CN110007094B

CN110007094B - Human D-dimer quantitative detection card and clinical application thereof

Info

Publication number: CN110007094B
Application number: CN201910354934.8A
Authority: CN
Inventors: 马永; 丁娜
Original assignee: ZONHON BIOPHARMA INSTITUTE Inc
Current assignee: ZONHON BIOPHARMA INSTITUTE Inc
Priority date: 2019-04-29
Filing date: 2019-04-29
Publication date: 2022-04-05
Anticipated expiration: 2039-04-29
Also published as: CN110007094A

Abstract

The invention relates to a colloidal gold quantitative detection card of a human D-dimer and clinical application thereof. The invention prepares various antibodies, and performs pairing screening to obtain an antibody combination with sensitivity and specificity meeting the requirements; meanwhile, the preparation method is convenient for mass production and can meet the requirement of large-scale clinical application in the future. The antibody combination is debugged and optimized for the detection system, and meanwhile, the artificially prepared recombinant antigen is adopted, so that the human D-dimer colloidal gold quantitative detection card is simple and convenient to operate, and the sensitivity, specificity and related detection performance can meet the detection requirements of human clinical samples.

Description

Human D-dimer quantitative detection card and clinical application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to two anti-human D-Dimer (D-Dimer) antibodies, a preparation method thereof and application of the antibodies in human D-Dimer detection.

Background

The D-Dimer (D-Dimer) is the final product of fibrin degradation (molecular weight about 180kDa) and contains degradation products of three polypeptide chains, cross-linked by disulfide bonds. The dimer structure is maintained by the formation of a cross-linked structure by two isopeptide bonds between the C-termini of the γ chains. Under normal state, the dynamic balance between plasmin and inhibitory enzyme is kept, so that blood circulation can be normally carried out. The fibrinolytic system in the human body plays an important role in maintaining the normal permeability of the vascular wall, maintaining the flowing state of blood and repairing tissues. In pathological conditions, when the body agglutinates, thrombin acts on fibrin to convert into cross-linked fibrin, and meanwhile, a fibrinolytic system is activated to degrade fibrin to form various fragments. The gamma chain connects two fragments containing D to form a D-dimer. The rise in D-dimer reflects secondary hyperfibrinolysis. Clinically, the detection of the D-dimer is mainly used for early exclusion diagnosis of deep vein thrombosis and pulmonary embolism; early diagnosis and dynamic monitoring of disseminated intravascular coagulation; monitoring of thrombus after surgical operation; monitoring pregnancy hypertension and premonitory consummation; early identification of malignant tumors and leukemia and monitoring of thrombus; monitoring liver diseases and kidney diseases; evaluation of thrombolytic therapy and monitoring of thrombus recurrence; evaluating the condition of the cardiovascular disease; cerebral infarction identification and treatment monitoring, etc.

At present, the application of D-dimer colloidal gold quantitative products at home and abroad is mainly in the field of point of care testing (POCT), and the D-dimer colloidal gold quantitative products are widely applied at home and abroad to outpatient service, emergency treatment, ICU wards and doctor clinics, are different from full-automatic biochemical analyzers or chemiluminescence meters and the like adopted in clinical testing centers and various large hospital clinical laboratory as a means for quickly diagnosing early disease treatment, and have the characteristics of simplicity, rapidness and accuracy. D-dimer colloidal gold quantitative products have been widely used in Europe and America.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an antibody capable of effectively and specifically binding to a human D-dimer. More specifically:

the first objective of the present invention is to provide two anti-human D-dimer antibodies.

A first anti-human D-dimer antibody (DD17),

the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO:2 and/or HCDR2 as shown in SEQ ID NO: HCDR3 shown at 3;

the light chain variable region sequence comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and/or LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.

Preferably, the amino acid sequence of the heavy chain variable region of the above antibody (DD17) is represented by SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 8.

A second anti-human D-dimer antibody (DD89),

the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 9, HCDR1 as shown in sequence SEQ ID NO: 10 and/or HCDR2 as set forth in sequence SEQ ID NO: HCDR3 shown in fig. 11;

the light chain variable region sequence comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 12, LCDR1 as shown in sequence SEQ ID NO: 13 and/or LCDR2 as set forth in sequence SEQ ID NO: LCDR3 shown at 14.

Preferably, the amino acid sequence of the heavy chain variable region of the above antibody (DD89) is represented by SEQ ID NO:15, and the amino acid sequence of the antibody light chain variable region is represented by SEQ ID NO: 16.

The second purpose of the invention is to provide two single-chain antibodies, wherein the amino acid sequence of the single-chain antibody (DD17) is shown as SEQ ID NO. 17; the amino acid sequence of the single-chain antibody (DD89) is shown in SEQ ID NO: 18.

The third purpose of the invention is to provide two nucleotide sequences for coding the single-chain antibody, wherein the nucleotide sequence for coding the single-chain antibody (DD17) is shown as SEQ ID NO. 19, and the nucleotide sequence for coding the single-chain antibody (DD89) is shown as SEQ ID NO. 20.

The fourth purpose of the invention is to provide an expression vector containing the nucleotide sequence.

