CN113999310A

CN113999310A - A kind of PLGF monoclonal antibody, test kit, its preparation method and application

Info

Publication number: CN113999310A
Application number: CN202011614752.9A
Authority: CN
Inventors: 张陆明
Original assignee: Jiangsu Proway Biotechnology Co ltd
Current assignee: Jiangsu Proway Biotechnology Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-02-01

Abstract

The invention is applicable to the field of biotechnology, and provides a PLGF monoclonal antibody, a kit, a preparation method and application thereof. The heavy chain variable region amino acid sequence of the PLGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; the light chain variable region amino acid sequence of the PLGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 5. The PLGF monoclonal antibody has high affinity and strong specificity; the detection kit can be used as an independent assessment factor for placenta insufficiency, and can also be used for predicting, identifying and monitoring treatment in preeclampsia.

Description

PLGF monoclonal antibody, kit, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a PLGF monoclonal antibody, a kit, a preparation method and application thereof.

Background

The placenta growth factor PLGF is one member of vascular endothelial growth factor family and has the molecular structure of glucoprotein homodimer. It is a glycoprotein with a gene located at 14q24q31, and is formed by connecting 1 alpha chain of 69kD and beta chain of 34kD through a disulfide bond to form a dimer. The base sequence of the polypeptide has high homology with PLGF. PLGF can be produced into 4 different subtypes by selective splicing of mRNA: PLGF-1, PLGF-2, PLGF-3 and PLGF-4.

Placental growth factor (PLGF), a highly specific marker, plays an important role in the vascularization of the placental chorion, and also in embryonic development. PLGF can be used to assess placental syncytiotrophoblast disease and its complications, placental villous crowding allows for oxygen supply pressure to syncytiotrophoblast cells, PLGF levels are significantly reduced, leading to placental dysplasia and placental insufficiency, difficulty in providing adequate nutrients and oxygen to the fetus, and may cause a range of diseases such as abortion, pregnancy hypertension, pre-eclampsia (PE), fetal growth restriction and preterm birth, and in severe cases, related complications such as stillbirth, eclampsia, HELLP syndrome and early paring of the placenta. Alere has developed a PLGF immunofluorescence kit for assessing placental insufficiency and for predicting, identifying and monitoring treatment of preeclampsia caused thereby.

Research and application of a labeling immunoassay technology have been rapidly developed in the last decade, and the labeling immunoassay technology has been widely applied to various fields of biomedical basic theory research and clinical disease diagnosis. The method for detecting serological indexes mainly comprises radioisotope immunoassay, enzyme-linked immunosorbent assay and chemiluminescence immunoassay. The methods can be used as a primary screening test and a confirmation test, wherein the chemiluminescence method has the advantages of wide detection linear range, simple detection instrument, convenient operation and the like.

Aiming at PLGF detection in the prior art, no similar products of a chemiluminescence method exist in China at present, and the specificity, affinity and other aspects of imported similar products have further possibility of improvement, so that the invention provides a PLGF monoclonal antibody, a kit, a preparation method and application thereof, wherein the PLGF monoclonal antibody is based on a chemiluminescence method and has high affinity and strong specificity.

Disclosure of Invention

The embodiment of the invention aims to provide a PLGF monoclonal antibody, a kit, a preparation method and application thereof, aiming at solving the problems in the prior art pointed out in the background technology.

The embodiment of the invention is realized by that, a PLGF monoclonal antibody,

the heavy chain variable region amino acid sequence of the PLGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1, or the amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1;

the light chain variable region amino acid sequence of the PLGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO.5, or the amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 5.

As another preferred scheme of the embodiment of the invention, the amino acid sequences of the heavy chain hypervariable region CDR-H1, the heavy chain hypervariable region CDR-H2 and the heavy chain hypervariable region CDR-H3 of the PLGF monoclonal antibody are respectively the amino acid sequences shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, or the amino acid sequences shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 are formed by replacing, deleting and/or adding one or more amino acid residues to form the amino acid sequences with the same functions.

As another preferred scheme of the embodiment of the invention, the light chain hypervariable region CDR-L1, the light chain hypervariable region CDR-L2 and the light chain hypervariable region CDR-L3 amino acid sequences of the PLGF monoclonal antibody are respectively the amino acid sequences shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, or the amino acid sequences shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8 are formed by replacing, deleting and/or adding one or more amino acid residues to form the amino acid sequences with the same functions.

