CN116203243A - PINP monoclonal antibody, kit containing PINP monoclonal antibody and application of PINP monoclonal antibody - Google Patents
PINP monoclonal antibody, kit containing PINP monoclonal antibody and application of PINP monoclonal antibody Download PDFInfo
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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
- G01N33/577—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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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
- G01N33/54326—Magnetic particles
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
PINP monoclonal antibody, kit containing the same and application thereof, wherein the antibody is prepared by the following method: firstly synthesizing genes and constructing vectors, and then inducing and expressing PINP proteins P1, P2 and P3 in escherichia coli and purifying to obtain recombinant antigens P1, P2 and P3; immunizing animals with recombinant antigens P2 and P3 to obtain immune cells; fusing the immune cells and fusion partner cells, and selecting positive fusion cells; subcloning the positive fusion cells to obtain cloned fusion cells, wherein the cloned fusion cells secrete monoclonal antibodies; wherein the antigen is recombinant antigens P1, P2 and P3, and the recombinant antigens P1, P2 and P3 respectively correspond to gene sequences of 23-161aa, 23-96aa and 97-161aa in the PINP sequence; the monoclonal antibodies are monoclonal antibodies derived from P1, P2 and P3; the method has the advantages of effectively improving the detection efficiency and sensitivity of PINP and easily controlling the difference between batches.
Description
Technical Field
The invention relates to the technical field of chemiluminescence immunodiagnosis, in particular to a PINP (type I procollagen amino terminal pro peptide) monoclonal antibody, a kit containing the same and application of the same in detecting human serum PINP.
Background
Bone type I collagen accounts for about 90% of bone mass, and is the most abundant form of collagen in humans. The type I collagen is mainly synthesized in bone tissue, and is in a procollagen form firstly, wherein an extension peptide is respectively arranged at the amino N end and the C end of the type I procollagen, and is respectively called type I procollagen N-terminal propeptide (PINP) and type I procollagen C-terminal propeptide (PICP); when procollagen type I is secreted extracellularly by osteoblasts, the N-and C-terminal extension peptides are cleaved by proteases, deposited in small amounts in the bone matrix, and mostly enter the blood circulation and are eventually cleared by liver metabolism. PINP in blood is in trimeric form (converted from trimeric collagen), but quickly becomes monomeric under thermal degradation. The PINP reagent detects all forms of PINP in the blood and is therefore referred to as total PINP (tPINP).
Under the condition of excluding the influence of liver diseases, the PINP content in blood can be detected to exactly reflect the activity of osteoblasts, so as to reflect the intensity of bone metabolism in vivo and guide the diagnosis and treatment of diseases caused by abnormal bone metabolism. In addition, PINP may also be used for growth process monitoring of Paget's bone disease, osteoblastic metastases, breast cancer, prostate cancer and childhood adolescents. PINP detection is currently divided into two types, namely full-segment PINP (INtact) and total PINP (Intact+Monomer), and besides the fact that the result of full-segment PINP is more accurate when detecting renal dysfunction patients, the correlation between the two is better under other conditions.
The detection method of the type I procollagen amino terminal peptide adopts an immunological method. Currently, most of the detection reagents for the type I procollagen amino terminal peptide approved to be marketed in China are imported, and most of the adopted detection methods are enzyme-free detection methods and chemiluminescent detection methods. The enzyme-free detection method is far less than the chemiluminescent detection method in terms of detection sensitivity, detection linear range and the like. In addition, the chemiluminescent detection method has the advantages of small substrate hazard, long effective period, few interference factors, high automation degree and the like. However, both enzyme-free and chemiluminescent assays are based on specific binding of antigen to antibody, and thus specific antibody pairs are the core materials for these assays.
However, there are various disadvantages to the current commercial antibody raw materials, and it is difficult to find a proper antibody pairing. Although the specificity of the monoclonal antibody prepared by using the polypeptide as an immunogen is better, the monoclonal antibody cannot meet the requirements of different methodologies due to single antigenic site. Meanwhile, the similarity between the polypeptide sequence and the natural protein structure is different to a certain extent, so that the binding force between the obtained monoclonal antibody and the natural protein is poor, and the sensitivity of the detection reagent is affected. In addition, the batch-to-batch differential control of polyclonal antibodies is also a current challenge in the development of in vitro diagnostic reagents.
Disclosure of Invention
The present application provides a PINP monoclonal antibody capable of effectively improving the detection efficiency and sensitivity of PINP, and easily controlling the difference between batches, aiming at the above-mentioned shortcomings of the prior art.
