buffer solution or MES buffer solution, 25g/L of magnesium chloride or potassium chloride, and a turbidity-increasing agent (polyethylene glycol 2000 or polyethylene glycol 600040 g/L, 2mL/L of thimerosal, 50g/L of sucrose, and 804 mL/L of tween-804, wherein the solvent is purified water and the pH is 7.7.
The latex reagent R2 comprises three latex reagent components formed by coupling HBP monoclonal antibody and latex microspheres, and each latex reagent component comprises a latex preservative solution and corresponding HBP monoclonal antibody-labeled latex microspheres which are preserved in the latex preservative solution and have the final concentration of 1.0m g/mL. Wherein, the latex preservative fluid comprises the following components in percentage by weight: 20g/L of MOPSO buffer solution, 15g/L of magnesium chloride or potassium chloride, 90g/L of sucrose, 1.0mL/L of thimerosal, 805 mL/L of tween-water and 7.7 of pH value, wherein the solvent is purified water.
Further, in the latex reagent R2, the mixing ratio of each latex reagent composition liquid obtained by coupling the HBP mab and the latex microspheres was 1:1: 1.
Further, in the latex reagent R2, the latex microspheres used were 300nm in diameter.
Example 3
This example is a kit for measuring HBP based on latex enhanced immunoturbidimetry, comprising reaction buffer R1 and latex reagent R2.
The reaction buffer R1 comprises the following components in parts by weight: 2.56g/L of MOPSO buffer solution, 15.4g/L of zinc chloride or calcium chloride, 800025 g/L of polyethylene glycol, 300 g/L of Proclin, 0.8mL/L of BSA, 36g/L of triton X-1001.5mL/L of solvent, purified water and pH 7.5.
The latex reagent R2 comprises two latex reagent compositions formed by coupling HBP monoclonal antibody and latex microspheres, and each latex reagent composition comprises a latex preservative solution and corresponding HBP monoclonal antibody-labeled latex microspheres which are preserved in the latex preservative solution and have a final concentration of 0.9m g/mL. Wherein, the latex preservative fluid comprises the following components in percentage by weight: 11.2g/L of MOPSO buffer solution, 8.9g/L of zinc chloride or calcium chloride, 20-90g/L of mannitol, 3000.5 mL/L of Proclin, X-1003.5mL/L of triton, purified water as a solvent and pH 7.6.
Further, in the latex reagent R2, the mixing ratio of each latex reagent composition liquid obtained by coupling the HBP mab and the latex microspheres was 1: 1.
Further, in latex reagent R2, the latex microspheres used were 150nm in diameter.
Example 4
This example is a method for preparing the kit for measuring HBP based on latex enhanced immunoturbidimetry in examples 1-3 above, comprising the following steps:
(1) preparing a reaction buffer R1:
according to the component content of the reaction buffer solution R1, all the components are mixed in the same container, and after being uniformly mixed, the mixture is filtered by a 0.22um filter membrane to prepare the reaction buffer solution R1.
(2) Formulation of latex reagent R2:
cleaning: taking 0.5mL of latex microspheres with the solid content of 10%, and adding the latex microspheres into 3.0mL of washing buffer solution; centrifuging at 20000r/min for 30min, and removing supernatant; the latex microsphere pellet was sonicated with 3.0mL of activation buffer to resuspend the pellet. In the step, phosphate buffer is used as the washing buffer and the activation buffer.
And (3) activation: weighing a certain amount of activating agent, adding the activating agent into the cleaned latex particles, uniformly mixing, and activating the latex particles; and centrifuging again, and carrying out resuspension ultrasonic treatment on the latex microsphere precipitate by using a coupling buffer solution to prepare the activated latex microspheres. In the step, phosphate buffer is adopted as coupling buffer; the activator adopts 0.1mL of 2 percent EDAC or 0.2mL of 4 percent NHS, and the activation time is 0.5 h; the ratio of the corresponding latex particulates to activator was 1: (10-20) (weight ratio).
Coupling: coupling at least two screened HBP monoclonal antibodies with strong specificity with activated latex particles to prepare coupled latex microspheres. In the step, the v/w ratio of the activated latex microspheres to each HBP monoclonal antibody is respectively 1 (5-6), and the coupling time is 2 h.
And (3) sealing: after the coupling time is over, the centrifugation is started, the centrifugation is carried out for 30min at 20000r/min, and the supernatant is removed; subsequently, 5.0mL of blocking buffer was taken to sonicate the resuspension of the latex microparticle pellet, followed by blocking for 1 h. In this step, one or more of BSA and glycine, preferably 1% BSA, is used as the blocking buffer.
