CN111303281A

CN111303281A - Method for preparing pancreatic stone protein antibody and establishing and detecting PSP

Info

Publication number: CN111303281A
Application number: CN201910396634.6A
Authority: CN
Inventors: 刘雨哲; 李宝民; 关恒; 刘斌; 王珺楠; 张蕾; 黄景林; 张金玲; 赵青; 刘戈; 仇淑园; 史永丰; 王贺; 朱道林
Original assignee: Changchun Hengxiao Biotechnology Co ltd
Current assignee: Changchun Hengxiao Biotechnology Co ltd
Priority date: 2019-05-14
Filing date: 2019-05-14
Publication date: 2020-06-19

Abstract

A method for preparing a pancreatic stone protein antibody and establishing and detecting PSP belongs to the technical field of genetic recombination of bioengineering. The invention aims to prepare a specific PSP immunogen, generate a PSP monoclonal antibody or a PSP polyclonal antibody, and prepare a pancreatic stone protein antibody and establish a PSP detection method for establishing a PSP detection method and a PSP in-vitro diagnostic kit. The invention constructs a label-PSP recombinant protein vector by using a gene recombination technology, expresses the label-PSP recombinant protein by using bacteria, yeast, insect cells, animal or human cells, and purifies the label-PSP recombinant protein to be used as PSP recombinant protein immunogen. The invention is used for detecting the increase of the PSP level in blood, can provide earlier, faster and more accurate diagnosis of the occurrence of bacterial septicemia, guide the treatment of the septicemia, monitor the progress of the septicemia and establish an immunological method for rapidly detecting the PSP kit.

Description

Method for preparing pancreatic stone protein antibody and establishing and detecting PSP

Technical Field

The present invention belongs to the field of gene recombination technology in bioengineering.

Background

Sepsis (Sepsis) is a potentially life-threatening condition that is mainly caused by bacterial, viral and fungal infections that trigger systemic reactions in the body, which further progresses to septic shock, which causes a large drop in blood pressure and rapid failure and death of vital organs.

Early diagnosis of sepsis is a life saving concern, with mortality rising 8% per hour of delayed treatment for sepsis, and there is a need to provide an earlier, faster, and more accurate diagnostic protocol for sepsis that will substantially reduce sepsis death.

Bacterial infection induces the production of host proteins, such as IL-6, PCT and CRP, which are tested to support the diagnosis of sepsis caused by bacterial infection. CRP is not specific for diagnosing sepsis because CRP levels are also elevated in inflammatory states of non-infectious etiology. PCT is a specific index for diagnosing septicemia, but partial patients without infection after operation have an increased PCT level in trauma patients; in the early stage of bacterial infection, the PCT level of some patients is not increased, which indicates that PCT can not be used as the only index for judging the occurrence of bacterial infectious septicemia, and a new bacterial induction host protein index needs to be searched for so as to improve the accuracy of early diagnosis of bacterial infectious septicemia.

Pancreatic Stone Protein (PSP) is a novel biomarker for diagnosing bacterial infectious sepsis in the blood circulation.

PSP is a protein extracted from pancreatic calculus and pancreatic juice of patients with chronic calcific pancreatitis, and 16KDa protein is coded by a regeneration gene (Regenerating gene), and belongs to C-lectin family. PSPs are mainly produced and secreted from acinar cells of the pancreas, and cells also secrete a small amount of PSPs in the stomach, intestinal tract, and the like.

Disclosure of Invention

The invention aims to prepare a specific PSP immunogen, generate a PSP monoclonal antibody or a PSP polyclonal antibody, and prepare a pancreatic stone protein antibody and establish a PSP detection method for establishing a PSP detection method and a PSP in-vitro diagnostic kit.

The invention constructs a label-PSP recombinant protein vector by using a gene recombination technology, expresses the label-PSP recombinant protein by using bacteria, yeast, insect cells, animal or human cells, and purifies the label-PSP recombinant protein as PSP recombinant protein immunogen; PSP protein amino acid sequence:

QEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA 60

FVASLIKESGTDDFNVWIGLHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYCVSLTS 120

STGFQKWKDVPCEDKFSFVCKFKN。

the invention synthesizes PSP epitope peptide-N, amino acid sequence

SEQ ID NO.1：CQNMNSGNLVSVLTQAEGAFVASLIKESGTDDF；

Synthesis of PSP epitope peptide-C, amino acid sequence

SEQ ID NO.2：LHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYC。

The PSP epitope peptide-N or C coupled carrier protein comprises Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Blue Carrier Protein (BCP) and the like, and is prepared into PSP epitope peptide-carrier protein immunogen.

The PSP recombinant protein immunogen of the invention can be used for immunizing animals including mice, rats, rabbits, goats, sheep, alpacas, donkeys and horses to produce PSP monoclonal antibodies or polyclonal antibodies.

The PSP epitope peptide-carrier protein immunogen of the invention can be used for immunizing animals including mice, rats, rabbits, goats, sheep, alpacas, donkeys and horses to produce PSP monoclonal antibodies or polyclonal antibodies.

The PSP recombinant protein of the invention is used for protein renaturation and is used as a calibrator for a PSP detection method.

The PSP monoclonal antibody or polyclonal antibody and the PSP calibrator establish a method for detecting the PSP, including ELISA, chemiluminescence, immunofluorescence, immunoturbidimetry, immunochromatography, immunoagglutination and the like.

The PSP monoclonal antibody or polyclonal antibody and the PSP calibrator establish a kit for detecting the PSP in vitro diagnosis, wherein one is a fast quantitative detection PSP immunochromatography test strip, and the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially connected and fixed on a PVC base plate.

The binding pad adsorbs PSP antibody labeled fluorescent microspheres; and a detection line (T line) formed by a PSP antibody and a quality control line (C line) formed by an anti-IgG antibody are fixed on the nitrocellulose membrane.

The preparation process of the PSP recombinant protein comprises the following steps:

(1) synthesizing PSPDNA; (2) constructing a pQE-1-PSP vector; (3) expressing the His-PSP protein; (4) separating and purifying His-PSP protein with Ni-NTAAgarose and ion chromatographic column; (5) carrying out His-PSP protein renaturation and concentration processes; (6) measuring the concentration of His-PSP protein, and freezing and storing at-80 ℃;

His-PSP recombinant protein amino acid sequence:

MKHHHHHHQLHAGAHQEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA

FVASLIKESGTDDFNVWIGLHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYCVSLTSSTGFQKWKDVPCEDKFSFVCKFKN。

the PSP is also an inflammatory factor, can induce the shedding of L-selectin in neutrophils and reduce the β integrin expression, activates the neutrophils, so the PSP is a marker for inflammation occurrence, the increase of the PSP level is closely related to the expression of CD11b and CD62L on the neutrophils, and the increase of the PSP level in blood circulation represents the occurrence of early bacterial infection.