The fifth object of the present invention is to provide a recombinant host cell containing the above expression vector. The host cell can be Escherichia coli, Pichia pastoris or mammalian cells, and is preferably Pichia pastoris.

It is a sixth object of the present invention to provide a method for producing the above single-chain antibody, comprising:

1) culturing the recombinant host cell under suitable conditions to express the antibody;

2) the antibody is then purified from the host cell and collected.

The seventh purpose of the invention is to provide the application of the anti-human D-dimer antibody in detecting the content of human D-dimer.

An eighth object of the present invention is to provide a set of antibody pairs DD17 and DD89 that can be paired and detect human D-dimer; the antibody has high detection sensitivity and good specificity to the combination.

The ninth purpose of the invention is to provide a colloidal gold immunochromatographic assay quantitative detection card for detecting human dimer by using the anti-human dimer antibody, which comprises a sample absorption pad, a gold label pad, a reaction membrane and a water absorption pad; the gold-labeled pad is sprayed with a colloidal gold particle-labeled antibody DD89, the reaction membrane is provided with a detection zone and a quality control zone, and the position of the detection zone is coated with an antibody DD 17.

The sub-blocking solution is 1-10% BSA, 0.01M PB solution, and pH 7.4.

The gold-labeled antibody complex solution is a solution of 1-10% BSA, 5% trehalose, 0.025% tween20 and 0.01M PB, and is pH 7.4.

The antibody coating solution is 0-0.25% BSA, 0-2.5% isopropanol, 0.01M PB solution, pH 7.4.

The sample dilutions were 0-2.5% BSA, 0-2.5% tween20, 0.01% -0.1% Proclin300, 0.01M PBS solution, pH 7.4.

The control band position is coated with an anti-His tag antibody or Protein L. The reaction membrane is preferably a nitrocellulose membrane. The anti-His tag antibody is preferably a murine anti-His antibody.

The invention prepares a plurality of antibodies, and performs pairing screening to obtain an antibody combination (DD17 and DD89) with sensitivity and specificity meeting the requirements; meanwhile, the preparation method is convenient for mass production and can meet the requirement of large-scale clinical application in the future. The antibody combination is debugged and optimized to obtain the colloidal gold quantitative detection test paper card which is simple and convenient to operate, and has sensitivity, specificity and related detection performance meeting the requirements of human clinical sample detection.

Drawings

FIG. 1 shows the specific detection effect of antibodies DD17 and DD89 (Western Blot),

lane 1 is standard protein (Marker); lane 2 is a human native D-dimer.

FIG. 1(a) detection antibody DD 17-HRP; FIG. 1(b) detection antibody DD 89-HRP.

FIG. 2 is a schematic diagram of the structure of the colloidal gold immunochromatographic assay quantitative detection card of the present invention. 1 is a sample pad, 2 is a reaction film, 3 is an absorption pad, 4 is a quality control line (C line), 5 is a detection line (T line), 6 is a gold label pad, and 7 is a PVC sheet.

FIG. 3 shows a D-dimer gold colloidal detection card detection range fitting curve.

FIG. 4. D-dimer colloidal gold assay card linear range fit curve.

FIG. 5 shows a fitting curve comparing the D-dimer colloidal gold test card with a control product.

Detailed Description

Definition of

"antibody", also known as immunoglobulin, is a large Y-shaped protein secreted by B lymphocytes, and is an immunoglobulin molecule capable of specifically binding to a target antigen, such as a protein, a sugar, a polynucleotide, a lipid, a polypeptide, a small molecule compound, etc., through complementary sites (antigen-binding sites) at the two bifurcated tips of the Y.

"Single chain antibody" (scFv) refers to the variable region of the heavy chain (V) of an antibody_H) And light chain variable region (V)_L) Is connected by 15-20 amino acid short peptides (linker)The linker used for connection is usually rich in glycine and serine, so that the stability and flexibility of the single-chain antibody are facilitated. The connection mode can be V_LIs connected to V_HC-terminal, or vice versa. Despite the removal of the constant region and the introduction of the linker, the single-chain antibody retains the specificity of the antibody to the antigen, and has the characteristics of small molecular weight, strong penetration, weak antigenicity and the like.

Complementary-determining regions (CDRs), also called hypervariable regions. Patterned at the amino acid end of the antibody monomer is the most critical region for binding of the target antigen to the antibody, and in immune network theory, the complementarity determining regions of each antibody are also called idiotypes or genotypes.

Example 1 preparation of anti-human D-dimer hybridoma cell lines

1. Animal immunization

BALB/c female mice (purchased from Kyoto Kavens laboratory animals Co., Ltd., Changzhou) were immunized with native human D-dimer (purchased from Meridian) following the general immunization protocol. For specific immunization, see "guidelines for antibody preparation and use". And tracking the serum titer of the immune mice by adopting an indirect ELISA method, selecting the immune mice with the highest serum titer, and performing fusion experiments on the spleen cells and myeloma cells of the mice.