And (3) dissolving: centrifuging at 20000r/min for 30min, and removing supernatant; adding 40mL of latex preservation solution to carry out resuspension ultrasonic treatment on the latex particles, and finally carrying out fixed dissolution to prepare respective latex reagent composition solutions; finally, mixing the prepared latex reagent composition liquid according to the proportion of 1: mixing at a ratio of 1 to obtain the required latex reagent R2.
Example 5
This example is a method of using the kit for measuring HBP based on latex enhanced immunoturbidimetry as described in examples 1-3 above.
A detection instrument: hitachi 7180.
Temperature: 37 ℃ is carried out.
A cuvette: 1 cm.
The analysis method comprises the following steps: two-point endpoint method.
Light spot measurement: 19-34.
Primary and secondary wavelengths: 600/0.
Sample size/R1/R2: 20uL/240uL/60 uL.
The reaction direction is as follows: (+).
The method comprises the following steps: as shown in table 1.
TABLE 1 procedure for use of the kit of the invention
The calibration mode is a Spline function Spline; and establishing a working curve by adopting a multipoint calibration mode and using purified water as a zero point.
The calculation method is that a calibration curve is fitted to corresponding △ A according to the concentration of the calibrator, and the concentration value of the sample is obtained through the calibration curve.
Example 6
This example is intended to evaluate the kit for the determination of HBP based on latex-enhanced immunoturbidimetry in the above examples:
(1) linear correlation verification
Linear correlation coefficient: the high value specimens close to the upper limit of the linear interval were diluted with low value specimens close to the lower limit of the linear interval in the range of [2.0ng/mL-300.0ng/mL ], mixed into specimens of at least 5 different concentrations (Xi), each of which was repeatedly measured 3 times, and the mean value (yi) of the measurement results was calculated, respectively, as shown in Table 2. And (3) calculating a linear regression equation by taking the dilution concentration (Xi) as an independent variable and the measured result mean value (yi) as a dependent variable, and calculating a correlation coefficient r of linear regression according to a formula (1), wherein the obtained result is more than or equal to 0.9900.
TABLE 2 comparison of the Linear correlation of the kit of the invention
FIG. 1 is a linear range linear regression plot of a kit of the invention, as shown in FIG. 1The straight line fitting curve of (1) is: 0.9888x +0.4202, R2＝0.9999R2>0.99, meeting the clinical requirements.
(2) Precision and repeatability verification
Under the repeated condition, samples (quality control products, calibration products or other fixed value samples) with the concentrations of (15.0 +/-3.0) ng/mL and (100.0 +/-20.0) ng/mL are taken, the same batch of reagents are used for repeated measurement for 10 times, the average value and the standard deviation of the measured values are respectively calculated, the intra-batch variation Coefficient (CV) is calculated according to the formula (2), and the obtained result is that the CV is less than or equal to 8.0 percent.
In the formula: SD is standard deviation, and the calculation formula is
dn is the deviation of the measured values at the same level, and the calculation formula is
xn is measured at the same level for each time;
is an average value and is calculated by the formula
The results are shown in Table 3.
TABLE 3 summary table of the repeatability test results of the kit of the present invention
As can be seen from table 3, the CV of the low value sample is 6.38%, and the CV of the high value sample is 2.6%, both of which are less than 8%, and satisfy the clinical requirements.
(3) Accuracy verification
Taking a high-level quality control product and a low-level quality control product, measuring according to the operating steps of the specification, repeatedly measuring 3 times by using the same batch of reagent, recording the test result as (xi), calculating the relative deviation (Bi) according to the formula (3), wherein the 3 times of results all accord with the measured value and the corresponding deviation of the target value of the quality control product is less than or equal to 15.0 percent; if 2 times of results in3 times of results meet the requirements and 1 time of results do not meet the requirements, continuously testing for 20 times again, and respectively calculating the relative deviation (Bi) according to the formula (3), and if the results are more than or equal to 19 times of results meet the requirements, the accuracy is verified to meet the requirement that the relative deviation between the measured value and the target value of the quality control product is less than or equal to 15.0 percent.
In the formula: xi is the result of the determination;
t is the target value.
The results are shown in Table 4.
TABLE 4 summary table of accuracy test results of the kit of the present invention
As can be seen from Table 4, the relative deviation of the low-value quality control is 0.7%, and the relative deviation of the high-value quality control is 0.5%, which are both less than 15%, so that the clinical requirements are met.