Up-regulating PSP expression during the onset of acute and chronic pancreatitis; systemic stress, in the absence of any pancreatic inflammation, also promotes increased pancreatic PSP expression and secretion, suggesting that PSP is also a systemic stress marker.

According to clinical researches, the PSP level (146 ng/ml) in serum of patients without pancreatic gland injury and patients with sepsis in the hospitalization process is higher than that of patients with local bacterial infection (111ng/ml), and the PSP level of patients with local bacterial infection is higher than that of patients without infection (22 ng/ml).

The sensitivity and specificity of the PSP detection index are superior to those of the currently used indexes such as PCT, CRP, leukocyte count and the like, the PSP is superior to the PCT in the aspect of distinguishing bacterial infection septicemia and non-infectious diseases, and the level of the PSP is measured within 24 hours after a patient enters an ICU, so that the death risk of the patient is more accurate than that of the PCT and the CRP. The detection of the increase of the PSP level in blood can provide earlier, faster and more accurate diagnosis of the occurrence of the bacterial septicemia, guide the treatment of the septicemia, monitor the progress of the septicemia and establish an immunological method for rapidly detecting the PSP kit.

Drawings

FIG. 1 is a side view of an immunochromatographic test strip; FIG. 1, bottom plate; 2. a sample pad; 3. a bonding pad; 4. a nitrocellulose membrane; 5. detecting lines; 6. a quality control line; 7. absorbent paper;

FIG. 2 is a front view of an immunochromatographic paper card; FIG. 5, detection line; 6. a quality control line; 8. a sample application hole; 9. detecting a window; 10. a housing; 11. a handle; 12. an anti-slip surface;

FIG. 3 shows the determination of His-PSP purity by silver staining;

FIG. 4 is a PSP concentration standard curve;

FIG. 5 is a plot of PSP calibrator concentration versus fluorescence intensity units.

Detailed Description

QEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA 60

FVASLIKESGTDDFNVWIGLHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYCVSLTS 120

STGFQKWKDVPCEDKFSFVCKFKN。

the invention synthesizes PSP epitope peptide-N, amino acid sequence

SEQ ID NO.1：CQNMNSGNLVSVLTQAEGAFVASLIKESGTDDF；

Synthesis of PSP epitope peptide-C, amino acid sequence

SEQ ID NO.2：LHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYC。

His-PSP recombinant protein amino acid sequence:

MKHHHHHHQLHAGAHQEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA

the present invention is described in further detail below:

in order to establish a method for detecting PSP, the invention provides a human PSP recombinant protein immunogen and a PSP epitope peptide immunogen which are used for generating a PSP monoclonal antibody or a PSP polyclonal antibody, and a method for detecting PSP and a PSP in-vitro diagnostic kit are established by using the PSP monoclonal antibody or the PSP polyclonal antibody.

Specifically, the invention discloses a preparation process of a PSP recombinant protein, which comprises the following steps:

(1) synthesizing PSPDNA; (2) constructing a pQE-1-PSP vector; (3) expressing the His-PSP protein; (4) separating and purifying His-PSP protein with Ni-NTAAgarose and ion chromatographic column; (5) carrying out His-PSP protein renaturation and concentration processes; (6) measuring the concentration of His-PSP protein, and freezing and storing at-80 ℃.

His-PSP recombinant protein amino acid sequence:MKHHHHHHQLHAGAHQEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA

FVASLIKESGTDDFNVWIGLHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYCVSLTSSTGFQKWKDVPCEDKFSFVCKFKN

the His-PSP recombinant protein is close to the PSP protein with a natural structure through protein renaturation, has the molecular weight of 16KDa, is suitable to be used as a protein immunogen, has high immunogenicity and specificity, and is used for immunizing animals (such as mice, rats, rabbits, goats, sheep, alpacas, donkeys, horses and the like) to generate a specific PSP monoclonal antibody or polyclonal antibody.

The invention discloses a PSP antibody preparation process, which comprises the following steps:

(1) His-PSP recombinant protein as immunogen; (2) immunizing an animal to produce a PSP antiserum; (3) determining PSP antiserum titer and specificity; (4) His-PSP coupled resin; (5) separating and purifying the PSP antibody by using a His-PSP-resin chromatographic column; (6) PSP antibody titers and specificities were determined.

Immunization of animals with His-PSP protein may include His antibody in the antiserum, which is bound with His peptide (MKHHHHHHQLHAGAHQE) to His antibody in His-PSP antiserum to form a His-peptide-Hisantibody complex, which is depleted of His antibody binding to His-PSP protein.

The invention discloses a preparation process of PSP epitope peptide-carrier protein immunogen, which comprises the following steps:

synthesizing PSP epitope peptide-N (SEQ ID NO.1), wherein the amino acid sequence is 64-96:

CQNMNSGNLVSVLTQAEGAFVASLIKESGTDDF;

synthesizing PSP epitope peptide-C (SEQ ID NO.2), wherein the amino acid sequence is 102-137:

LHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYC。

the PSP epitope peptide-N or C has hydrophilicity and strong antigenicity, and is coupled with carrier protein to prepare PSP epitope peptide-carrier protein immunogen. The immunogen has high immunogenicity and specificity, and can be used for immunizing animals to produce PSP monoclonal antibodies or polyclonal antibodies. In the present invention, carrier proteins that may be used include Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Blue Carrier Protein (BCP), and the like. The activated BCP can be effectively combined with the terminal-SH group of the PSP epitope peptide, the BCP has strong immunogenicity and good immune effect, has a far relationship with immune animals, and is not easy to cause cross reaction by taking the BCP as carrier protein, so the BCP carrier protein is preferred.

The invention discloses a process for preparing a PSP monoclonal antibody by using a PSP epitope peptide-carrier protein immunogen, which comprises the following steps:

1. immunization

The invention uses PSP epitope peptide-N or C coupled BCP protein carrier, after purifying PSP-N/C-BCP, PSP epitope peptide immunogen is obtained, and then mice are immunized, and ELISA is used for detecting mice with high PSP antibody titer, and the mice are used for cell fusion.

2. Cell fusion

Fusing mouse spleen cells and Sp2/0 cells by using polyethylene glycol (PEG) as a fusion agent, screening hybridoma cells by using HAT culture solution, and detecting hybridoma cell wells secreting PSP monoclonal antibodies by using ELISA.