Another object of the embodiments of the present invention is to provide a method for preparing a PLGF monoclonal antibody as described above, comprising the steps of:

firstly, preparing a PLGF protein antigen;

secondly, preparing a PLGF polyclonal antibody;

thirdly, preparing a PLGF monoclonal antibody;

fourthly, screening monoclonal antibodies: establishing a dose response curve, and screening an optimal monoclonal antibody;

fifthly, the monoclonal antibody gene in the hybridoma is regulated:

extracting total RNA of the hybridoma cells;

carrying out reverse transcription synthesis by taking OligodT as a primer to obtain cDNA;

carrying out PCR amplification by taking the synthesized cDNA as a template;

obtaining a gene sequence for coding a heavy chain variable region and a gene sequence for coding a light chain variable region;

carrying out codon optimization and synthesis on the heavy chain variable region gene sequence, the encoding light chain variable region gene sequence and the constant region of the antibody to respectively obtain a heavy chain and a light chain of the complete antibody gene;

cloning the obtained heavy chain and light chain genes into pMD18-T expression vectors respectively;

transfecting the obtained plasmid into a competent cell;

collecting cell supernatant, purifying and concentrating to obtain the PLGF monoclonal antibody.

As another preferred embodiment of the present invention, the PCR amplification conditions are: denaturation at 95 deg.C for 5 min; 95 ℃, 1min, 55 ℃, 2min, 72 ℃, 1min, 35 cycles; extension at 72 ℃ for 10 min.

The other purpose of the embodiment of the invention is to provide a PLGF detection kit, which contains the PLGF monoclonal antibody.

Another object of the embodiments of the present invention is to provide a method for preparing the PLGF detection kit, comprising the following steps:

diluting the PLGF monoclonal antibody with a buffer solution, and adding streptavidin magnetic beads for reaction;

washing a buffer solution;

adding PBS confining liquid containing fetal calf serum, performing magnetic separation after reaction, and removing supernatant;

after being washed by buffer solution, the magnetic particles are suspended in the preservation buffer solution of PBS containing bovine serum albumin to prepare PLGF coated magnetic beads;

diluting a plurality of gradients by PLGF protein antigen according to a proportion, and subpackaging to prepare a PLGF calibrator;

adding the PLGF polyclonal antibody into an acridine ester solution, and carrying out a light-resistant reaction;

taking out after the reaction is finished, adding a lysine salt solution, and continuing the reaction in a dark place;

purifying to obtain a PLGF acridine ester standard antibody;

adding a serum sample and a calibrator into the PLGF coated magnetic beads, adding a PLGF acridine ester standard antibody, and incubating to form an antibody-antigen-labeled antibody compound;

adding HNO respectively₃、H₂O₂Immediately putting the luminescence excitation liquid A, NaOH and Triton-100 luminescence excitation liquid B into a chemiluminescence immunoassay instrument, and detecting the luminescence intensity of each hole;

and calculating the content of the PLGF in the sample according to the reaction curve.

As another preferred embodiment of the present invention, PBS, tween-20 washing buffer was used.

As another preferred embodiment of the present invention, the PLGF acridinium ester standard antibody is obtained by purification using a G-25 desalting column.

The embodiment of the invention also aims to provide application of the PLGF detection kit in preparation of a detection kit for assessing placental insufficiency and monitoring preeclampsia.

The PLGF monoclonal antibody has high affinity, strong specificity and good biological activity in vitro; the detection kit containing the PLGF monoclonal antibody has the advantages of simple detection method, high accuracy and low cost. The detection kit containing the PLGF monoclonal antibody has the premature rupture of fetal membranes with the coincidence rate of 99 percent, can be used as an independent assessment factor of placental insufficiency, and can also be used for predicting, identifying and monitoring treatment of preeclampsia caused by the premature rupture of fetal membranes.

Drawings

FIG. 1 is a schematic diagram of a PLGF detection dose-response system.

FIG. 2 is an absolute bias plot of the detection data for two PLGF kits.

FIG. 3 is a graph showing the relative bias of the detection data of two PLGF kits.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Specific implementations of the present invention are described in detail below with reference to specific embodiments.