In conclusion, the kit for determining HBP based on latex enhanced immunoturbidimetry and the preparation and use methods thereof provided by the invention can effectively improve the sensitivity and specificity of the HBP detection reagent. The kit for detecting HBP is prepared by adopting an innovative technology of coupling at least two monoclonal antibodies with strong specificity through the marked latex particles and then mixing the two monoclonal antibodies together, realizes automatic detection by using a full-automatic biochemical analyzer, effectively improves the sensitivity and specificity of the reagent, and has extremely high application value and wide market prospect.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A kit for measuring HBP based on latex enhanced immunoturbidimetry is characterized by comprising a reaction buffer R1 and a latex reagent R2;
the reaction buffer R1 comprises the following components in parts by weight: 0.5-5g/L of buffer solution, 10-25g/L of inorganic salt, 15-40g/L of turbidity enhancer, 0.5-2mL/L of preservative, 25-50g/L of stabilizer, 0.5-4mL/L of surfactant and purified water as solvent;
the latex reagent R2 comprises at least two latex reagent constitutional solutions formed by coupling HBP monoclonal antibody and latex microspheres, and each latex reagent constitutional solution comprises a latex preservative solution and corresponding HBP monoclonal antibody-labeled latex microspheres which are preserved in the latex preservative solution and have a final concentration of 0.8-1.0m g/mL; the latex preservation solution comprises the following components in percentage by weight: 5-20g/L of buffer solution, 4-15g/L of inorganic salt, 20-90g/L of stabilizing agent, 0.2-1.0mL/L of preservative, 1-5mL/L of surfactant and purified water as solvent; in the latex reagent R2, the mixing ratio of each latex reagent composition liquid formed by coupling HBP monoclonal antibody and latex microspheres is 1: 1.
2. The kit for the latex-enhanced immunoturbidimetry-based measurement of HBP according to claim 1, wherein in the reaction buffer R1:
the buffer solution is one of a tris buffer solution, a glycine buffer solution, a2- (N-morpholine) ethanesulfonic acid buffer solution and a 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution;
the inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride, zinc chloride and calcium chloride;
the turbidity increasing agent is one or more of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000 and polyethylene glycol 8000;
the preservative is one or more of Proclin300 and thimerosal;
the stabilizer is one of BSA and sucrose;
the surfactant is one or more of Tween-20, Tween-80 and Triton X-100.
3. The kit for the latex-enhanced immunoturbidimetry-based measurement of HBP according to claim 1, wherein the pH of the reaction buffer R1 is 7.3-7.7.
4. The kit for the latex-enhanced immunoturbidimetry-based measurement of HBP according to claim 1, wherein in the latex preservative solution:
the buffer solution is 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution;
the inorganic salt is one or more of sodium chloride, magnesium chloride, potassium chloride, zinc chloride and calcium chloride;
the stabilizer is one or more of BSA, sucrose and mannitol;
the preservative is one or more of Proclin300 and thimerosal;
the surfactant is one or more of Tween-20, Tween-80 and Triton X-100.
5. The kit for the latex-enhanced immunoturbidimetry-based measurement of HBP according to claim 1, wherein the pH of the latex stock solution is 7.4 to 7.7.
6. The kit for measuring HBP based on latex-enhanced immunoturbidimetry according to claim 1, wherein the latex microspheres used in the HBP monoclonal antibody-labeled latex microspheres have a diameter of 100-300 nm.
7. The kit for the latex-enhanced immunoturbidimetry-based measurement of HBP according to any one of claims 1 to 6, wherein the latex reagent R2 is prepared by:
① adding latex microsphere with solid content of 10% into washing buffer solution, centrifuging, removing supernatant, ultrasonic resuspending the latex microsphere precipitate with activating buffer solution, activating with activating agent, centrifuging again, and ultrasonic resuspending the latex microsphere precipitate with coupling buffer solution to obtain activated latex microsphere;
②, at least two HBP monoclonal antibodies are taken and respectively mixed with the prepared activated latex microspheres according to a specific proportion, coupling is carried out for a certain time, and the coupled latex microspheres are prepared, wherein the v/w ratio of the activated latex microspheres to each HBP monoclonal antibody is respectively 1 (5-6), and the coupling time is 2 h;
③ adding blocking buffer solution into the coupled latex microspheres for blocking, centrifuging again, removing supernatant, adding latex preservative solution, resuspending and ultrasonically treating to obtain latex reagent composition liquid, and mixing the latex reagent composition liquids to obtain the required latex reagent R2.