2. Cell fusion

(1) Preparation of spleen cells

Immunized mice, eyeballs are picked and blood is taken, after cervical vertebra is cut off, the immunized mice are placed in 75% (v/v) alcohol for soaking for 10 minutes, spleens of the immunized mice are taken out from a sterile operating platform, the spleens are placed in a cell screen, cells are fully ground, the cells are screened, the spleen is centrifugally washed for a plurality of times by using sterile 1640 culture medium (purchased from Gibco company), and then the cells are resuspended to prepare single cell suspension, and the single cell suspension is counted for standby.

(2) Preparation of feeder cells

Taking one female BALB/c mouse 8-10 weeks old, picking an eyeball to obtain negative serum, and immersing the negative serum in 75% (v/v) alcohol for 10 minutes after the cervical vertebra is cut off; the abdominal skin was aseptically peeled, the peritoneum was exposed, and about 10mL of 1640HT medium (purchased from SIGMA) was injected into the abdominal cavity of the mouse with a syringe, and the abdomen was gently massaged and air-blown several times. Sucking the culture medium containing the macrophages and injecting the culture medium into 20% 1640HAT culture medium for later use;

taking one female BALB/c mouse with the age of 2-3 weeks, and immersing the mouse in 75% (v/v) alcohol for 10 minutes after the mouse dies after cervical vertebra breakage; aseptically placing thymus into a cell screen, grinding, sieving to obtain thymocytes, and placing the thymocytes into the 20% 1640HAT culture medium containing macrophages for later use.

(3) Cell fusion

Mouse myeloma cell line SP2/0 was selected at the logarithmic growth phase and collected and counted. Get about 10⁸The above spleen cells were combined with 2X 10⁷Each of the above SP2/0 cell lines was mixed in a fusion tube, centrifuged at 1000rpm for 10 minutes, and the supernatant was discarded (discarded as clean as possible), and the fusion tube was gently rubbed back and forth on the palm of the hand to loosen the pellet. 1mL of preheated PEG1450 (polyethylene glycol 1450, available from SIGMA) was added slowly and quickly over 60 seconds, 30mL of 1640HT medium was added and stopped, centrifuged at 1000rpm for 10 minutes, the supernatant was removed, the precipitate was loosened by gentle rubbing, and added to 20% of 1640HAT medium obtained in step 2.

Mixing the HAT culture medium, subpackaging at 200 μ L/well into 96-well cell culture plate, standing at 37 deg.C and 5% CO₂Cultured in a cell culture box. After one week, 20% 1640HAT medium was replaced with 10% 1640HT medium, and after 3 days, the supernatant was examined.

3. Screening of anti-human D-dimer specific hybridoma

(1) Preparation of the test plate: diluting natural human D-dimer (purchased from Meridian company) to 1 mu g/mL by using CB coating solution, coating a 96-hole ELISA plate with 100 mu L/hole, coating overnight at 2-8 ℃, washing and patting dry once; PBST buffer containing 2% bovine serum albumin was blocked (200 ul/well) for 2 hours at 37 ℃; patting dry for later use.

(2) Screening of positive clones: adding 100 μ L/well of cell culture supernatant to be detected into the detection plate, acting at 37 deg.C for 30min, washing, drying, adding 100 μ L/well HRP-labeled goat anti-mouse IgG, acting at 37 deg.C for 30min, washing, drying, adding 100 μ L/well TMB color developing solution, and developing at 37 deg.C in dark for 15min50 μ L of 2M H was added to each well₂SO₄The reaction was stopped and the value read at OD 450. Positive well determination principle: OD450 value/negative control value is not less than 2.1. Selecting positive clone strains to carry out cell cloning screening. After three to four rounds of cloning screening, the positive rate of the monoclonal cell strain is determined to be a stable cell strain with 100 percent of positive rate, and the cell strain is determined. The hybridoma cell strains DD17 and DD89 both have higher titer, and then the antibody variable region sequence sequencing analysis is further carried out on the hybridoma cell strains.

Example 2 determination of variable region sequences of antibodies of hybridoma cell lines

The sequences of the variable regions of the antibodies of the hybridoma cell lines DD17 and DD89 were determined.

Extraction of RNA: the hybridoma cell strains DD17 and DD89 were subjected to total RNA extraction and immediately reverse transcription with reference to the instructions of a cell total RNA extraction kit (purchased from Roche Co.);

reverse transcription of RNA into DNA: performing reverse transcription on the total RNA extracted in the previous step by referring to Thermo Scientific reversed First strand cDNA Synthesis Kit (purchased from Thermo company), preparing cDNA, and freezing and storing at-20 ℃ for later use;

c. PCR amplification and recovery of variable region sequences: performing PCR amplification on variable region sequences of a heavy chain and a light chain by using cDNA obtained in the previous step as a template and a universal primer of a mouse IgG subtype monoclonal antibody variable region sequence as a primer, and recovering a PCR product by using a DNA gel recovery kit (purchased from TIANGEN company);

d. cloning and sequencing of variable region sequences: according to the specification of cloning vector pMD18-T kit (purchased from Takara), the heavy chain and light chain variable region genes were ligated with pMD18-T vector, respectively, to transform E.coli DH 5. alpha. positive clones were picked up and submitted to Nanjing Kingsry Biotech Ltd for sequencing.