In order to solve the technical problems, the technical scheme adopted in the application is as follows: a PINP monoclonal antibody, which is prepared by the following method, the preparation process comprising: firstly synthesizing genes and constructing vectors, and then inducing and expressing PINP proteins P1, P2 and P3 in escherichia coli and purifying to obtain recombinant antigens P1, P2 and P3; immunizing animals with recombinant antigens P2 and P3 to obtain immune cells; fusing the immune cells and fusion partner cells, and selecting positive fusion cells; subcloning the positive fusion cells to obtain cloned fusion cells, wherein the cloned fusion cells secrete monoclonal antibodies; wherein the antigen is recombinant antigens P1, P2 and P3, and the recombinant antigens P1, P2 and P3 respectively correspond to gene sequences of 23-161aa, 23-96aa and 97-161aa in the PINP sequence; the monoclonal antibodies are P1, P2 and P3-derived monoclonal antibodies.
Further, the amino acid sequences corresponding to P1, P2 and P3 are as follows:
P1:QEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAP；
P2:QEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCP
P3:DGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAP。
further, the preparation of the recombinant antigen comprises: the gene sequences of the 23-161aa, the 23-96aa and the 97-161aa are respectively digested with BamH1 and Xho 1; and respectively connecting the gene sequences of the 23-161aa, the 23-96aa and the 97-161aa which are subjected to double digestion with pet28a plasmid which is subjected to digestion in the same digestion mode, converting the plasmid into competent cells of escherichia coli, and expressing and purifying the competent cells to obtain the recombinant antigens P1, P2 and P3.
Further, the positive fusion cells were selected by the indirect Elisa method.
Further, the preparation method of the antibody further comprises the following steps: adding recombinant antigen P1 into the capture antibody, performing immune reaction, washing, adding a labeled antibody, washing, adding SA-HRP (horse radish peroxidase labeled strepitavidin also called horse radish peroxidase labeled Streptavidin), and performing color development to obtain the pairing success.
The application also provides a tPINP in-vitro diagnosis kit, which comprises a luminescent reagent, a solid-phase reagent, a calibrator and a quality control product.
Further, the preparation raw materials of the solid phase reagent compriseMagnetic particles, wherein the magnetic particles are Fe 2 O 3 Or Fe (Fe) 3 O 4 The composite of the magnetic nano particles and the organic polymer material contains one or more active functional groups on the surface of the magnetic particles.
Further, the solid phase reagent is a magnetic particle suspension coated by PINP monoclonal antibody, the concentration of the magnetic particles is 0.1-1 mg/ml, and the solid phase reagent also contains free PINP monoclonal antibody, and the concentration of the free PINP monoclonal antibody is 50-1000 ng/ml.
Further, the luminous reagent is working solution of PINP monoclonal antibody with acridinium ester marked according to proportion, and the concentration is 0.1-1.0 mug/ml.
The invention also provides a preparation method of the tPINP in-vitro diagnostic kit, which comprises the following steps:
preparing solid phase reagent diluent: sequentially dissolving buffer solution, inorganic salt, metal chelating agent, protein protectant, preservative and surfactant into purified water, regulating pH to be within the range of 6.0-8.0, fixing volume according to the preparation amount, and filtering with a 0.22 μm filter membrane for later use;
preparing a luminescent reagent diluent: sequentially dissolving buffer solution, inorganic salt, metal chelating agent, protein protectant, preservative and surfactant into purified water, regulating pH to be within the range of 5.0-7.0, fixing volume according to the preparation amount, and filtering with a 0.22 μm filter membrane for later use;
preparing a calibrator diluent: sequentially dissolving buffer solution, inorganic salt, metal chelating agent, protein protectant, preservative and surfactant into purified water, regulating pH to be within the range of 6.0-8.0, fixing volume according to the preparation amount, and filtering with a 0.22 μm filter membrane for later use;
preparing a calibrator: adding PINP recombinant antigen into calibrator diluent according to a proportion to prepare calibrator solutions with the concentrations of 100ng/ml and 800ng/ml respectively, and respectively marking the calibrator solutions as CAL1 and CAL2;
preparing a quality control product: the PINP antigen recombinant antigen is added into the calibrator diluent according to the proportion to prepare quality control product solutions with the concentration of 60ng/ml and 150ng/ml respectively, and the quality control product solutions are respectively marked as QC1 and QC2;
preparing a solid phase reagent: coating the PINP antibody on the surface of magnetic particles to prepare an intermediate product, and then adding a proper amount of the intermediate product of the magnetic particles into solid-phase reagent diluent to prepare a suspension with the working concentration of 0.1-0.8 mg/ml;
preparing a luminescent reagent: the acridinium ester is marked on the PINP antibody according to the proportion to prepare a luminous reagent intermediate product, and then a proper amount of the luminous reagent intermediate product is added into luminous reagent diluent to prepare luminous reagent working solution with the working concentration of 0.1-1.0 mug/ml.