8. A method of preparing a kit for the latex-enhanced immunoturbidimetry-based measurement of HBP according to any of claims 1 to 7, comprising the steps of:
(1) preparing a reaction buffer R1:
according to the component content of the reaction buffer solution R1, mixing all the components in the same container, and uniformly mixing to obtain a reaction buffer solution R1;
(2) formulation of latex reagent R2:
① adding latex microsphere with solid content of 10% into washing buffer solution, centrifuging, removing supernatant, ultrasonic resuspending the latex microsphere precipitate with activating buffer solution, activating with activating agent, centrifuging again, and ultrasonic resuspending the latex microsphere precipitate with coupling buffer solution to obtain activated latex microsphere;
②, at least two HBP monoclonal antibodies are taken and respectively mixed with the prepared activated latex microspheres according to a specific proportion, coupling is carried out for a certain time, and the coupled latex microspheres are prepared, wherein the v/w ratio of the activated latex microspheres to each HBP monoclonal antibody is respectively 1 (5-6), and the coupling time is 2 h;
③ adding blocking buffer solution into the coupled latex microspheres for blocking, centrifuging again, removing supernatant, adding latex preservative solution, resuspending and ultrasonically treating to obtain latex reagent composition liquid, and mixing the latex reagent composition liquids to obtain the required latex reagent R2.
9. The method for preparing the kit for measuring HBP according to claim 8, wherein in step ①, the washing buffer, the activating buffer and the coupling buffer are phosphate buffers, the activating agent is EDAC/NHS, the ratio of the corresponding latex particles to the activating agent is 1 (10-20), the blocking buffer is one or more of BSA and glycine, and the blocking time is 1 h.
10. Use of a kit for the latex-enhanced immunoturbidimetry-based measurement of HBP according to any of claims 1 to 7, comprising the following specific steps:
(1) pipetting 20. mu.L of the sample, adding 240. mu.L of reagent R1, and incubating at 37 ℃ for 5 min;
(2) adding 60 μ L reagent R2, and incubating at 37 deg.C;
(3) and (3) after incubation for 30s, reading the absorbance A1, incubating for 4.5min, reading the absorbance A2, finally calculating delta A, wherein △ A is A2-A1, and calculating the HBP content in the sample according to the delta A.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911069850.6A CN110716057A (en) | 2019-11-05 | 2019-11-05 | Kit for determining HBP (hepatitis B protein) based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911069850.6A CN110716057A (en) | 2019-11-05 | 2019-11-05 | Kit for determining HBP (hepatitis B protein) based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110716057A true CN110716057A (en) | 2020-01-21 |
Family
ID=69214761
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911069850.6A Pending CN110716057A (en) | 2019-11-05 | 2019-11-05 | Kit for determining HBP (hepatitis B protein) based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110716057A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111596072A (en) * | 2020-06-11 | 2020-08-28 | 安徽大千生物工程有限公司 | Kit for determining PTH based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
| CN111856026A (en) * | 2020-02-28 | 2020-10-30 | 安徽大千生物工程有限公司 | Kit for determining III C based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
| CN111999501A (en) * | 2020-08-20 | 2020-11-27 | 安徽伊普诺康生物技术股份有限公司 | Kit for measuring human serum lipoprotein phospholipase A2 and preparation and use methods thereof |
| CN112595853A (en) * | 2020-12-04 | 2021-04-02 | 安徽大千生物工程有限公司 | IL-6 immunoturbidimetry detection kit prepared based on recombinant monoclonal antibody and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108008130A (en) * | 2017-11-24 | 2018-05-08 | 海格德生物科技（深圳）有限公司 | Kit based on latex enhancing immune turbidimetry for Determination Lp-PLA2 and preparation method thereof |
| CN108152512A (en) * | 2017-12-25 | 2018-06-12 | 苏州康和顺医疗技术有限公司 | Heparin-binding protein detection kit and preparation method thereof |
| CN108956978A (en) * | 2018-04-11 | 2018-12-07 | 中翰盛泰生物技术股份有限公司 | A kind of heparin-binding protein assay kit and measuring method using latex immunoturbidimetry |
| CN109613259A (en) * | 2018-12-20 | 2019-04-12 | 北京贝尔生物工程股份有限公司 | A kind of people's heparin-binding protein assay kit of highly sensitive, wide detection range |