The amino acid sequence of the heavy chain variable region of the antibody of the hybridoma cell strain DD17 is shown as SEQ ID NO 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8. The Vbase2 database analyzes the above sequences, and the amino acid sequences of the complementarity determining regions of the heavy chain variable region are: as shown in sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO:2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3; the amino acid sequence of each complementarity determining region of the light chain variable region is: as shown in sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.

The amino acid sequence of the heavy chain variable region of the antibody of the hybridoma cell strain DD89 is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 16. The Vbase2 database analyzes the above sequences, and the amino acid sequences of the complementarity determining regions of the heavy chain variable region are: as shown in sequence SEQ ID NO: 9, HCDR1 as shown in sequence SEQ ID NO: 10 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown in fig. 11; the amino acid sequence of each complementarity determining region of the light chain variable region is: as shown in sequence SEQ ID NO: 12, LCDR1 as shown in sequence SEQ ID NO: 13 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown at 14.

Example 3 recombinant expression and purification of Single chain antibodies

According to the sequencing result in example 2, a connecting peptide (GGGGS)3 is added between the heavy chain and light chain variable regions of the antibodies of hybridoma cell strains DD17 and DD89, six histidines are introduced, the whole genes of the six histidines are directly fused, and the recombinant expression of the single chain antibody is carried out by using a pichia pastoris expression system. The expressed antibodies were designated as antibodies DD17 and DD89, respectively. The recombinant expression of the single-chain antibody is specifically as follows:

a) construction of Single chain antibody Gene expression vector

The gene sequence of the single-chain antibody DD17 is shown as SEQ ID NO. 19, and the amino acid sequence is shown as SEQ ID NO. 17; the gene sequence of the single-chain antibody DD89 is shown in SEQ ID NO. 20, and the amino acid sequence is shown in SEQ ID NO. 18. Introducing XhoI restriction sites at the upstream of single-chain antibody DD17 and DD89 gene fragments and DNA sequences behind the XhoI sequence in a pPICZ alpha A vector, introducing histidine tags and XbaI restriction sites at the downstream, carrying out whole-gene synthesis, and constructing into a pUC57 plasmid (purchased from Nanjing Kingsry Biotech, Ltd.) to obtain a long-term storage plasmid, wherein the plasmids are named as pUC57-DD17-scFv and pUC57-DD 89-scFv. Performing PCR amplification, wherein

The upstream primer P1 is TGT AAA ACG ACG GCC AGT;

the downstream primer P2 is CAG GAA ACA GCT ATG AC.

After a conventional PCR procedure, agarose gel electrophoresis analysis revealed that the product size was consistent with the expected size. After recovery and purification of the PCR-derived gene products, they were digested simultaneously with XhoI (# R0146S, available from New England Biolabs) and XbaI (# R0145V, available from New England Biolabs), ligated to pPICZ. alpha.A (V19520, available from Invitrogen) plasmid using T4 ligase, transformed into DH 5. alpha. competent cells, and cultured overnight at 37 ℃ in LB plates containing Zeocin (R250-01, available from Invitrogen). Screening positive clone bacteria for sequencing, comparing and completely conforming to expected sequences in the next day to obtain expression vectors of single-chain antibodies DD17 and DD89, which are marked as pPICZ alpha-DD 17-scFv and pPICZ alpha-DD 89-scFv.

b) Construction, screening and expression of single-chain antibody gene in pichia host engineering strain

YPDS solid medium preparation: refer to the Invitrogen company EasySelect Pichia Expression Kit Specification; pichia competent cells: refer to the easy select Pichia Expression Kit Specification; preparing a BMGY culture medium: refer to the Multi-Copy Pichia Expression Kit Specification by Invitrogen; preparing a BMMY culture medium: refer to the Multi-Copy Pichia Expression Kit Specification by Invitrogen.

The pPICZ alpha-DD 17-scFv and pPICZ alpha-DD 89-scFv plasmids were linearized by SacI restriction endonuclease. After ethanol precipitation, the linearized vector is electrically transformed into X-33 competent yeast cells, spread on YPDS solid medium containing Zeocin and cultured at 30 ℃ for 3-5 days, and then positive clones are generated.

Inoculating the obtained recombinant single-chain antibody gene engineering strains DD17 and DD89 in BMGY culture medium, culturing at 30 deg.C and 220rpm until the thallus density reaches OD₆₀₀Methanol was added every 24 hours to a final concentration of 1.0% (v/v) 2.0 to 6.0. After one week, the fermentation broth was collected.

c) Single chain antibody purification

Single-chain antibodies DD17 and DD89 were purified using a histidine-tagged affinity column, and HisTrap HP was selected as the pre-packed column, and the specific steps were as follows:

(1) impurity removal pretreatment of fermentation liquor: the supernatant of the single-chain antibody DD17 and DD89 fusion protein fermentation liquor obtained by the expression is centrifuged to collect the supernatant, and binding buffer is added to ensure that the final concentration of the supernatant is 300mM NaCl, 20mM NaH2PO4 and 10mM Imidazole, the pH is adjusted to 7.5, and the mixture is filtered by a 0.45 mu m filter membrane.