Example 1

The embodiment provides a PLGF monoclonal antibody, wherein the heavy chain variable region amino acid sequence of the PLGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; or an amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1;

the amino acid sequences of a heavy chain hypervariable region CDR-H1, a heavy chain hypervariable region CDR-H2 and a heavy chain hypervariable region CDR-H3 of the PLGF monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; or the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 is formed into an amino acid sequence with the same function by replacing, deleting and/or adding one or more amino acid residues;

the light chain variable region amino acid sequence of the PLGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 5; or an amino acid sequence with the same function formed by replacing, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 5;

the light chain hypervariable region CDR-L1, the light chain hypervariable region CDR-L2 and the light chain hypervariable region CDR-L3 of the PLGF monoclonal antibody have amino acid sequences shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively; or the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 is formed into an amino acid sequence with the same function by replacing, deleting and/or adding one or more amino acid residues.

SEQ ID NO.1：

SEQ ID NO.2：

SEQ ID NO.3：

SEQ ID NO.4：

SEQ ID NO.5：

SEQ ID NO.6：

SEQ ID NO.7：

SEQ ID NO.8：

Example 2

This embodiment provides a method for preparing a PLGF monoclonal antibody, comprising the steps of:

firstly, preparation of PLGF protein antigen:

(1) a pair of primers was designed based on the sequence of human PLGF (CAL49880.1) in GenBank database:

the upstream primer is vF: 5'-CGGCGACGGAGCGGGCTGGCATGTGGGGGG-3', respectively;

the downstream primer is vR: 5'-CCAGCTCTTGCAGCACTCATCCCATCCCTG-3', respectively;

the sites of the upstream primer and the downstream primer are anastomosed with the corresponding multiple cloning sites on the mammalian cell high-efficiency expression plasmid vector pSec/WG;

the cloning plasmid containing human PLGF gene fragment is synthesized by whole gene, and the specific amplification of PLGF gene is carried out by Pyrobest DNA polymerase with the plasmid as a template, and the PCR amplification conditions are as follows: denaturation at 95 deg.C for 5 min; 95 ℃, 1min, 55 ℃, 2min, 72 ℃, 1min, 35 cycles; extending for 10min at 72 ℃;

carrying out gel recovery, chloroform extraction, ethanol precipitation and TE dissolution on the amplified PCR product to obtain pSec/WG plasmid for later use;

carrying out Pci I and Xho I double enzyme digestion on the recovered PLGF gene and pSec/WG plasmid respectively, and recovering by a gel electrophoresis method;

respectively purifying the PCR enzyme digestion product and the recovery product of the vector again, mixing PLGF and pSec/WG according to the molar ratio of 1:1, dissolving with TE, reacting for 12 hours at 16 ℃, and placing at 70 ℃ for 10min to terminate the reaction;

connecting the reaction product with DH-5 alpha competent cells, screening out positive clones by an ampicillin (1mg/mL) antibody, amplifying, extracting and recombining PLGF/pSec/WG plasmids, and carrying out enzyme digestion and sequencing identification for later use;

(2) transfecting the recombinant PLGF/pSec/WG plasmid to a eukaryotic expression cell line Flp-In CHO by using a liposome reagent Lipofectamine 2000, and placing the transfected cells In a culture medium containing hygromycin for culture and screening;

untransfected Flp-In CHO cell lines were cultured In complete Hams F12 medium (containing 10% FBS, 2 mM L-glutamine) supplemented with 1% penicillin/streptomycin and 100. mu.g/mL Zeocin-transfected recombinant plasmid PLGF/pSec/WG Flp-In CHO cells because the insertion of the PLGF gene inactivated the Zeocin resistance gene In the host cell genome but simultaneously carried In the hygromycin resistance gene (Hyg +), so that the host cells transfected with the recombinant plasmid could be cultured In hygromycin-containing medium, whereas the untransfected host cells could not survive; culturing in complete Hams F12 culture medium containing 800 μ g/mL hygromycin, and screening out corresponding recombinant gene expression clone in 6-7 days;

culturing Flp-In CHO cells transfected with the PLGF/pSec/WG recombinant plasmid In an UltraCHO serum-free culture medium, and collecting cell culture supernatant for 1 time every 3-4 days;

centrifuging the collected culture supernatant at 10000r/min for 5 minutes, and removing the precipitate;adding 0.02% NaN into the supernatant₃And all possible cell debris was removed by filtration through a 0.22 μm filter;