Further, the buffer solution is selected from one or more of phosphate buffer solution, tris buffer solution, 3- (N-morpholino) propane sulfonic acid buffer solution, 2- (N-morpholino) ethane sulfonic acid buffer solution, borate buffer solution, glycine buffer solution or hydroxyethyl piperazine ethane sulfuric acid buffer solution, wherein the concentration of the buffer solution is 0.05-0.2mol/L, and the pH value is 6-9.
Further, the inorganic salt is selected from one or more of sodium chloride, potassium chloride and calcium chloride, and the concentration of the inorganic salt is 50-500 mM/L.
Further, the metal chelating agent is selected from one or more of disodium ethylenediamine tetraacetate, hydroxyethyl ethylenediamine triacetic acid and polyacrylic acid, and the concentration of the metal chelating agent is 0.1-1% (mass percent).
Further, the protein protectant is one or more selected from bovine serum albumin, bovine IGG and fish skin gelatin, and the concentration of the protein protectant is 0.1-1% (mass percent).
Further, the preservative is selected from one or more of sodium azide, N-methyl-isothiazolone, N '-methylene-bis [ N' - (3-hydroxymethyl-2, 5-dione-4-imidazolidinyl) ] urea or ProClin 300, and the concentration of the preservative is 0.1-0.5% (mass percent).
Further, the surfactant is selected from Tween 20, an ethylene oxide-propylene oxide block copolymer or polyethylene glycol octyl phenyl ether, and the concentration of the surfactant is 1-10% (mass percent).
The concentrations of the buffer, inorganic salt, metal chelator, protein protectant, preservative, and surfactant described herein are the final concentrations of the respective reagents after formulation.
Further, the magnetic particles coated with the PINP antibody are a compound formed by magnetic particles with different particle diameters and surface active functional groups and the PINP antibody, the surface active functional groups of the magnetic particles are selected from p-toluenesulfonyl or carboxyl, and the particle diameter of the magnetic particles is 0.1-5 mu m.
The invention provides a detection method of human serum PINP, which uses the tPINP in-vitro diagnosis kit, wherein a measuring instrument is MS-i3080 or other series of full-automatic chemiluminescence instruments, and the PINP detection method comprises the following steps: adding a sample incubation, a solid phase reagent and a luminescent reagent into a reaction tube, incubating for 15 minutes at 37 ℃, and measuring a luminescent value RLU after cleaning; taking the concentration of the calibrator as an abscissa, and taking the luminescence value RLU corresponding to each concentration calibrator as an ordinate, and performing a logic-log (5 p) function curve; after the calibration is successful, the measuring instrument automatically calculates the corresponding concentration value.
The application has the advantages and beneficial effects that:
1. the monoclonal antibody obtained by the application is used for a magnetic particle chemiluminescence immunodiagnosis reagent, wherein the PINP antibody in a solid phase reagent is used as a capture antibody, the PINP antibody in a luminescent reagent is used as a detection antibody, and a classical double-antibody sandwich method is used for capturing an object to be detected in serum, so that the sensitivity is high, the specificity is strong, the detection range is wide, and the detection efficiency is high.
2. The PINP monoclonal antibody prepared by the method can effectively control the batch-to-batch difference, and is beneficial to meeting the requirements of batch production and clinical diagnosis of diagnostic reagents.
3. The kit provided by the invention has the advantages that the free PINP monoclonal antibody is added in the solid phase component, part of the object to be detected can be neutralized, the sample is not required to be diluted, the testing speed is increased, and the accuracy of the testing result is improved; if the sample is directly detected without dilution without addition of free antibody, the hook effect can result in high value sample misalignment.
4. The kit can be suitable for MS-i3080 (produced by Meikang Cheng De) and other series of full-automatic chemiluminescence apparatuses, realizes full automation of a series of processes of sample injection, reagent addition, cleaning, detection, calculation and the like, and improves the detection efficiency.
Drawings
FIG. 1 is an electrophoretogram of recombinant antigen P1 of the present application.
FIG. 2 is an electrophoretogram of recombinant antigens P2 and P3 of the present application.
FIG. 3 tPINP in vitro diagnostic kit correlation detection results.
FIG. 4 tPINP in vitro diagnostic kit linear assay results.
Fig. 5 tPINP in vitro diagnostic kit quality control on-board stability test results.