3. Screening out hybridoma cells secreting PSP monoclonal antibody

And (3) carrying out subcloning screening on the cells with the positive detection holes by using a limiting dilution method to obtain a hybridoma cell strain secreting the monoclonal antibody, carrying out mass amplification, and carrying out long-term subculture to obtain the hybridoma cell strain stably secreting the monoclonal antibody.

4. Characterization of PSP monoclonal antibodies by ELISA

Identifying IgG class PSP monoclonal antibodies using ELISA; PSP monoclonal antibody affinity was determined by ELISA.

5. Preparation of Large amounts of PSP monoclonal antibodies

Amplification culture of secreted PSP monoclonal antibody hybridHybridoma cell line, collecting cell supernatant, and treating with (NH)₄)₂SO₄Precipitating the monoclonal antibody in the supernatant to obtain a monoclonal antibody concentrated solution; separating and purifying the ProteinA-Agarose to obtain a PSP monoclonal antibody, measuring the concentration of the monoclonal antibody, and storing at-20 ℃; the binding capacity and specificity of the purified PSP monoclonal antibody and PSP are determined by ELISA.

The invention discloses a preparation process of a PSP calibrator, which comprises the following steps:

(1) synthesizing PSPDNA; (2) constructing a pQE-1-PSP vector; (3) expressing and purifying the His-PSP protein; (4) performing His-PSP protein renaturation; (5) concentrating the His-PSP protein; (6) detecting the concentration of His-PSP protein, and freezing and storing at-80 ℃.

The invention further establishes a method for detecting PSP by using the PSP monoclonal antibody or polyclonal antibody and a PSP calibrator, and the method comprises an enzyme-linked immunosorbent assay (ELISA), a Chemiluminescence assay (CLIA), an immunofluorescence assay (FIA), an immunoturbidimetric assay (Turbidimetric Immunoassay), an Immunochromatography assay (immunochromagraphy), an immunoagglutination assay (immuno Agglutination) and the like.

The invention discloses a process for establishing a PSP (phosphosilicate protein) detection in-vitro diagnostic kit, in particular to a PSP immunochromatographic test strip for rapid quantitative detection, which comprises the following steps:

the preparation process of the PSP antibody-fluorescent microsphere comprises the following steps:

(1) carbodiimide (EDC) and N-hydroxyl Sulfo-succinimide (Sulfo-NHS) activated carboxyl modified fluorescent microspheres; (2) adding a PSP antibody to perform coupling reaction; (3) terminating the coupling reaction; (4) suspending the PSP antibody-fluorescent microspheres by using Tris buffer solution, and storing at 4 ℃ for later use.

The invention discloses a preparation process of a binding pad adsorbed with PSP antibody-fluorescent microsphere, which comprises the following steps:

and (3) diluting the concentration of the PSP antibody-fluorescent microspheres to the required concentration by using a Tris buffer solution, spraying the fluorescent microspheres on a bonding pad at the speed of 4-10ul/cm, drying for 2-8 hours at the temperature of 30-37 ℃, and drying and sealing for later use.

The invention discloses a preparation process of a T line and a C line on a nitrocellulose membrane, which comprises the following steps:

diluting PSP antibody (T line liquid) and anti-IgG antibody (C line liquid) with Tris buffer solution to required concentration, spraying the T line liquid and the C line liquid onto nitrocellulose membrane at the speed of 0.4-1.0ul/cm to form T lines and C lines at the interval of 4-6mm at 30-37 deg.C, drying for 2-6 hr, drying and sealing for use.

The invention discloses a test strip assembling process:

sticking a nitrocellulose membrane in the middle of the PVC bottom plate, and sticking absorbent paper at one end, wherein the absorbent paper is positioned above the nitrocellulose membrane; the other end is stuck with a bonding pad and is positioned above the nitrocellulose membrane; the other end of the bonding pad is adhered with a sample pad and is positioned above the bonding pad to form a test paper board, and the test paper board is cut into test paper strips with the width of 3-5mm (figure 1).

The invention discloses a test paper card assembling process, which comprises the following steps:

the invention comprises a shell which is composed of a shell surface and a shell base. And (3) placing the test strip on a shell base, and closing the shell surface to form the test strip card. A detection window is arranged on the shell surface corresponding to the position of the nitrocellulose membrane, and a sample adding hole is arranged on the shell surface corresponding to the position of the sample pad, as shown in figure 2.

The invention adopts the principle of double-antibody sandwich immunochromatography: PSP in a sample moves forwards through chromatography, is combined with PSP antibody-fluorescent microspheres adsorbed by a combination pad to form a PSP-PSP antibody-fluorescent microsphere compound, enters a nitrocellulose membrane under the action of capillary power, and is combined with PSP antibody fixed on a T line of the nitrocellulose membrane to form a fluorescent microsphere-PSP antibody-PSP-PSP antibody compound, and is gathered on the T line, free PSP antibody-fluorescent microspheres are not combined at the T line, continue to move forwards and are combined with anti-IgG antibody fixed on a C line, and unbound components continue to move to a water absorption paper position. The quantity of fluorescent microspheres captured by the T line is positively correlated with the concentration of PSP in the sample, and the fluorescent microspheres captured by the C line indicates that the immunochromatography process is completed. And scanning the signals of the T line and the C line by an immunofluorescence analyzer, and obtaining the concentration of the PSP in the sample according to the positive correlation between the intensity of the signals of the T line and the concentration of the PSP in the sample.

The invention provides a PSP immunochromatography test paper card operation process:

adding the PSP calibrator into a sample adding hole of a PSP immunochromatography test paper card, reacting for 5-10 minutes, measuring the fluorescence intensity of a T line and a C line on the test paper card by using an immunofluorescence analyzer, preparing a PSP concentration and fluorescence intensity unit correlation curve, setting an immunofluorescence analyzer automatic PSP concentration detection system after further setting the PSP concentration and fluorescence intensity unit correlation parameters of the immunofluorescence analyzer, and further detecting the PSP concentration in a sample.

The principles and results of the present invention are further illustrated in conjunction with specific experiments, which set forth the multi-step experimental procedures of the present invention.