the column was packed with l.0mL of resin and equilibrated with approximately 10 volumes of PBS (pH 7.2); passing the supernatant containing the target Protein through a Protein G-Sepharose 4B column for 2 times; 20-30 volumes of PBS (pH 7.2) column wash; 28 μ L of 1.25M Tris-HCl (pH 8.0) was added to each collection vial beforehand; eluting with 100mM Glycine-HCl (pH 3.0) eluent, and collecting in the collection tubules at a rate of 1.0rnl per tube to neutralize the eluent in the collection tubules immediately; usually the protein is eluted in the 3 rd to 10 th tube, with peaks around 3, 4, 5; collecting all the eluate with A280 > 0.01, and concentrating with centrifugal filter column of Ultrafree 15(MWCO 10000, Millipore); the expression level is 50mg/L, SDS-PAGE electrophoresis identification is carried out, and the purity of the purified PLGF protein is more than 98 percent; after the protein is quantified, filtering and sterilizing through a 0.22 mu m filter membrane, and storing at-20 ℃ for later use;

preparation of PLGF polyclonal antibody

(1) Using male big ear rabbit as immune animal, injecting 10mg BCG vaccine to stimulate animal, using PLGF protein as immune antigen, and starting immunity after one week; 4-6 points of subcutaneous injection are injected below feet, and Freund's complete adjuvant is used as an immunologic adjuvant to immunize animals for four times; 1mg of PLGF protein is taken each time, equivalent Freund's complete adjuvant and antigen solution are respectively sucked into two injectors for full emulsification for 1 hour, and foot subcutaneous injection is carried out, and two weeks are separated each time;

taking auricular venous blood to detect titer by an ELISA method, performing carotid bleeding when the ratio reaches 1:40000, centrifuging at 5000rpm to take serum, and purifying by DEAE ion exchange to obtain crude serum polyclonal antibody for later use;

(2) diluting the crude serum polyclonal antibody to 1mg/mL by using 0.5mol/L PBS (pH 7.5), preparing 3mL of CNBr-Sepharose 4B agarose gel, coupling 9mg of PLGF protein antigen by using a coupling agent, reacting at room temperature for 4 hours to prepare a PLGF antibody affinity chromatographic column, purifying the crude serum polyclonal antibody on the column, eluting and collecting, determining the OD value of the antibody at the wavelength of 280nm by using an ultraviolet-visible spectrophotometer, dividing the obtained OD value by 1.35 to obtain the concentration of the determined antibody, adding 40-50% of glycerol, and placing at-20 ℃ for long-term storage;

preparation of PLGF monoclonal antibody

(1) Culturing the stably expressed PLGF/pSec/WG recombinant plasmid Flp-In CHO cells, immunizing 5 female BALA/c mice, and injecting 1 × 10 subcutaneously into each BALB/c mouse⁷Cells, immunised 4 times consecutively, each time with 2 weeks intervals; collecting blood 7 days after immunization, detecting serum titer by CLIA method, selecting mice with highest titer, and injecting into spleen for 1 × 10⁶Boosting the immunity of each cell, taking the spleen of the mouse 3 days later, grinding the spleen, and counting the spleen cells for later use;

(2) fusing splenocytes and bone marrow cells Sp2/0 according to the ratio of cell count 5:1, inducing by PEG1200, adding the fused cells into a 96-well plate containing a feeder layer for culturing, changing the culture solution by half amount by using HAT culture medium after one week, and observing the cell state after fusion;

screening positive hybridoma cell strains by an indirect CLIA method, selecting 1 strain of hybridoma cell (2H2) with continuous secretion positive rate of PLGAb being more than 98% for amplification culture, and preparing a mouse for intraperitoneal injection, wherein the mouse is intraperitoneally injected with 500 mu L of liquid paraffin 1 week before;

collecting hybridoma cells by centrifugation, suspending with incomplete culture medium, mixing, and adjusting cell number to 1 × 10⁹Performing inoculation on mice at the beginning, performing intraperitoneal injection on each mouse at 500 mu L, extracting ascites from mice with obviously swollen abdomens after 1 week, centrifuging the obtained ascites at 3000r/min for 3 minutes, and collecting supernatant;