FIG. 6 tPINP in vitro diagnostic kit thermostability assay results.
Detailed Description
The following description of the embodiments of the present invention will be made with reference to the accompanying drawings, in which the embodiments mentioned are only some embodiments of the present invention, and not all embodiments are represented. Based on the embodiments of the present invention, one of ordinary skill in the art may make various inventive improvements and modifications to the specific technical solutions of the embodiments of the present invention without departing from the scope or spirit of the invention.
EXAMPLE 1 preparation of PINP monoclonal antibodies
1. Recombinant antigen preparation
1. Gene synthesis and vector construction
Synthesizing Gene sequences P1, P2 and P3 corresponding to 23-161aa, 23-96aa and 97-161aa in a COL1A1 (Gene ID: 1277), respectively adding BamH1 and Xho1 enzyme cutting sites at two ends of N, C of P1, P2 and P3, and cloning to a pet-28a vector corresponding site, wherein the structures of expression products are 6 xHis-P1-6 xHis, 6 xHis-P2-6 xHis and 6 xHis-P3-6 xHis respectively;
QEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAP (specifically shown in a sequence table 1 of the application, namely SEQ ID NO. 1);
QEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCP (specifically shown in a sequence table 2 of the application, namely SEQ ID NO. 2);
DGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAP (specifically shown in a sequence table 3 of the application, namely SEQ ID NO. 3);
the P1, P2 and P3 sequences are the corresponding parts selected from PINP proteins respectively and are expressed as recombinant proteins respectively;
2. inducible expression and purification
The 3 plasmids with correct construction are respectively transformed into competent cells of escherichia coli BL21 (DE 3), the single clones are selected and inoculated into 1L LB culture medium (Kan+), the culture is carried out at 37 ℃ until the OD600 is about 0.8, and the final concentration of 0.4mM IPTG (isopropyl thiogalactoside) is added for overnight induction culture at 20 ℃.
Collecting the induced solid bacteria, washing twice with PBS at 7500rpm for 15min, and re-dissolving according to 1g of wet bacteria in 20ml of balance buffer (20mM Tris 8.0, 150mM NaCl,20mM imidazole); crushing in a homogenizer, adding 50mg/L nuclease and 1.5mM PMSF protein inhibitor before crushing, and emulsifying. Crushing pressures are respectively 300bar, 600bar, 1000bar and 1000bar, centrifuging is carried out after crushing, and supernatant is taken out at 12000rpm for 30 min.
The Ni column was equilibrated with equilibration buffer (20 mM Tris 8.0, 150mM NaCl), loaded and equilibrated, and protein purity was measured by elution with a 50mM, 100mM, 150mM, 250 mM, 500mM imidazole gradient, respectively, and the target peaks were collected and SDS gel was used. Purified protein 10mM PBS7.4 is dialyzed for 3 times, BCA is measured for concentration, and the split package is frozen for standby.
Referring to fig. 1, which is an electrophoresis diagram of recombinant antigen P1, and fig. 2, which is an electrophoresis diagram of recombinant antigens P2 and P3, it can be seen from the electrophoresis diagram that the antigen prepared in this example has high purity, and meets the experimental requirements.
2. Immunization of animals
The recombinant antigens P2 and P3 are respectively used as immunogens, and the immunized mice are female Balb/c mice which are 6-8 weeks old and are healthy; at the time of primary immunization, the immunization dose is 100 ug/mouse, freund's complete adjuvant is used for mixing, and subcutaneous multipoint injection is carried out; boosting was performed 3 weeks later, 50 ug/mouse, mixed with Freund's incomplete adjuvant, injected subcutaneously at multiple points, and thereafter boosted again at 2 week intervals; 3 booster immunizations were performed after the first immunization. After 1 week of first, third and fourth, orbital blood collection was performed, and the ELISA plate was coated with the corresponding immunogen, and the titer of the mouse antiserum was detected by indirect ELISA. After 10 days of the last booster, 100 ug/mouse without adjuvant was used for impact underarm injection; fusion experiments were performed 3 days after the impact.