(2) HisTrap HP affinity column purification: the pretreated single-chain antibody DD17 and DD89 fermentation broth was subjected to affinity purification using a fully automated intelligent protein purification system (AKTA avant150, available from GE healthcare Co.), and the column was HisTrap HP (17-5248-02, available from GE healthcare Co.). The binding buffer was 300mM NaCl, 20mM NaH₂PO₄10mM Imidazole, pH7.5, and elution buffer 300mM NaCl, 20mM NaH₂PO₄500mM Imidazole, pH 7.5. Linear elution was performed during elution and the individual elution peaks were collected. The purity of the purified protein reaches more than 95 percent; the collection tubes meeting the requirements are combined, the buffer solution is changed into a PBS solution, ultrafiltration concentration (1mg/ml) is carried out, and the mixture is filtered and sterilized and stored at the temperature of minus 20 ℃ for standby.

Example 4 evaluation of antibody Performance

1. Western blot identification of antibodies DD17 and DD89

a. Polyacrylamide gel electrophoresis: preparing 7.5% separation gel and 5% concentration gel, loading (non-reducing) standard protein and human natural D-dimer (purchased from Hytest company), and performing electrophoresis at constant pressure for 1 hr;

b. film transfer: the membrane was spun for 80 minutes under constant flow (35 mA/membrane) to transfer the proteins on the polyacrylamide gel to nitrocellulose. Staining SDS-PAGE gel subjected to membrane transfer by Coomassie brilliant blue G250, and observing the residual condition of protein;

c. and (3) sealing: TBST buffer containing 5% skimmed milk was blocked (blocking solution) overnight at 4 ℃; washing with a washing solution (TBST, for details, TBST buffer of TaKaRa) once for 10 minutes after blocking;

d. antigen-antibody reaction: diluting a confining liquid (according to a volume ratio of 1: 1000), and adding a horseradish peroxidase marker DD17(DD17-HRP, 1mg/mL, labeled by a classic sodium periodate method in the company, the same below) and a horseradish peroxidase marker DD89(DD89-HRP, 1mg/mL, labeled by a classic sodium periodate method in the company, the same below) into the two cellulose nitrate membranes respectively, and reacting at room temperature for 1 hour; TBST washes 5 times for 10 minutes each;

e. and (3) color development and photographing: sucking up residual liquid on the nitrocellulose membrane, adding a mixed solution (purchased from Thermo company) of 2mL of a stable peroxidase solution (1mL) and a luminol/enhancer solution (1mL) into the nitrocellulose membrane, uniformly wetting the surface of the nitrocellulose membrane, carrying out a reaction at room temperature in a dark place for one minute, and then photographing in a gel imaging system (purchased from GE company) (figure 1) to obtain a result.

As seen in the detection results, the antibodies DD17 and DD89 both have good specificity, and can specifically detect human D-dimer.

2. Evaluation of single-chain antibodies DD17 and DD89 on colloidal gold detection platform

The antibodies purified in example 3 were paired and combined to detect D-dimer (D-dimer) as coating antibody or labeled antibody, respectively, as follows:

1) diluting DD17 or DD89 to 1.5mg/ml with antibody coating solution, and streaking on nitrocellulose membrane;

2) diluting colloidal gold labeled DD17 or DD89 with gold labeled antibody suspension, and spraying onto the bonding pad;

3) pasting, slitting and clipping the film as shown in the figure (see example 5 for concrete preparation)

4) Diluting a D-dimer standard substance (manufactured by Zhonghong) with a sample diluent until the concentration is 5mg/L and 1mg/L, respectively adding 50ul of the two concentration standard substances and a zero concentration standard substance (namely the sample diluent) into a colloidal gold detection card (DD 17-DD 89 mark or DD 89-DD 17 mark), and after 10min, placing the detection card on a reading instrument for reading. The results are shown in the following table:

from the above results, it can be seen that the double antibody sandwich detection system comprising DD17 as a coating antibody and DD89 as a labeled antibody can be applied to a colloidal gold detection platform for detecting D-dimers.

EXAMPLE 5 preparation of anti-D-dimer colloidal gold immunoassay card

1. Solution preparation

1)0.01M PB buffer preparation: weighing Na₂HPO₄·12H₂O 3.22g，NaH₂PO₄·2H₂0.15g of O, 1000ml of purified water is added, the mixture is stirred by a rotor until dissolved, the pH value is measured by a ph meter and is 7.4 +/-0.1, and the mixture is filtered by a 0.45um filter membrane.

2) Preparing a sealing liquid: bovine Serum Albumin (BSA) 5g was weighed, and 50ml of 0.01M PB solution (pH 7.4. + -. 0.1) was added thereto, and the mixture was stirred with a rotor until dissolved.

3) Preparation of antibody coating solution: 0.2mL of isopropanol was weighed and added to 9.8mL of 0.01M PB solution (pH 7.4. + -. 0.1) and stirred on a rotor for 5-10 min.