(3) purifying ascites with protein G purifying column; balancing the purification column with 0.02mol/L PB buffer solution, adding ascites for sampling, eluting with 0.1mol/L glycine hydrochloric acid buffer solution (pH 2.7), collecting with an EP tube, dialyzing with 0.05mol/LPB, concentrating to obtain PLGF monoclonal antibody, and freezing at-20 deg.C;

fourth, monoclonal antibody screening

(1) Coating the prepared PLGF monoclonal antibody, and diluting the antibody to 2-5 mug/mL by 0.5mol/L PBS; adding 100 mu L/hole into an enzyme label plate for coating, after overnight at 2-8 ℃, washing with 0.9% NaCl for 3 times, adding a blocking solution containing 1% BSA, blocking at 150 mu L/hole, and airing for later use after overnight at 2-8 ℃;

(2) screening the optimal monoclonal antibody: diluting the prepared PLGF protein antigen, diluting 8 gradients (0, 10, 50, 100, 200, 400, 1000 and 2000pg/mL) according to a proportion, sequentially adding the diluted PLGF protein antigen into the prepared enzyme label, adding a biotinylated polyclonal antibody and streptavidin marked with horseradish peroxidase into each hole with the volume of 50 mu L, incubating for 1 hour at 37 ℃, washing for 5 times by PBST, adding a chromogenic substrate solution, and detecting an OD value by using an enzyme label instrument;

dose-response curves were constructed with PLGF concentration as X-axis and OD as Y-axis (see FIG. 1). As can be seen from FIG. 1, the dose-response curve established by the PLGF detection kit has a linear coefficient R of 0.99, a detection range of 0-600pg/ml and excellent analysis performance; finally, selecting the PLGF monoclonal antibody with the best evaluation index; the monoclonal antibody as an evaluation standard of a sandwich ELISA detection method simultaneously meets the requirements that an OD value of S0 is less than 0.1, an OD value of S7/S1(P/N) is the largest, a dose-response curve correlation coefficient is more than 0.99, and the detection rate of 30 cases of quality control serum is more than 90%;

fifth, the retrieval of monoclonal antibody genes in hybridomas

(1) Extraction with Trizol reagent 5X 10⁶Total RNA of selected hybridoma cells of (2H 2);

then using OligodT as a primer, carrying out reverse transcription by AMV reverse transcriptase at the temperature of 37 ℃ for 15 minutes, and synthesizing to obtain cDNA;

taking the synthesized cDNA as a template, and carrying out nested PCR amplification aiming at a primer of an RNA sequence and a gene specific primer GSP, wherein the PCR conditions are as follows: denaturation at 95 deg.C for 5 min; 95 ℃, 1min, 55 ℃, 2min, 72 ℃, 1min, 35 cycles; extending for 10min at 72 ℃;

(2) identifying the PCR product by 1% agarose gel electrophoresis, cutting and recovering a target gel strip, calling a target gene, connecting the target gene to a pGEM-T vector, carrying out EcoRI enzyme digestion to identify a positive recombinant plasmid, and determining a DNA sequence to respectively obtain a coding heavy chain variable region gene sequence and a coding light chain variable region gene sequence;

carrying out codon optimization and synthesis on the obtained heavy chain and light chain variable region gene sequences and the constant region of the antibody to respectively obtain a heavy chain and a light chain of a complete antibody gene, and respectively cloning the obtained heavy chain and light chain genes into a pMD18-T expression vector; respectively extracting 150 mu g of plasmids, removing endotoxin (less than 1EU/mg), co-transfecting the obtained 2 plasmids 1:1 into suspension competent TG1 cells with the volume of 50mL, wherein PEI is adopted as a transfection reagent, and the ratio of the plasmids to the PEI is 1: 2;

after 3-7 days of transfection, collecting cell supernatant, detecting whether the antibody expression is correct by SDS-PAGE, purifying by a ProteinA column, and concentrating to obtain qualified PLGF monoclonal antibody with the purity of more than 98%.

Example 3

This example provides a PLGF detection kit comprising the PLGF monoclonal antibody described in example 1.

Example 4

The embodiment provides a preparation method of a PLGF detection kit, which comprises the following steps:

(1) using streptavidin magnetic beads as an immunoreaction carrier, diluting the PLGF monoclonal antibody to 2.2 mu g/mL by using 0.02mol/L Tris-HCl coupling buffer solution, adding the streptavidin magnetic beads, and reacting for 30min in a shaker at 37 ℃;

washing with 0.01mol/L PBS and 0.05% tween-20 washing buffer solution for about 3 times;

adding 1ml of 0.01mol/L PBS blocking solution containing 2% fetal calf serum, reacting in a shaking table for 2h at 37 ℃ and 180rpm, performing magnetic separation, and removing supernatant;

washing with 1ml of 0.01mol/L PBS buffer solution containing 0.1% tween-20 and pH7.4 for 3 times, suspending the magnetic particles in 1ml of 0.01mol/L PBS buffer solution containing 1% bovine serum albumin and pH7.4 to obtain PLGF-coated magnetic beads, and keeping at 4 deg.C;