3. Cell fusion and monoclonal antibody preparation
The impacted mice were sacrificed at cervical amputation and sterilized by immersion in 75% ethanol. In an ultra clean bench, the spleens of mice were removed under aseptic conditions, pressed with sterilized ground glass flakes and gently ground to isolate spleen cells, spleen cells were counted. Mix in a ratio of SP2/0 cells to spleen cells 1:5, centrifuge 500g for 5min at room temperature, discard supernatant. 1.5ml of PEG was added and the PEG and cells were thoroughly and homogeneously mixed, and this process was completed within 3 minutes. 50ml of fresh DMEM medium was added, centrifuged at 500g for 5 minutes at room temperature, and HAT medium was resuspended and gently mixed. 100 ul/well was added to a 96-well plate and placed in an incubator for cultivation. After 4-5 days, when the cells in the 96-well plate grow to 1/3 bottom area, HT culture medium is replaced, recombinant antigen P1 and corresponding immunogen are coated at 1ug/ml, and the double-positive clone is selected by indirect ELISA detection; and performing subcloning for 3-4 times, ensuring that the monoclonal antibody is amplified and cultured when the monoclonal antibody is formed, performing ascites cell injection, and purifying to obtain the monoclonal antibody.
4. Antibody labeling and pairing
1. Antibody labelling
Monoclonal antibody was diluted with 1xPBS to a concentration of 1mg/ml, biotin (EZ-Link NHS-PEG4-Biotin, thermo) was added in a molar ratio of 1:5 (antibody: biotin) per 2mg of antibody and mixed rapidly. After mixing, the centrifuge tube containing the mixture was placed on a vertical stirrer for reaction for 30min, or overnight at 4 ℃. The biotinylated antibody was dialyzed 3 times against 1xPBS and changed every 4 hours. Obtaining the biotin-labeled monoclonal antibody.
2. Antibody pairing
2. Mu.g/ml x 100. Mu.l of unlabeled mab of P2 immune origin per well was coated by incubation at 37℃for 1 h; 200 μL 2% BSA blocked, incubated at 37℃for 1h, and PBST washed 3 times; 1ug/ml of recombinant antigen P1 was added to each well, negative controls without antigen were set for each pairing, incubated for 1h at 37℃and PBST washed 3 times; adding P3 immune source biotin-labeled monoclonal antibody (1:200), incubating at 37 ℃ for 1h, and washing with PBST for 3 times; SA-HRP 1:2000, 100 μl, incubation at 37deg.C for 30min, PBST wash 4 times; color development, OD value was read at 450 nm. OD is 2.1 times greater than that of the negative control hole, positive pairing is judged, and an antibody with high signal to noise ratio is selected for preparing a chemiluminescent reagent for sample testing.
2. Mu.g/ml x 100. Mu.l of unlabeled mab of P3 immune origin per well was coated by incubation at 37℃for 1 h; 200 μL 2% BSA blocked, incubated at 37℃for 1h, and PBST washed 3 times; 1ug/ml of recombinant antigen P1 was added to each well, negative controls without antigen were set for each pairing, incubated for 1h at 37℃and PBST washed 3 times; adding P2 immune source biotin-labeled monoclonal antibody (1:200), incubating at 37 ℃ for 1h, and washing with PBST for 3 times; SA-HRP 1:2000, 100 μl, incubation at 37deg.C for 30min, PBST wash 4 times; color development, OD value was read at 450 nm. OD is 2.1 times greater than that of the negative control hole, positive pairing is judged, and an antibody with high signal to noise ratio is selected for preparing a chemiluminescent reagent for sample testing.
Example 2 preparation of PINP detection kit
1. Preparation of solid phase reagent Diluent
Sequentially dissolving buffer solution, inorganic salt, metal chelating agent, protein protectant, preservative and surfactant into purified water, regulating pH to be within the range of 6.0-8.0, fixing volume according to the preparation amount, and filtering with a 0.22 μm filter membrane for later use.
2. Preparation of solid phase reagents
Coating a PINP antibody on the surface of a magnetic particle to prepare a magnetic particle intermediate product, and then adding a proper amount of the magnetic particle intermediate product into a solid-phase reagent diluent to prepare a working solution with the concentration of 0.1-0.8 mg/ml;
3. preparation of luminescent reagent Diluent
Sequentially dissolving buffer solution, inorganic salt, metal chelating agent, protein protectant, preservative and surfactant into purified water, regulating pH to be within the range of 5.0-7.0, fixing volume according to the preparation amount, and filtering with a 0.22 μm filter membrane for later use;
4. preparation of luminescent reagents
The acridinium ester is marked on the PINP antibody according to the proportion to prepare a luminous reagent intermediate product, and then a proper amount of the luminous reagent intermediate product is added into luminous reagent diluent to prepare luminous reagent working solution with the concentration of 0.1-1.0 mug/ml.
5. Preparation of a calibrator dilution
Sequentially dissolving buffer solution, inorganic salt, metal chelating agent, protein protectant, preservative and surfactant into purified water, regulating pH to be within the range of 6.0-8.0, fixing volume according to the preparation amount, and filtering with a 0.22 μm filter membrane for later use.