Preparation of PSP recombinant protein

Synthesis of PSPDNA (nucleic acid sequence 67-501)

67caagaggccca

78gacagagttg ccccaggccc ggatcagctg cccagaaggc accaatgcct atcgctccta

138ctgctactac tttaatgaag accgtgagac ctgggttgat gcagatctct attgccagaa

198catgaattcg ggcaacctgg tgtctgtgct cacccaggcc gagggtgcct ttgtggcctc

258actgattaag gagagtggca ctgatgactt caatgtctgg attggcctcc atgaccccaa

318aaagaaccgc cgctggcact ggagcagtgg gtccctggtc tcctacaagt cctggggcat

378tggagcccca agcagtgtta atcctggcta ctgtgtgagc ctgacctcaa gcacaggatt

438ccagaaatgg aaggatgtgc cttgtgaaga caagttctcc tttgtctgca agttcaaaaa

498ctag

Synthesizing PSPDNA to express PSP protein amino acid sequence:

QEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA 60

FVASLIKESGTDDFNVWIGLHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYCVSLTS 120

STGFQKWKDVPCEDKFSFVCKFKN

catcaagaggcc ……………… aaa aactagg

tcgagtagttctccgg……………… ttt ttgatccagct

SacI SalI。

(II) construction of pQE-1-PSP vector

pQE-1 vector DNA 3ul (3 ug)

Cutsmart buffer 6ul

SacI 3ul

SalI 3ul

H20 45ul

Mixing, reacting at 37 deg.C for 2 hr

2. Placing in 1% Agarose/TBE gel electrophoresis hole, performing electrophoresis, performing DNA staining by EB, observing 3.5kb carrier DNA strip under ultraviolet, cutting DNA strip, placing in a microcentrifuge tube

3. DNA in the Agarose band was extracted using the QIAquick Gel Extraction Kit (QIAGEN),

4. determination of DNA concentration with Nanodrop Spectrophotometer

5. T4 DNA ligase (New England Biolabs.), was used to ligate the PSP DNA into the pQE-1 vector,

adding the following substances into a microcentrifuge tube

PSPDNA 50ng

50ng of vector DNA (SacI/SalI)

10X T4 DNA LigaseBuffer 1.0 ul

T4 DNA Ligase 0.5ul

H2O to 10ul

Mixing evenly, reacting for 4 hours at 25 ℃,

6. ligation of DNA into competent bacteria

7. Bacterial Plasmid DNA was extracted using the PureLink Quick Plasmid Miniprep Kits (Life Technologies Inc.)

8. Identifying pQE-1-PSP vector for expressing His-PSP recombinant protein.

(III) expression of His-PSP recombinant protein

pQE-1-PSP vector transfer into competent bacteria (M15 bacterial strain)

2. 50ul of the above-mentioned bacterial solution was plated on 25ug/ml Kanamycin, 100ug/ml Ampicillin LB-agar plate, and cultured overnight at 37 deg.C

3. Individual colonies were picked and inoculated into 5ml of 25ug/ml Kanamycin, 100ug/ml Ampicillin LB medium, 37 ℃ with shaking overnight

4. Diluting the bacterial solution to 100ml, shaking at 37 deg.C for 2-3 hr, OD₆₀₀At a value of 0.2 to 0.5,

5. adding 200ul IPTG (1.0M) into the bacterial liquid, shaking at 25 deg.C for 3-5 hr

6. Centrifuging, collecting bacteria, washing with PBS once

7. Adding PBS (pH7.5), containing 0.1% NP-40,1.0mM DTT,1.0mM PMSF,10ug/ml Leupepptin, 10ug/ml aprotinin into the centrifuge tube, suspending the bacteria

8. After disruption of the bacteria by sonication, centrifugation was carried out at 13000rpm for 20 minutes at 4 ℃ and the supernatant was transferred to a fresh centrifuge tube.

(IV) purification of His-PSP recombinant protein

1. Preparation of SP-Sepharose column chromatography using Buffer A (20mM NaPO)₄pH8.0,100mM NaCl,0.1% CTAB) equilibrium SP-Sepharose column chromatography

2. Adding the bacterial supernatant (containing His-PSP, pI = 6.5) into SP-Sepharose column, passing through SP-Sepharose column, collecting filtrate, adding into SP-Sepharose column, repeating for 5 times, and collecting filtrate (containing His-PSP protein)

Tris buffer wash Ni-NTAAgarose (QIAGEN)

4. Adding Ni-NTA Agarose into the filtrate, and reacting at 4 deg.C for 1 hr

5. Centrifuging at 4 deg.C for 2 min at 3000rpm, and removing supernatant

6. Add 50mM Tris buffer, 300mM NaCl,10mM imidazole,0.05% NP-40 to Ni-NTA Agarose centrifuge tubes and wash 4 times

7. Elution of the bound protein was performed 2 times by adding an eluent (50mM Tris, pH8.0, 200mM NaCl, 500mM MIDdazole) to a Ni-NTA Agarose centrifuge tube

8. The mixed eluate was transferred into a dialysis bag (Spectra/Pro), and placed in a dialysate (50mM Tris, pH7.6,150mM NaCl, 5% Glycerol, 1.0mM DTT,1.0mM EDTA, 1.0mM PMSF) under slow agitation at 4 ℃ overnight

9. Collecting protein liquid in the dialysis bag and transferring the protein liquid into a centrifuge tube

10. Preparation of Q-Sepharose column chromatography on Buffer B (50mM K)₂PO₄pH8.0,100mM NaCl, 0.2% CTAB) equilibrium Q-Sepharose column

11. Adding the dialysate into a Q-Sepharose chromatographic column, passing through the Q-Sepharose chromatographic column, collecting filtrate,

adding into Q-Sepharose chromatographic column, repeating for 5 times (His-PSP protein binding to Q-Sepharose chromatographic column)

12. Adding Buffer B to the Q-Sepharose column, and slowly passing through the Q-Sepharose column

13. Eluent (0.5M K)₂PO₄pH4.5, 300mM NaCl,0.1% CTAB) to a Q-Sepharose column, passing the eluate slowly through the Q-Sepharose column, and collecting the eluate (containing His-PSP protein)

14. Transferring the eluate into dialysis bag (Spectra/Pro), and adding dialysate (20mM NaPO)₄pH7.5,100mM NaCl, 5% Glycerol, 1.0mM DTT,1.0mM EDTA, 1.0mM PMSF) at 4 deg.C with slow agitation and passing through the solution

15. Transferring the dialyzed protein solution in the dialysis bag into a microcentrifuge tube, centrifuging at 13000rpm for 10 minutes at 4 DEG C

16. Adding the dialyzed protein solution into a protein concentration and purification centrifugal column (Microcon-3 KDa centrifugal Filter), and concentrating and purifying the dialyzed protein solution in the centrifugal column

17. And (4) measuring the concentration of the protein solution, and freezing and storing at-80 ℃.

(V) silver staining method for determining His-PSP protein purity (FIG. 3)

1. Placing the frozen protein liquid centrifuge tube into a water bath at 37 ℃, quickly dissolving, and placing into ice blocks

2.1.0ul protein solution, 5ul PBS, 4ul 4X protein sample solution (50mM Tris, pH 6.8, 2% SDS,10% Glycerol,1% β -mercaptoethanol, 12.5mM EDTA, 0.02% Bromophenol blue), 3 minutes at 100 deg.C