4) Preparing a gold-labeled antibody re-solution: weighing 1g bovine serum albumin and 5g trehalose, adding 100ml 0.01M PB solution (pH7.4 + -0.1), stirring with rotor until dissolved, adding 25ul Tween-20, and stirring with rotor for 5-10min

5) Preparation of sample diluent: adding 18.75g bovine serum albumin into 750ml 0.01M PB solution (pH7.4 + -0.1), stirring with rotor until dissolved, adding 0.75ml Proclin300, stirring with rotor for 5-10min, and filtering with 0.45um filter membrane.

2. Preparation of D-dimer colloidal gold immunoassay card

1) Labeling of colloidal gold

Colloidal gold labeling of antibody DD89 (exemplified with 1ml of colloidal gold solution): by K₂CO₃Adjusting the pH value of the colloidal gold (5 ul of 0.2M K is added into each 1ml of the colloidal gold₂CO₃) Stirring for 5-10min, slowly adding antibody DD89 (8 ug antibody DD89 is added into each 1ml of colloidal gold), and stirring at low speed for 30 min; adding 100ul of confining liquid, and stirring for 20 minutes; transferring the colloidal gold solution into a centrifuge tube, and centrifuging at 1500rpm and 2-8 ℃ for 15 min; transferring the supernatant into another centrifuge tube, and centrifuging at 12000rpm and 2-8 deg.C for 30 min; removing supernatant, and re-dissolving the precipitate with 70ul gold-labeled antibody re-solution to obtain the colloidal gold-labeled DD89 antibody.

2) Gold label pad and reaction film preparation

Spraying the DD89 gold-labeled antibody on the gold-labeled pad 6, and drying for later use;

the antibody DD17 is diluted to 1.5mg/ml by using an antibody diluent and coated on the position of the T line 5 of the reaction membrane 2 (a nitrocellulose membrane); after the anti-His tag antibody is diluted to 0.2mg/ml with an antibody diluent, the antibody is coated on the position of the C line 4 of the reaction membrane 2 (nitrocellulose membrane), and the reaction membrane is dried for later use.

3) Assembling big card, cutting strip and assembling

The sample pad 1, the gold-labeled pad 6, the nitrocellulose membrane 2 coated with the antibody and the absorbent pad 3 are sequentially pasted on a PVC bottom plate from left to right (as shown in figure 2), and the T line 5 of the nitrocellulose membrane coated with the antibody is arranged on the left, and the C line 4 is arranged on the right.

Cutting the large plate into small strips with the width of 2.92 mm; placing the cut strips in the clamping grooves of the bottom card, wherein the position of a sample adding hole of the panel corresponds to the sample pad; the panel is involuted with the bottom plate and is forcibly pressed to enable the panel to be inosculated with the bottom plate; when the card is pressed, the card is vertically arranged on a conveying belt of the shell pressing machine to be pressed in sequence.

4) Kit assembly

Packing the assembled detection card and drying agent into an aluminum foil bag, sealing the aluminum foil bag by a heat sealing machine, and labeling;

subpackaging the sample diluent according to 0.45 ml/tube, filling into a self-sealing bag according to the specification of the kit, and labeling;

according to the specification of a finished product, putting a certain number of parts of inner bags, 1 self-sealing bag containing sample diluent, 1 part of specification and 1 qualified label into a packaging box, and sticking the label outside the packaging box.

3. Method for using D-dimer colloidal gold detection card

1) The outer package was opened and the test card was removed from the sealed aluminum foil pouch and placed on a flat table.

2) A30. mu.l sample of plasma was aspirated and mixed well with 0.45ml of sample diluent.

3) 50ul of the treated sample was taken and added to the well of the test card, and allowed to stand at room temperature for 10 min.

4) The detection card is put into an immunochromatography quantitative analyzer, the detection is started by pressing a 'quick detection' key, and the detection card is automatically scanned by the analyzer.

5) The detection result is read/printed from the display screen of the immunochromatographic quantitative analyzer.

4. Evaluation of detection effect of D-dimer colloidal gold detection card

1) Precision: the DD17 (coated) -DD89 (labeled) detection card detects the D-dimer reference substance of 5mg/L and 1mg/L respectively for 10 times of repeated measurement according to the using method of the detection card, and the precision of the detection card is calculated after the outlier is removed. The experimental result shows that the coefficient of variation CV of the detection result is less than 12%.

Concentration (mg/L)	5	1
			CV	10.44％	9.92％

2) Detection range: the DD17 (coated) -DD89 (labeled) detection card is used for detecting D-dimer recombinant protein with different concentrations of 0.3/0.625/1.25/2.5/5/10mg/L, and the fitted curve and the detection range are 0.3-10mg/L (shown in figure 3).

3) Linear range: preparing 5 series concentration samples of 0.3/0.625/2.5/5/10mg/L from the high-value sample and the sample diluent, detecting by using a DD17 (coating) -DD89 (marking) detection card, detecting each sample for 3 times, performing regression statistics on the result and the theoretical concentration, and judging whether the concentration is linear in the concentration range. The linear range is 0.3-10mg/L (as shown in figure 4).

4) Accuracy: the DD17 (coating) -DD89 (marking) detection card detects the D-dimer reference substance of 5mg/L and 1mg/L respectively for 3 times according to the using method of the detection card, the relative deviation of the average value and the theoretical value is calculated, and the experimental result shows that the relative deviation B of the detection results of three concentrations is less than 15%.