6. Preparation of a calibration Material
PINP recombinant antigen was added to calibrator dilutions in a ratio to prepare calibrator solutions at concentrations of 100ng/ml and 800ng/ml, respectively, designated CAL1 and CAL2.
7. Preparation of quality control product
PINP recombinant antigen is added into calibrator diluent according to proportion to prepare quality control substance solutions with the concentration of 60ng/ml and 150ng/ml respectively, which are respectively marked as QC1 and QC2.
Example 3 application of PINP detection kit
The PINP recombinant antigen is subjected to gradient dilution, detection and reassignment are carried out by using an import registration kit A (a kit sold by Roche production in the United states), 2 points in the range of 5-1000ng/mL are selected as calibration points, and a self-assembled luminescent reagent and a solid-phase reagent are used for detection and calibration of a CL120 chemiluminescent immunoassay analyzer, so that the test conditions are as follows: in one step, 40. Mu.L of the luminescent reagent, 40. Mu.L of the solid phase reagent, 20. Mu.L of the sample are incubated at 37℃for 15min.
1. Correlation: 40 samples were collected, the theoretical value covered by the linear range 5-1000ng/mL, and the test was performed with a certain entry registration kit A, referring to the instruction manual. Test conditions: in one step, 40. Mu.L of luminescent reagent, 40. Mu.L of solid phase reagent, 20. Mu.L of sample are incubated at 37℃for 15min, and the test results are recorded. And calculating the test correlation coefficient R2 of the kit and the imported kit A.
Referring to fig. 3, the detection result of the present kit is shown, wherein the abscissa of fig. 3 is the test concentration of the inlet kit a, and the ordinate is the measured concentration of the present kit, and the correlation coefficient R2 is 0.988.
2. Sensitivity: the blank sample was prepared from 5% bovine serum and was continuously assayed 25 times under the same conditions as above. Recording test results, respectively calculating an average value AV and a standard deviation SD, and calculating a lowest detection line LOB=AV+2SD; 5 low-concentration samples with the concentration of about 3ng/ml are respectively added by taking 5% bovine serum as a matrix, each sample is continuously measured for 5 times, the test conditions are the same, and the test results are recorded.
Referring to Table 1, the LOB of the kit is 0, and the lowest detection limit LOD is less than 3ng.
TABLE 1 PINP detection kit LOB and LOD test results
| Sensitivity of | Batch 1 (ng/ml) | Batch 2 (ng/ml) | Batch 3 (ng/ml) |
| |
0 | 0 | 0 |
| LOD | 2.89 | 2.98 | 3.03 |
3. Specificity: the sample containing 50pg/ml parathyroid hormone and 15ng/ml osteocalcin is detected by using a self-prepared luminous reagent and a solid-phase reagent, the test conditions are the same, and the test result is recorded.
Referring to Table 2, the test results of the sample containing 50pg/ml parathyroid hormone and 15ng/ml osteocalcin with the kit are all 0, which shows that PINP has no cross reaction with parathyroid hormone and osteocalcin.
TABLE 2 PINP assay kit specificity test results
4.
5. Within lot repeatability: 10 samples with the concentration of 20ng/ml and 100ng/ml are continuously tested, the test conditions are the same, and the test results are recorded. The average value AV and standard deviation SD are calculated, respectively, and the lowest detection line cv=sd/AV is calculated.
Referring to Table 3, samples with the concentration of 20ng/ml and 100ng/ml are continuously tested by the kit, and CV is calculated to meet the requirement of less than or equal to 5 percent, and can be controlled within 3 percent.
TABLE 3 in-batch reproducibility test results for PINP assay kits
5.
6. Linearity: mixing a high-concentration sample near the upper limit of the linear range and a low-concentration sample near the lower limit of the linear range to obtain 8-11 diluted concentrations, testing 3 times for each diluted concentration, and recording test results under the same test conditions. And (3) taking the dilution concentration as an independent variable, taking a detection result as a dependent variable to calculate a linear regression equation, and calculating a linear regression coefficient (r).
Referring to FIG. 4, the linear regression coefficient meets the requirement that r meets ≡0.99.
7. On-board stability: and (3) putting the PINP detection kit into an instrument after opening the bottle, enabling the instrument to be in a starting state so as to keep the temperature of the reagent disk, measuring a quality control product 3 times every working day, taking an average value of results, continuously monitoring for 28 days (except for holidays), comparing the measured concentration with a target value of the quality control product, testing the same as above, and recording a test result.
Referring to FIG. 5, the accuracy of the quality control product can still meet the requirement of deviation less than or equal to + -10% after the on-board continuous monitoring for 28 days without calibration.