3. Adding 10ul protein sample solution into 10% SDS-PAGE gel hole, and performing electrophoresis

4. After completion of the electrophoresis, SDS-PAGE gels were taken and silver stained, as follows:

i. 100ml of 50% Methanol was added to a container of SDS-PAGE gel, and the mixture was slowly shaken at room temperature for 15 minutes,

removal of Methanol liquid

Ii, adding 100ml of 5% Methanol into the SDS-PAGE gel container, slowly shaking at room temperature for 10 minutes, removing the Methanol liquid, and washing the SDS-PAGE gel 1 time with water

Adding 10ul of DTT to a SDS-PAGE gel container containing 200ml of water, shaking slowly at room temperature for 10 minutes, removing DTT liquid, washing the SDS-PAGE gel 1 time with water

Iv addition of 100ml of 0.1% AgNO₃Placing in SDS-PAGE gel container, slowly shaking at room temperature for 15min, removing AgNO₃Liquid, washing SDS-PAGE gel 2 times with water

V. adding 100ml of 3% Na₂CO₃&0.05% Formaldehyde was allowed to react at room temperature in SDS-PAGE gel containers until clear protein bands were observed

Vi, adding Critic Acid to SDS-PAGE gel container, and stopping reaction

Vii, after the SDS-PAGE gel was washed with water, the SDS-PAGE gel was dried, and the purity of the His-PSP was judged to be 99% or more.

(VI) determination of purified His-PSP concentration by Bradford method (FIG. 4)

1. 1ml of concentrated Dye Reagent (Bio-Rad Protein Assay Dye Reagent Concentrate) was taken together with 4ml of H₂O

Mixing uniformly to obtain the diluted dye reagent

2. BSA standard solutions were prepared at concentrations of 0, 0.25, 0.5, 1.0, 2.0, 4.0mg/ml

3. Adding 79ul PBS into the well of the microtiter plate, adding 1.0ul BSA standard solution with different concentrations, and purifying 1.0ul His-PSP protein

Liquid, 3 wells per sample

4. Add 80ul of diluted dye reagent to microtiter plate wells at room temperature and allow to stand for 5 minutes

5. Placing the microtiter plate into a biochemical analyzer, selecting the wavelength of 495nm, and measuring the OD value

6. Preparing BSA concentration standard curve, and calculating the concentration of the purified His-PSP protein

The mean OD of the purified PSP was 1.903

His-PSP protein concentration =1.4476X 1.903-0.6203 =2.13mg/ml

。

Second, preparation of PSP antibody

(one) production of PSP antiserum

1. (Day 0) Collection of 5ml goat blood

2. (Day 1)0.5ml PSP (1.0mg/ml) was mixed with 0.5ml Freund's complete adjuvant (CFA), and injected 20 points subcutaneously (50 ul per point) into goats

3. (Day 14)0.25ml PSP (1.0mg/ml) was mixed with 0.25ml Freund's incomplete adjuvant (IFA) and injected 10 points subcutaneously (50 ul per point) into goats

(Day 28)0.25ml PSP (1.0mg/ml) was mixed with 0.25ml IFA and injected 10 points subcutaneously (50 ul per point) into goats

5. (Day 42) goat blood 5ml was collected and PSP antiserum titer and specificity were determined

6. (Day 45) 0.25ml PSP (1.0mg/ml) was mixed with 0.25ml IFA and injected 10 points subcutaneously (50 ul per point) into goats

7. (Day 59) Collection of 200ml goat blood

8. (Day 60) 0.25ml PSP (1.0mg/ml) was mixed with 0.25ml IFA and injected 10 points subcutaneously (50 ul per point) into goats

(Day 74) Collection of 200ml goat blood

10. Repeat 8-9 steps 2-3 times, terminate the test.

(III) determination of PSP antiserum titre and specificity

1. His-PSP (10. mu.g/ml), BSA (10. mu.g/ml) coating solution was prepared with 50mM carbonate buffer (pH9.6), and 1OOul coating solution was added to the well of the enzyme reaction plate, and washed 2 times with PBS overnight at 4 ℃

2. Add 200ul blocking PBS (0.5% BSA) per well, wash 2 times in PBS at room temperature for 2 hours

PSP antiserum (1.0mg/ml His-peptide) was diluted to different concentrations (1: 10, 1:50, 1:200, 1: 1000), 100ul was added to the wells, the wells were shaken slowly at room temperature for 60 minutes, and the reaction solution was aspirated

4. The enzyme reaction plate was washed 5 times with PBST (0.02% Tween 20) for 5 minutes each

5. Add 1:4000 dilution 100ul anti-goat IgG antibody-HRP (Promega) to the wells, shake slowly at room temperature for 60 min,

aspirating antibody fluids

6. The enzyme reaction plate was washed 5 times with PBST for 5 minutes each

7. 100ul HRP Substrate (TMB Substrate buffer, Santa Cruz Biotech Inc.) was added and reacted for 10 minutes

8. Add 50ul 2N H₂SO₄Terminating the reaction

9. Placing the enzyme reaction plate into a MULTISKAN FC analyzer, selecting a wavelength of 450nm, and measuring OD value

。

(IV) His-PSP coupled agarose

1.6.0mg His-PSP protein, 200mg NHS Activated Agarose (NHS-Activated Agarose) was purchased from ThermoFisher scientific, dissolved 2ml in PBSpH7.5, and shaken for 2 hours

2. Transferring the reaction solution into a centrifuge tube, centrifuging for 2 minutes at 3000g, and removing the liquid

3. Adding 5ml PBSpH7.5 into centrifuge tube, centrifuging for 2 min at 3000g, removing liquid, and repeating step 3 once

4.5 ml of stop solution (1M Ethanolamine) was added to the tube and shaken for 30 minutes

5.3000g, centrifuge for 2 minutes, remove liquid

6. Adding 5ml PBS pH7.5 into centrifuge tube, 3000g, centrifuging for 2 min, removing liquid, and repeating step 6 three times

7. Adding 5ml of preservation solution (PBS, pH7.5, 0.05% NaN)₃) Centrifuging at 3000g for 2 min in a centrifuge tube to remove liquid

8. Adding 2ml of preservation solution (PBS, pH7.5, 0.05% NaN)₃) Placing the mixture in a centrifuge tube, and standing the mixture at 4 ℃ for later use.