(2) diluting the prepared PLGF protein antigen with 1% BSA/0.5mol/L PBS, diluting according to a proportion by 6 gradients (10, 50, 100, 200, 400, 600pg/mL), and subpackaging to prepare a PLGF calibrator;

adding 50 μ l of a 0.5mM acridinium ester (4- (2-succinimidylcarboxyl) phenyl-10-methylacridine-9-carboxylate fluorosulfonate) solution to the biotinylated PLGF polyclonal antibody, and reacting in a shaker at 25 deg.C and 180rpm in the absence of light for 20 min;

taking out after the reaction is finished, adding 100 mu l of 10% lysine salt solution, keeping out of the sun at 25 ℃, reacting for 30min at 180rpm in a shaking table;

purifying by using a G-25 desalting column to obtain a PLGF acridine ester standard antibody;

(3) adding 50 mu L/hole of serum sample and calibrator into PLGF-coated magnetic beads, adding 50 mu L/hole of PLGF acridine ester standard antibody, incubating at 37 ℃ for 1h to form a solid phase antibody-antigen-labeled antibody compound, washing for 5 times at 0.01mol/LPBS (low pressure polystyrene), and removing unbound PLGF polyclonal antibody and acridine ester;

respectively adding 0.1mol/L HNO₃，0.1％H₂O₂Immediately putting 50 mul of luminescence excitation liquid A, 0.25mol/L NaOH and 50 mul of 2% Triton-100 luminescence excitation liquid B into a chemiluminescence immunoassay instrument, and detecting the luminescence intensity (RLU) of each hole within the accumulated time of 1.5 s;

the RLU value of the sample increases along with the increase of the concentration of the PLGF, and the content of the PLGF in the sample can be calculated according to the reaction curve.

Experimental examples Performance of detection kit for detecting PLGF

(1) Performance analysis of PLGF detection kit

Repeatability: the intra-batch Coefficient of Variation (CV) of the PLGF detection kit is not more than 10 percent; the inter-batch Coefficient of Variation (CV) is not more than 15%;

analysis sensitivity: the lowest detection limit of the kit is not more than 5 pg/mL;

analysis of specificity: the detection result of specific substances (VEGF and EGF) at a certain concentration is not more than 50 pg/mL;

linear range: in the concentration range of 0-600pg/mL, the linear correlation coefficient r is not less than 0.9900.

(2) Clinical Performance of PLGF detection kit

2.1 test of 122 clinical specimens, 43 normal persons, and 76 patients with fetal membrane injury and placental insufficiency were performed. The normal reference value PLGF of the kit is less than or equal to 87pg/mL, and the detection result of the kit is as follows:

TABLE 1 percent of agreement and confidence intervals

TABLE 2 symmetry metric

a. A null hypothesis is not assumed.

b. The null hypothesis is assumed using progressive standard error.

2.2 choose the PLGF detect reagent box of Alere company as the contrast reagent box, the invention reagent box as the test reagent box, compare the measured value, the result is shown in fig. 2 relative bias chart and fig. 3 absolute bias chart.

TABLE 3 test results

It can be seen that neither the test kit of the present invention nor the comparative kit exceed both the mean absolute difference value of 4 times and the mean relative difference value of 4 times. The results of both kits showed no outliers.

2.3 correlation analysis

TABLE 4 correlation analysis results

Significant correlation at the.01 level (double-sided).

As can be seen from Table 4, the correlation coefficient of the detection results of the comparison kit and the test kit is 0.996, and the result of the hypothesis test performed on the correlation coefficient is P < 0.05, which indicates that the detection results of the two reagents have a linear correlation relationship; the correlation coefficient r is close to the maximum possible value 1, which indicates that the detection results of the two reagents have strong correlation.

2.4 regression equation of comparison kit and test kit and 95% confidence interval of equation slope b

TABLE 5 95% confidence intervals for equation slope b

Coefficient of performance^a

a. Dependent variable-measured value of kit of the invention

From the above table of SPSS output, it can be seen that the regression equation between the assay results of the clinical test kit and the assay results of the contrast agent is Y ═ 1.004x-0.494, and the slope b of the equation is within the 95% confidence interval.