8. Thermal stability: and (3) simultaneously detecting the calibrator and 40 samples by using the reagent which is placed at 37 ℃ for 7 days and is balanced at 2-8 ℃ for 2 days and the reagent placed at 2-8 ℃, wherein the test conditions are the same as the above, and recording the test result.
Referring to table 4 and fig. 6, the two-point calibration values and target values of the two groups of reagent tests are within 10%, and the results of the test samples after calibration are basically free of deviation.
Table 4 thermal stability test results of PINP assay kit
Finally, it should be noted that: the above embodiments are merely exemplary of the present invention, and are not intended to limit the scope of the present invention. However, if a person skilled in the art modifies or replaces the technical solution of the foregoing embodiments, the modification or the equivalent replacement is within the scope of the claims of the present invention.
Claims (10)
1. A PINP monoclonal antibody, characterized in that: the antibody is prepared by the following steps: firstly synthesizing genes and constructing vectors, and then inducing and expressing PINP proteins P1, P2 and P3 in escherichia coli and purifying to obtain recombinant antigens P1, P2 and P3; immunizing animals with recombinant antigens P2 and P3 to obtain immune cells; fusing the immune cells and fusion partner cells, and selecting positive fusion cells; subcloning the positive fusion cells to obtain cloned fusion cells, wherein the cloned fusion cells secrete monoclonal antibodies; wherein the antigen is recombinant antigens P1, P2 and P3, and the recombinant antigens P1, P2 and P3 respectively correspond to gene sequences of 23-161aa, 23-96aa and 97-161aa in the PINP sequence; the monoclonal antibodies are P1, P2 and P3-derived monoclonal antibodies.
2. The PINP monoclonal antibody of claim 1, wherein:
P1:QEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAP；
P2:QEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCP；
P3:DGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAP。
3. the PINP monoclonal antibody of claim 1, wherein: the preparation process of the antigen comprises the following steps: the gene sequences of the 23-161aa, the 23-96aa and the 97-161aa are respectively digested with BamH1 and Xho 1; respectively connecting the gene sequences of the 23-161aa, the 23-96aa and the 97-161aa which are subjected to double digestion with pet28a plasmid which is subjected to digestion in the same digestion mode, converting the gene sequences into competent cells of escherichia coli, and expressing and purifying the competent cells to obtain recombinant antigens P1, P2 and P3; the positive fusion cells are selected by an indirect Elisa method.
4. A PINP monoclonal antibody according to any one of claims 1-3, characterized in that: the method also comprises the following steps: adding recombinant antigens P1, P2 or P3 into the capture antibody, performing immune reaction, washing, adding a labeled antibody, washing, adding SA-HRP, and developing to obtain the pairing success.
5. The tPINP in-vitro diagnosis kit is characterized by comprising a luminous reagent, a solid-phase reagent, a calibrator and a quality control product.
6. The tPINP in vitro diagnostic kit according to claim 5, wherein: the preparation raw materials of the solid phase reagent comprise magnetic particles, wherein the magnetic particles are Fe 2 O 3 Or Fe (Fe) 3 O 4 The composite of the magnetic nano particles and the organic polymer material contains one or more active functional groups on the surface of the magnetic particles.
7. The tPINP in vitro diagnostic kit according to claim 6, wherein: the solid phase reagent is magnetic particle suspension coated by PINP monoclonal antibody, the concentration of the magnetic particles is 0.1-1 mg/ml, and the solid phase reagent also comprises free PINP monoclonal antibody, and the concentration is 50-1000 ng/ml; the luminous reagent is working solution containing acridinium ester labeled PINP monoclonal antibody according to a certain proportion, and the concentration is 100-500 ng/ml.