(V) PSP-Sepharose column separation of PSP antibody

1.5ml PSP antiserum (containing 1.0mg/ml His-peptide) was mixed with 5ml PBS pH7.5 and passed through a 0.22um filter

2. 10ml of the above PSP antiserum dilution was added to a PSP-agarose centrifuge tube and shaken for 1 hour

3.3000 g, centrifuge for 2 minutes, remove liquid

4. 10ml of washing solution (20mM Tris, pH7.5, 0.5M NaCl, 0.02% NP-40) was added to the PSP-agarose centrifuge tube,

shaking for 15 minutes

5.3000g, centrifuge for 2 minutes, remove the liquid, repeat the 4-5 steps three times

6. Adding 1ml eluent (100mM Glycine, pH2.8) into a centrifuge tube, centrifuging at 4000g for 1 min, collecting supernatant,

add to 50ul 2.0M Tris (pH9.0) microcentrifuge tubes, mix well and repeat step 6 five times.

7. Transferring the mixed eluate into dialysis bag, and adding dialysate (20mM NaPO)₄(pH7.5),150mM NaCl,1.0mM EDTA,

1.0mM DTT,1.0mM PMSF, 10% Glycerol), 4 deg.C, overnight.

8. Adding the PSP antibody solution into a protein concentration and purification centrifugal column (Microcon-10 kDa centrifugal Filter),

concentrated PSP antibodies

9. Measuring the concentration of PSP antibody, and storing at-20 deg.C for use.

(VI) determining the antibody titer and specificity of PSP

1. His-PSP (10. mu.g/ml), BSA (10. mu.g/ml) coating solution was prepared with 50mM carbonate buffer (pH9.6), and 1OOul was added

The solution was plated onto enzyme reaction plate wells, incubated overnight at 4 ℃ and washed 2 times with PBS

3. 100ul of PSP antibody (0, 1, 5, 20, 100 ng/ml) was added to the wells at various concentrations, and at room temperature,

slowly shake for 60 minutes, and suck out the reaction solution

5. Add 1:2500 dilution 100ul anti-goat IgG antibody-HRP (Promega) to the wells, shake slowly at room temperature for 60 min,

aspirating antibody fluids

6. The enzyme reaction plate was washed 5 times with PBST for 5 minutes each

8. Add 50ul 2N H₂SO₄Terminating the reaction

。

Thirdly, preparing PSP-N monoclonal antibody

(I) preparation of PSP epitope peptide-N-carrier protein immunogen

PSP epitope peptide-N (SEQ ID NO.1) CQNMNSGNLVSVLTQAEGAFVASLIKESGTDDF is a peptide from 64 th to 96 th positions of human PSP protein and contains 33 amino acids. The peptide segment has hydrophilicity and strong antigenicity, becomes PSP-N-carrier protein immunogen after being connected with carrier protein, and is inoculated to animals to generate specific PSP antibodies.

(II) PSP peptide-N coupled blue carrier protein

1. Carrier protein liquid: maleimide activated Blue Carrier Protein (BCP), available from Thermo Scientific Inc., 5mg BCP dissolved in 0.5mlddH₂O(10mg/ml)

2. PSP-N peptide solution 4mg of synthetic PSP peptide-N (> 95%) was dissolved in 0.5ml of PBS, pH7.2 (8mg/ml)

3.0.5 ml BCP carrier protein solution is mixed with 0.5ml PSP-N peptide solution and reacted for 2 hours at room temperature

4. Adding the reaction solution to a Microcon-10KDa Centrifugal Filter

5. Centrifuging at 14000g for 2-8 min, and reducing the reaction solution to 50-100ul

3. Removing liquid from the collection tube, adding 400ul PBS (pH7.5) to the protein concentration and purification spin column

4. Centrifuging at 14000g for 2-8 min, and reducing the liquid volume in the centrifugal column to 50-100ul

5. Repeating the steps for more than 5 times for 3-4 times

6. Sucking out about 50-100ul PSP-N-BCP solution in the protein concentration and purification centrifugal column

7. Measuring the concentration of PSP-N-BCP, and freezing and storing at-80 ℃.

(III) preparation of PSP-N monoclonal antibody

Immunization

The method comprises the steps of (1) emulsifying PSP-N-BCP and Freund's complete adjuvant (0.1mg/ml), performing subcutaneous multi-point injection and Intraperitoneal (IP) primary immunization on BALB/c mice at about 8 weeks, and after 2-3 weeks, emulsifying PSP-N-BCP/Freund's incomplete adjuvant (0.1mg/ml), and performing subcutaneous multi-point injection and IP secondary booster immunization on BALB/c mice.

After 2.6-8 weeks, BALB/c mouse blood (0.1-0.4 ml) was obtained using retro-orbital bleeding, and BALB/c mice with high anti-PSP-N antibody titer were selected for cell fusion by ELISA assay using PSP-N coated reaction plates.

After 3.8-9 weeks, PSP-N-BCP/PBS (0.1mg/ml) was mixed and selected BALB/c mice were injected subcutaneously into the extremities at multiple points and immunized with IP for the third booster immunization.

4. 2-3 days before cell fusion, 0.1mg PSP-N-BCP/PBS mixture with volume of 0.1ml is injected into selected BALB/c rat tail vein for boosting.

Cell fusion

1. Murine myeloma cells (Sp2/0) were cultured in DMEM solution with 8-azaguanine to achieve the best growth state 2 weeks before fusing the cells.

2. Spleens from BALB/c mice were removed rapidly, splenocytes isolated, and pooled with 5: 1 ratio with Sp2/0 cells, polyethylene glycol (PEG4000) as a fusogenic agent, fusing splenic cells with Sp2/0 cells.

3. The fused cells are suspended in HAT culture solution containing FBS, then equal volume of feeder cells is added, the mixture is evenly mixed and then is distributed in a 96-well cell plate (200 ul/well), the cell plate is placed in 5% C02 to be cultured at 37 ℃, after 5 days, the solution is half reserved and changed, and after 10 days, hybridoma cell culture supernatant in the 96-well cell culture plate is detected by ELISA.

Screening for Positive hybridoma cells

1. The coating solution of PSP peptide-N (10. mu.g/ml), BSA (10. mu.g/ml) was prepared in 50mM carbonate buffer (pH9.6), and 1OOul was added

Coating solution is put into a well of an enzyme-linked reaction plate, kept at 4 ℃ overnight, and washed 2 times with PBS

3. Adding 100ul hybridoma cell culture supernatant to the wells, shaking slowly at room temperature for 60 min, and removing the reaction solution

5. Add 1:5000 dilution 100ul anti-mouse IgG antibody-HRP (Promega) to the wells, shake slowly at room temperature for 60 min,

aspirating antibody fluids

6. The enzyme reaction plate was washed 5 times with PBST for 5 minutes each

8. Add 50ul 2N H₂SO₄Terminating the reaction

10. As a result of observation, cells producing PSP-N monoclonal antibodies of the IgG class were confirmed.

Screening out hybridoma cells secreting PSP-N monoclonal antibody

1. Carrying out subclone screening by using a limiting dilution method, carrying out positive detection on cells in a hole of an IgG PSP-N monoclonal antibody, diluting the cells to 1 cell in each hole by using an HTDMEM culture medium, culturing the cells on a 96-hole cell culture plate, observing the actual cell number of each hole under a microscope within 5 hours, recording a single cell hole, identifying that the antibody is positively secreted by using ELISA after the cells grow into a clone, thus obtaining a hybridoma cell strain secreting the monoclonal antibody, and carrying out mass amplification and cryopreservation.