-
2019
- 2019-11-05 CN CN201911069850.6A patent/CN110716057A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108008130A (en) * | 2017-11-24 | 2018-05-08 | 海格德生物科技（深圳）有限公司 | Kit based on latex enhancing immune turbidimetry for Determination Lp-PLA2 and preparation method thereof |
| CN108152512A (en) * | 2017-12-25 | 2018-06-12 | 苏州康和顺医疗技术有限公司 | Heparin-binding protein detection kit and preparation method thereof |
| CN108956978A (en) * | 2018-04-11 | 2018-12-07 | 中翰盛泰生物技术股份有限公司 | A kind of heparin-binding protein assay kit and measuring method using latex immunoturbidimetry |
| CN109613259A (en) * | 2018-12-20 | 2019-04-12 | 北京贝尔生物工程股份有限公司 | A kind of people's heparin-binding protein assay kit of highly sensitive, wide detection range |
Non-Patent Citations (2)
| Title |
|---|
| 任玮等: "肝素结合蛋白、降钙素原和白细胞计数在脓毒症早期的临床对比初探", 《国际检验医学杂志》 * |
| 张国辉: "血液HBP,CRP和WBC联合检测对儿童脓毒血症早期诊断价值研究", 《现代检验医学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111856026A (en) * | 2020-02-28 | 2020-10-30 | 安徽大千生物工程有限公司 | Kit for determining III C based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
| CN111596072A (en) * | 2020-06-11 | 2020-08-28 | 安徽大千生物工程有限公司 | Kit for determining PTH based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
| CN111999501A (en) * | 2020-08-20 | 2020-11-27 | 安徽伊普诺康生物技术股份有限公司 | Kit for measuring human serum lipoprotein phospholipase A2 and preparation and use methods thereof |
| CN112595853A (en) * | 2020-12-04 | 2021-04-02 | 安徽大千生物工程有限公司 | IL-6 immunoturbidimetry detection kit prepared based on recombinant monoclonal antibody and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110716057A (en) | Kit for determining HBP (hepatitis B protein) based on latex enhanced immunoturbidimetry and preparation and use methods thereof | |
| CN101377492B (en) | Bladder chalone C determining reagent kit | |
| CN111122874A (en) | Kit for determining LN based on latex enhanced immunoturbidimetry, and preparation and use methods thereof | |
| CN101699287B (en) | Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof | |
| US20050227223A1 (en) | Method of judging viral infection | |
| CN110687286A (en) | Latex enhanced immunoturbidimetry kit | |
| CN111239418A (en) | Kit for determining ANA based on latex enhanced immunoturbidimetry and preparation and use methods thereof | |
| CN110806487A (en) | Kit for detecting human heparin binding protein and preparation method thereof | |
| CN111856009A (en) | Kit for determining MMP-3 based on latex enhanced immunoturbidimetry, and preparation and use methods thereof | |
| CN111596072A (en) | Kit for determining PTH based on latex enhanced immunoturbidimetry and preparation and use methods thereof | |
| CN112255416A (en) | Kit for quantitatively detecting HBP (hepatitis B protein) by using magnetic particle chemiluminescence as well as preparation and detection methods thereof | |
| CN112098647A (en) | Kit for determining CK-MB based on latex enhanced immunoturbidimetry and preparation and use methods thereof | |
| CN112858698B (en) | D-dimer latex enhanced immunoturbidimetry kit and preparation method thereof | |
| CN108627652B (en) | It is a kind of to detect based on simple grain diameter and simultaneously the kit of RBP ELISA in serum and urine specimen | |
| CN110412292B (en) | Cystatin C determination kit for simply and effectively removing rheumatoid factor interference in sample | |
| CN111239404B (en) | Detection kit capable of simultaneously detecting retinol binding protein in urine sample and serum sample | |
| CN109738644B (en) | Anti-mullerian hormone immune turbidimetry quantitative detection reagent | |
| CN111856039A (en) | Immunoturbidimetric methodology kit for determining procalcitonin in whole blood | |
| CN111856026A (en) | Kit for determining III C based on latex enhanced immunoturbidimetry and preparation and use methods thereof | |
| CN109580931A (en) | A kind of α 1- microglobulin detection kit | |
| CN115561450A (en) | Kit for detecting haptoglobin content | |
| CN111912990B (en) | Neutrophil gelatinase-associated lipocalin assay kit | |
| CN114137204A (en) | KL-6 determination kit and preparation and detection methods thereof | |
| CN112485444A (en) | Kit for determining D-dimer based on latex enhanced immunoturbidimetry and preparation method thereof | |
| CN112763724A (en) | Reagent combination and kit for simultaneously determining content of retinol binding protein in serum and urine |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200121 |