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.

Sequence listing

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Claims

1. a PLGF monoclonal antibody, is characterized in that,

The heavy chain variable region amino acid sequence of the PLGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1;

The amino acid sequence of the light chain variable region of the PLGF monoclonal antibody is the amino acid sequence shown in SEQ ID NO.5.

2. PLGF monoclonal antibody according to claim 1, is characterized in that,

The amino acid sequences of the heavy chain hypervariable region CDR-H1, the heavy chain hypervariable region CDR-H2, and the heavy chain hypervariable region CDR-H3 of the PLGF monoclonal antibody are SEQ ID NO.2, SEQ ID NO.3, and SEQ ID respectively. The amino acid sequence shown in NO.4.

3. PLGF monoclonal antibody according to claim 1, is characterized in that, light chain hypervariable region CDR-L1, light chain hypervariable region CDR-L2, light chain hypervariable region CDR-L3 amino acid of PLGF monoclonal antibody The sequences are the amino acid sequences shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

4. a preparation method of the PLGF monoclonal antibody as described in any one of claim 1～3, is characterized in that, comprises the following steps:

1. Preparation of PLGF protein antigen;

2. Preparation of PLGF polyclonal antibody;

3. Preparation of PLGF monoclonal antibody;

4. Monoclonal antibody screening: establish a dose-response curve and screen the optimal monoclonal antibody;

5. Extraction of monoclonal antibody genes from hybridomas:

Extract the total RNA of hybridoma cells;

Using OligodT as a primer, reverse transcription was used to synthesize cDNA;

PCR amplification was carried out using synthetic cDNA as a template;

Obtain the gene sequence encoding the variable region of the heavy chain and the gene sequence encoding the variable region of the light chain;

The heavy chain variable region gene sequence and the light chain variable region gene sequence encoding the constant region of the antibody are codon-optimized and synthesized to obtain the heavy chain and light chain of the complete antibody gene respectively;

The obtained heavy chain and light chain genes were cloned into pMD18-T expression vector;

Transfect the obtained plasmid into competent cells;

The cell supernatant was collected, purified and concentrated to obtain PLGF monoclonal antibody.

5. The preparation method of PLGF monoclonal antibody according to claim 4, characterized in that, the conditions of PCR amplification are: 95 ℃ of denaturation 5min; 95 ℃, 1min, 55 ℃, 2min, 72 ℃, 1min, 35 Cycle; 72°C, extension 10min.

6 . A PLGF detection kit, characterized in that it contains the PLGF monoclonal antibody according to any one of claims 1 to 3 . 7 .

7. a preparation method of PLGF detection kit as claimed in claim 6, is characterized in that, comprises the following steps:

Dilute the PLGF monoclonal antibody with buffer, add streptavidin magnetic beads to react;

wash buffer;

Add the PBS blocking solution containing fetal bovine serum, carry out magnetic separation after the reaction, and remove the supernatant;

After washing with buffer, the magnetic particles were suspended in the storage buffer of PBS containing bovine serum albumin to prepare PLGF-coated magnetic beads;

Using PLGF protein antigen, dilute multiple gradients in proportion to make PLGF calibrator;

Add the PLGF polyclonal antibody to the acridine ester solution to avoid light reaction;

After the reaction is completed, take it out, add lysine salt solution, and continue the reaction in the dark;

Purified to obtain PLGF acridine ester-labeled antibody;

Serum samples and calibrators were added to the PLGF-coated magnetic beads, and then PLGF acridine ester-labeled antibodies were added to form antibody-antigen-labeled antibody complexes after incubation;

Add HNO ₃ , H ₂ O ₂ luminescence excitation solution A, NaOH, and Triton-100 luminescence excitation solution B respectively, and immediately put them into the chemiluminescence immunodetector to detect the luminescence intensity of each well;

According to the reaction curve, the content of PLGF in the sample was calculated.

8. The preparation method of the PLGF detection kit according to claim 7, characterized in that, using PBS, tween-20 washing buffer.

9. the preparation method of PLGF detection kit according to claim 7, is characterized in that, with G-25 desalting column purification, obtains PLGF acridine ester-labeled antibody.

10. The application of a PLGF detection kit as claimed in claim 6 in preparing a detection kit for assessing placental insufficiency and monitoring preeclampsia.