2. After long-term subculture of the monoclonal antibody hybridoma cell strain, subcloning identification is carried out by using a limiting dilution method, so that the hybridoma cell strain capable of stably secreting the monoclonal antibody is obtained.

3. And carrying out amplification culture on the stable monoclonal antibody hybridoma cell strain to generate a certain amount of antibody for identifying the characteristics of the monoclonal antibody.

Production and purification of PSP-N monoclonal antibodies

1. Amplifying and culturing PSP-N monoclonal antibody hybridoma cell strain with a rotary bottle, collecting cell supernatant, and filtering the supernatant through a 0.45um filter membrane.

2. With (NH)₄)₂SO₄The monoclonal antibody in the supernatant was precipitated and dialyzed to remove (NH)₄)₂SO₄And obtaining the monoclonal antibody concentrated solution.

Reacting ProteinA-Agarose with the monoclonal antibody concentrated solution, washing with buffer solution, eluting with 0.1 Mcricacid (pH3.0) to bind PSP-N monoclonal antibody, dialyzing the monoclonal antibody, concentrating and purifying the monoclonal antibody, determining the concentration of the monoclonal antibody, packaging, and storing at-20 deg.C.

Determination of binding force and specificity of purified monoclonal antibody and PSP

3. 100ul of PSP-N monoclonal antibody (0, 1, 5, 20, 100 ng/ml) at various concentrations was added to the wells, and at room temperature,

slowly shake for 60 minutes, and suck out the reaction solution

5. Add 1:2500 dilution 100ul anti-mouse IgG antibody-HRP (Promega) to the wells, shake slowly at room temperature for 60 min,

aspirating antibody fluids

6. The enzyme reaction plate was washed 5 times with PBST for 5 minutes each

8. Add 50ul 2N H₂SO₄Terminating the reaction

。

Determination of the affinity constant of PSP-N monoclonal antibodies

Coating solution was put into the well of the enzyme reaction plate, and washed 2 times with PBS overnight at 4 deg.C

3.10^-8MHis-PSP (a) mixed with 20ng/ml PSP-N monoclonal antibody (A), 20ng/ml PSP-N monoclonal antibody fluid

（A₀) 100ul of the reaction solution was added to each well at room temperature for l hours, and PBST was washed for 5min for a total of 5 times

4. Adding 100ul 1:2500 diluted anti-mouse IgG antibody-HRP to each well, reacting at room temperature for l hours, washing with PBST for 5min, 5 times

5. Adding 100ul substrate developing solution into each well, reacting at room temperature in the dark for 1O-15min, adding 50ul stop solution, and measuring OD value

Calculating the affinity constant (K) formula of the PSP-N monoclonal antibody:

A₀/(A₀–A)=1+K/a

1.586/（1.586-0.275）=1+K/10^-8M

K=2.0 X 10^-9M。

fourthly, preparing the PSP-C monoclonal antibody

(I) preparation of PSP epitope peptide-N-carrier protein immunogen

PSP epitope peptide-N (SEQ ID NO.2) LHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYC is a peptide segment containing 36 amino acids from positions 102 to 137 of human PSP protein. The peptide segment has hydrophilicity and strong antigenicity, is connected with carrier protein and then is used as PSP-C-carrier protein immunogen to inoculate animals to generate specific PSP antibody.

Preparing PSP-C monoclonal antibody by using the PSP-N monoclonal antibody preparation synchronous process.

ELISA (enzyme-Linked immuno sorbent assay) is used for determining binding force and specificity of purified PSP-C monoclonal antibody and PSP

。

ELISA determination of PSP-C monoclonal antibody affinity constant (K)

Calculating the affinity constant (K) formula of the PSP-C monoclonal antibody:

A₀/(A₀–A)=1+K/a

1.408/（1.408-0.291）=1+K/10^-8M

K=2.6 X 10^-9M。

fifthly, preparing a PSP calibrator

PSP recombinant protein preparation in the same procedure, PSP calibrator was prepared by transferring Q-Sepharose eluate (containing His-PSP protein) into a dialysis bag (Spectra/Pro), adding into dialysate (20mM Tris, pH7.5,100mM NaCl,10mM KCl,2.0mM MgCl2, 5% Glycerol, 1.0mM DTT,1.0mM EDTA, 1.0mM PMSF), and slowly stirring and passing through the dialysate at 4 ℃.

Transferring the dialyzed protein solution in the dialysis bag into a microcentrifuge tube, centrifuging at 13000rpm for 10 minutes at 4 ℃, collecting supernatant, adding the supernatant into a protein concentration and purification Centrifugal column (Microcon-3 KDa Centrifugal Filter), concentrating and purifying His-PSP protein solution in the Centrifugal column, measuring the concentration of the His-PSP protein, and freezing and storing at-80 ℃.

Sixthly, preparing an immunochromatographic assay PSP test strip

Coupling PSP antibody to europium chelate fluorescent microsphere

In the embodiment of the invention, Europium Chelate (Europium chemical) fluorescence labeling carboxyl modified particles (Europium chemical PS-COOH) are selected, and the diameter of the Europium Chelate fluorescence labeling carboxyl modified particles is 100-300nm, which is purchased from Bangs Laboratories Inc.