8. A preparation method of a tPINP in-vitro diagnosis kit is characterized by comprising the following steps: the method comprises the following steps:
preparing solid phase reagent diluent: sequentially dissolving buffer solution, inorganic salt, metal chelating agent, protein protectant, preservative and surfactant into purified water, regulating pH to be within the range of 6.0-8.0, fixing volume according to the preparation amount, and filtering with a 0.22 μm filter membrane for later use;
preparing a luminescent reagent diluent: sequentially dissolving buffer solution, inorganic salt, metal chelating agent, protein protectant, preservative and surfactant into purified water, regulating pH to be within the range of 5.0-7.0, fixing volume according to the preparation amount, and filtering with a 0.22 μm filter membrane for later use;
preparing a calibrator diluent: sequentially dissolving buffer solution, inorganic salt, metal chelating agent, protein protectant, preservative and surfactant into purified water, regulating pH to be within the range of 6.0-8.0, fixing volume according to the preparation amount, and filtering with a 0.22 μm filter membrane for later use;
preparing a calibrator: adding PINP recombinant antigen into calibrator diluent according to a certain proportion to prepare calibrator solutions with the concentrations of 100ng/ml and 800ng/ml respectively, and respectively marking as CAL1 and CAL2;
preparing a quality control product: adding PINP antigen recombinant antigen into calibrator diluent according to a certain proportion to prepare quality control product solutions with the concentration of 60ng/ml and 150ng/ml respectively, and marking the quality control product solutions as QC1 and QC2 respectively;
preparing a solid phase reagent: coating the PINP antibody on the surface of magnetic particles to prepare an intermediate product, and then adding a proper amount of the intermediate product of the magnetic particles into solid-phase reagent diluent to prepare a suspension with the working concentration of 0.1-0.8 mg/ml;
preparing a luminescent reagent: the acridinium ester is marked on the PINP antibody according to a certain proportion to prepare a luminous reagent intermediate product, and then a proper amount of the luminous reagent intermediate product is added into luminous reagent diluent to prepare luminous reagent working solution with working concentration of 0.1-1.0 mug/ml.
9. The method for preparing the tpin vitro diagnostic kit according to claim 8, wherein:
the buffer solution is one or more selected from phosphate buffer solution, tris buffer solution, 3- (N-morpholino) propane sulfonic acid buffer solution, 2- (N-morpholino) ethane sulfonic acid buffer solution, borate buffer solution, glycine buffer solution or hydroxyethyl piperazine ethane sulfuric acid buffer solution, wherein the concentration of the buffer solution is 0.05-0.2mol/L, and the pH value is 6-9;
the inorganic salt is selected from one or more of sodium chloride, potassium chloride and calcium chloride, and the concentration of the inorganic salt is 50-500 mM/L;
the metal chelating agent is selected from one or more of disodium ethylenediamine tetraacetate, hydroxyethyl ethylenediamine triacetic acid and polyacrylic acid, and the concentration of the metal chelating agent is 0.1-1%;
the protein protecting agent is one or more selected from bovine serum albumin, bovine IGG and fish skin gelatin, and the concentration of the protein protecting agent is 0.1-1%;
the preservative is selected from one or more of sodium azide, N-methyl-isothiazolone, N '-methylene-bis [ N' - (3-hydroxymethyl-2, 5-diketone-4-imidazolidinyl) ] urea or ProClin 300, and the concentration of the preservative is 0.1% -0.5%;
the surfactant is selected from Tween 20, an ethylene oxide-propylene oxide block copolymer or polyethylene glycol octyl phenyl ether, and the concentration of the surfactant is 1% -10%;
the magnetic particles coated with the PINP antibody are a compound formed by selecting magnetic particles with different particle diameters and surface active functional groups and the PINP antibody, the active functional groups on the surfaces of the magnetic particles are selected from p-toluenesulfonyl or carboxyl, and the particle diameter of the magnetic particles is 0.1-5 mu m;
the calibrator is a liquid calibrator with 2 different concentration points, wherein the concentration of the liquid calibrator is within the range of 0-2000 ng/ml, and the liquid calibrator is prepared from PINP antigen; the quality control product is a liquid quality control product prepared from PINP antigen, wherein the concentration of the liquid quality control product is in the range of 0-500 ng/ml and is 2 different concentration points;
the PINP antibody in the solid phase reagent is used as a capturing antibody, the PINP antibody in the luminescent reagent is used as a detection antibody, and when the capturing antibody in the solid phase reagent is an antibody derived from a recombinant antigen P2, the detection antibody in the luminescent reagent is an antibody derived from a recombinant antigen P3; correspondingly, when the captured antibody in the solid phase reagent is an antibody derived from the recombinant antigen P3, the detection antibody in the luminescent reagent is an antibody derived from the recombinant antigen P2.
10. The detection method of human serum tPINP is characterized in that a tPINP in-vitro diagnosis kit is used, a measuring instrument is MS-i3080 or other series of full-automatic chemiluminescence instruments, and the detection method comprises the following steps: adding a sample incubation, a solid phase reagent and a luminescent reagent into a reaction tube, incubating for 15 minutes at 37 ℃, and measuring a luminescent value RLU after cleaning; taking the concentration of the calibrator as an abscissa, and taking the luminescence value RLU corresponding to each concentration calibrator as an ordinate, and performing a logic-log (5 p) function curve; after the calibration is successful, the measuring instrument automatically calculates the corresponding concentration value.
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