Concentration (mg/L)	5	1
			B	0.13％	9.33％

5. Accuracy-methodological comparison

And selecting a commercialized D-dimer colloidal gold detection card which obtains good credit in the market in the same kind of products as comparison verification of the products. 30 clinical patient specimens were selected, numbered in the order of 1 to 30, and the test was performed simultaneously with the control product and the colloidal gold test card DD17 (coated) -DD89 (labeled) to be evaluated, in the order of 1, 2, 3. Correlation coefficient R of detection results of comparison and product to be evaluated²The result obtained by the two methods is better correlated to 0.987.

6. Formulation screening

In addition to the above-mentioned best preparation example 1, the applicant tried various preparation schemes, for example, the following sets of test cards were prepared and applied as follows:

SEQUENCE LISTING

<110> Jiangsu Zhonghong bioengineering institute of drug creation Limited

<120> human D-dimer quantitative detection card and clinical application thereof

<130> human D-dimer quantitative detection card and clinical application thereof

<160> 20

<170> PatentIn version 3.3

<210> 1

<211> 8

<212> PRT

<213> Mus musculus

<400> 1

Gly Tyr Thr Leu Ser Asp Asp Trp

1 5

<210> 2

<211> 8

<212> PRT

<213> Mus musculus

<400> 2

Ile Leu Pro Gly Asn Gly Leu Thr

1 5

<210> 3

<211> 13

<212> PRT

<213> Mus musculus

<400> 3

Ala Arg Gly Lys Val Arg Arg Val Tyr Tyr Phe Asp Tyr

1 5 10

<210> 4

<211> 5

<212> PRT

<213> Mus musculus

<400> 4

Ser Ser Val Ser Tyr

1 5

<210> 5

<211> 3

<212> PRT

<213> Mus musculus

<400> 5

Ser Thr Ser

1

<210> 6

<211> 9

<212> PRT

<213> Mus musculus

<400> 6

His Gln Trp Ser Ser Tyr Pro Cys Thr

1 5

<210> 7

<211> 120

<212> PRT

<213> Mus musculus

<400> 7

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Leu Ser Asp Asp

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Asn Gly Leu Thr Asn Tyr Asn Glu Arg Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Ser Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Lys Val Arg Arg Val Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 8

<211> 106

<212> PRT

<213> Mus musculus

<400> 8

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Glu Ile Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Asp Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Cys Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 8

<212> PRT

<213> Mus musculus

<400> 9

Val Phe Leu Phe Ser Asp Ala Trp

1 5

<210> 10

<211> 10

<212> PRT

<213> Mus musculus

<400> 10

Val Arg Asn Lys Pro Asn Asn His Ala Thr

1 5 10

<210> 11

<211> 11

<212> PRT

<213> Mus musculus

<400> 11

Thr Gly Thr Tyr Gly Asn Tyr Asn Phe Asp Tyr

1 5 10

<210> 12

<211> 6

<212> PRT

<213> Mus musculus

<400> 12

Gln Asp Val Ser Thr Ala

1 5

<210> 13

<211> 3

<212> PRT

<213> Mus musculus

<400> 13

Trp Ala Ser

1

<210> 14

<211> 9

<212> PRT

<213> Mus musculus

<400> 14

Gln Gln His Tyr Ser Thr Pro Pro Thr

1 5

<210> 15

<211> 120

<212> PRT

<213> Mus musculus

<400> 15

Glu Leu Ser His Met Val Asp Leu Gln Ala Ala Ala Asn Ser Leu Val

1 5 10 15

Tyr Pro Arg Leu Trp Asp Gly Ala Gly Val Phe Leu Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ser Pro Glu Arg Gly Leu Glu Trp Val

35 40 45

Ala Glu Val Arg Asn Lys Pro Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Thr Tyr Gly Asn Tyr Asn Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 16

<211> 107

<212> PRT

<213> Mus musculus

<400> 16

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 17

<211> 241

<212> PRT

<213> Artificial sequence

<400> 17

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Leu Ser Asp Asp

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Asn Gly Leu Thr Asn Tyr Asn Glu Arg Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Ser Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Lys Val Arg Arg Val Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala

130 135 140

Ile Met Ser Ala Ser Leu Gly Glu Glu Ile Thr Leu Thr Cys Ser Ala

145 150 155 160

Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr

165 170 175

Ser Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Phe Tyr Ser Leu Thr

195 200 205

Ile Ser Ser Val Glu Ala Glu Asp Ala Ala Asp Tyr Tyr Cys His Gln

210 215 220

Trp Ser Ser Tyr Pro Cys Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210> 18

<211> 242

<212> PRT

<213> Artificial sequence

<400> 18

Glu Leu Ser His Met Val Asp Leu Gln Ala Ala Ala Asn Ser Leu Val

1 5 10 15

Tyr Pro Arg Leu Trp Asp Gly Ala Gly Val Phe Leu Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ser Pro Glu Arg Gly Leu Glu Trp Val

35 40 45

Ala Glu Val Arg Asn Lys Pro Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Thr Tyr Gly Asn Tyr Asn Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser His Lys

130 135 140

Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala

145 150 155 160

Ser Gln Asp Val Ser Thr Ala Val Thr Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly

180 185 190

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

195 200 205

Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys Gln

210 215 220

Gln His Tyr Ser Thr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 19

<211> 723

<212> DNA

<213> Artificial sequence

<400> 19

caggttcagc tgcagcagtc tggacctgag ctgatgaagc ctggggcctc agtgaagata 60

tcctgcaagg ccactggcta cacgctcagt gacgactgga tagagtgggt aaagcagagg 120

cctggacatg gccttgagtg gattggagag attttacctg gaaatggtct tactaactac 180

aatgagagat tcaagggcaa ggccaccttc actgcagatt catcctccaa cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgccgtct attactgtgc aagggggaag 300

gtacgacggg tctactactt tgactattgg ggccaaggca ccactctcac agtctcctca 360

ggtggtggtg gatccggagg tggtggttct ggtggtggtg gttctcaaat tgttctcacc 420

cagtctccag caatcatgtc tgcatctcta ggggaggaga tcaccctgac ctgcagtgcc 480

agctcgagtg tgagttacat gcactggtac cagcagaagt caggcacttc tcccaaactc 540

ttgatttata gcacatccaa cctggcttct ggagtccctt ctcgcttcag tggcagtggg 600

tctgggacct tttattctct cacaatcagc agtgtggagg ctgaagatgc tgccgattat 660

tactgccatc agtggagtag ttatccatgc acgttcggag gggggaccaa gctggaaata 720

aaa 723

<210> 20

<211> 726

<212> DNA

<213> Artificial sequence

<400> 20

gagctctccc atatggtcga cctgcaggcg gccgcgaatt cactagtgta tccaagatta 60

tgggatggag cgggggtctt tctctttagt gacgcctgga tggactgggt ccgccagtct 120

ccagagaggg ggcttgagtg ggttgctgaa gttagaaaca aacctaataa tcatgcaaca 180

tactatgctg agtctgtgaa agggaggttc accatctcaa gagatgattc caaaagtagt 240

gtctacctgc aaatgaacag cttaagagct gaagacactg gcatttatta ctgtaccggg 300

acctatggta actacaactt tgactactgg ggccaaggca ccactctcac agtctcctca 360

ggtggtggtg gatccggagg tggtggttct ggtggtggtg gttctgacat tgtgatgacc 420

cagtctcaca aattcatgtc cacatcagta ggagacaggg tcagcatcac ctgcaaggcc 480

agtcaggatg tgagtactgc tgtaacctgg tatcaacaaa aaccagggca atctcctaaa 540

ctactgattt actgggcatc cacccggcac actggagtcc ctgatcgctt cacaggcagt 600

ggatctggga cagattatac tctcaccatc agcagtgtgc aggctgaaga cctggcactt 660

tattactgtc agcaacatta tagcactcct ccgacgttcg gtggaggcac caagctggag 720

atcaaa 726

Claims

1. The human D-dimer colloidal gold quantitative detection card comprises a sample absorption pad, a gold label pad, a reaction membrane and a water absorption pad; the gold label pad is sprayed with a second antibody marked by colloidal gold particles, the reaction membrane is provided with a detection zone and a quality control zone, and the position of the detection zone is coated with the first antibody;

the first antibody, wherein the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO:2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3; the light chain variable region sequence comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown at 6;

the second antibody, whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 9, HCDR1 as shown in sequence SEQ ID NO: 10 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown in fig. 11; the light chain variable region sequence comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 12, LCDR1 as shown in sequence SEQ ID NO: 13 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown at 14.

2. The human D-dimer colloidal gold assay card according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the first antibody is represented by SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 8.

3. The human D-dimer colloidal gold quantitative assay card according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the second antibody is represented by SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 16.

4. The human D-dimer colloidal gold assay card of claim 1, wherein the first antibody is a single chain antibody having an amino acid sequence as shown in SEQ ID NO. 17.

5. The human D-dimer colloidal gold quantitative assay card according to claim 1, wherein the second antibody is a single-chain antibody having an amino acid sequence represented by SEQ ID NO. 18.

6. The human D-dimer colloidal gold quantitative determination test paper card according to any one of claims 1 to 5, characterized in that: the blocking solution used for the preparation is 1% -10% BSA, 0.01MPB solution, pH 7.4.

7. The human D-dimer colloidal gold quantitative determination test paper card according to any one of claims 1 to 5, characterized in that: the antibody complex solution is 1-10% BSA, 5% trehalose, 0.025% tween20, 0.01MPB solution, pH 7.4.

8. The human D-dimer colloidal gold quantitative determination test paper card according to any one of claims 1 to 5, characterized in that: the antibody coating solution is 0-0.25% BSA, 0-2.5% isopropanol, 0.01MPB solution, pH 7.4.

9. The human D-dimer colloidal gold quantitative determination test paper card according to any one of claims 1 to 5, characterized in that: the sample dilutions were 0-2.5% BSA, 0-2.5% tween20, 0.01% -0.1% Proclin300, 0.01MPBS solution, pH 7.4.