In the embodiment of the invention, EDC and Sulfo-NHS are used for coupling PSP-N or C monoclonal antibody to europium chelate fluorescent-labeled carboxyl modified microspheres (diameter is 200 nm) by the following steps:

1. 0.4ml of Europium Chemate PS-COOH (10mg/ml) with a diameter of 200nm was put into a 1.5ml centrifuge tube and centrifuged

Heart, 13000rpm, 6 min, remove supernatant

2. Adding 1.0ml of 50mM MES buffer (pH6.0) into the centrifuge tube, and suspending the microspheres

3. Centrifuging at 13000rpm for 6 min, removing supernatant, and repeating 2-3 steps twice

4. 0.5ml of 50mM MES buffer (pH6.0) containing 5mM EDC and 10mM Sulfo-NHS was added to the centrifuge tube and mixed,

reacting at 25 ℃ for 30 minutes

5. Centrifuging at 13000rpm for 6 minutes, and removing the reaction solution

6. Adding 1.0ml of 50mM phosphate buffer (pH7.5) into the centrifuge tube

7. Centrifuging at 13000rpm for 6 min, removing supernatant, and repeating 6-7 steps once

8. Adding 0.4ml PSP-N or C monoclonal antibody (0.4 mg/ml) into the centrifuge tube, reacting at 25 deg.C for 2 hr

9. 50ul of 0.2M Tris (pH7.5) was added to the centrifuge tube at 25 ℃ for 30 minutes

10. Centrifuging at 13000rpm for 6 minutes, removing supernatant,

11. adding 1.0ml of 0.2% BSA, 0.01% Tween-2050 mM phosphate buffer (pH7.5) into the centrifuge tube

12. Centrifuging at 13000rpm for 6 min, removing supernatant, repeating 11-12 steps twice

13. Buffer (10 mM Tris, pH8.0, 0.2% BSA, 10% Sucrose, 0.1% PVP, 0.2% Tween20,0.01% NaN)₃) Suspending the PSP-N or C monoclonal antibody-fluorescent microspheres to the required concentration, and storing at 4 ℃ for later use.

(II) processing sample pad and conjugate pad

The invention selects glass fiber MF1 sample pad (GE Healthcare) to be soaked in sample pad treatment fluid (20mM Tris, pH7.5, 2% Sucrose,0.5% BSA, 0.5% Tween-20, 0.01% NaN)₃) Drying at room temperature for 1 hr, drying at 30 deg.C for 8 hr, and sealing.

The invention selects glass fiber GFDX combined pad (EMD Millipore) to be soaked in combined pad treatment fluid (10 mM Tris, pH8.0, 0.2% PVP, 15% Sucrose,0.5% BSA, 0.5% Tween-20, 0.01% NaN₃) Drying at room temperature for 1 hr and 30 deg.C for 8 hr, and sealing.

(III) spraying the conjugate pad and nitrocellulose Membrane

1. Buffer 1 (10 mM Tris, pH8.0, 5.0% Sucrose, 0.2% PVP, 0.2% Tween20,0.01% NaN)₃) Diluting PSP-N or C antibody-europium chelate fluorescent microsphere liquid to 0.2mg/ml, spraying PSP-N or C antibody-fluorescent microsphere on a binding pad (GFDX) by using a Bio-DotXYZ3060 instrument in a non-contact type micro quantitative nozzle mode at the speed of 6ul/cm, drying for 6 hours at the temperature of 30 ℃, adding a drying agent, and sealing for later use.

2. Buffer 2 (5 mM Tris, pH8.0, 2.0% Trehalo, 0.1% PVP, 0.01% Tween20,0.01% NaN)₃) Diluting PSP antibody to 2.0mg/ml, diluting anti-mouse IgG antibody to 2.0mg/ml, placing the nitrocellulose membrane on a Bio-DotXYZ3060 instrument, spraying the PSP antibody solution and anti-mouse IgG antibody solution onto the nitrocellulose membrane in a non-contact micro-quantitative nozzle manner at a speed of 0.8ul/cm, drying at 30 ℃ for 3 hours at a distance of 5mm between two lines, and adding a drying agent for sealing for later use.

(IV) assembling immunochromatographic test paper card

The test strip is assembled in a room with the humidity of less than 30 percent and the temperature of 30-37 ℃. An immunochromatographic test strip, as shown in figure 1, comprises a PVC base plate, a sample pad (25 mm) adhered on the PVC base plate, a binding pad (8 mm), a nitrocellulose membrane (25 mm) and absorbent paper (30 mm), wherein the nitrocellulose membrane is adhered to the middle part of the base plate, a T line is formed by a PSP antibody coating at intervals, a C line is formed by an anti-mouse IgG antibody coating, and PSP-N or C monoclonal antibody-europium chelate fluorescent microspheres are sprayed on the binding pad.

In the embodiment of the invention, the T line and the C line are arranged in parallel, and the distance between the T line and the C line is 5 mm.

The absorbent paper is positioned at one end of the nitrocellulose membrane close to the line C, is partially overlapped with the nitrocellulose membrane, and has the overlapping length of 2 mm; the absorbent paper was overlaid on top of the nitrocellulose membrane. The combination pad is positioned at one end of the nitrocellulose membrane close to the T line; partially overlapping the nitrocellulose membrane, wherein the overlapping length is 2 mm; the conjugate pad was overlaid on top of the nitrocellulose membrane. The sample pad is positioned at the outer side of the combination pad and partially overlapped with the combination pad, the overlapping length is 3mm, and the sample pad is overlapped above the combination pad. And (3) assembling each group of parts into a test paper board, and cutting the test paper board into test paper strips with the width of 4 mm.

Putting the test strip into a shell base, closing the shell surface to form a test strip card (figure 2), wherein the shell surface is provided with a sample adding hole (8) and a detection window (9) to expose a local area of the test strip; the sample adding hole is opened at the upper part of the sample pad (2) to expose part of the sample pad area; the observation window is opened on the upper part of the nitrocellulose membrane (4) to expose all the T line (5) and the C line (6).

Seventh, the process of detecting PSP concentration by immunofluorescence analyzer

(I) immunochromatographic test paper card operating process

When the PSP in a sample is quantitatively detected by using a PSP immunochromatography test paper card, PSP calibrator (5ul), serum/slurry (5ul) and whole blood (10 ul) are added into a sample hole, then 100 chromatographic solution (PBS, pH7.5,0.5% BSA, 0.5% Tween-20) is added into the sample hole, the reaction is carried out for 5-10 minutes, and corresponding fluorescent signals are generated on a T line and a C line, and the detection is carried out by using an immunofluorescence analyzer.

(II) establishing a program for detecting the concentration of PSP by an immunofluorescence analyzer

1. Establishing a PSP concentration correlation curve

The PSP calibrator was prepared with His-PSP recombinant protein as calibrator and PBS (pH7.5) containing 0.5% BSA at concentrations of 0,10, 20, 50, 100, 200, 400 ng/ml. Adding 5ul of PSP calibrator into sample wells of PSP immunochromatographic test paper cards, adding 100ul of chromatography liquid into the sample wells, performing membrane chromatography reaction, after 5-10 minutes, selecting an excitation wavelength of 365nm and a detection wavelength of 610nm by using an immunofluorescence analyzer, and measuring the fluorescence intensity of T lines and C lines of the test paper strip cards. Taking the concentration of the PSP calibrator as an ordinate and the fluorescence intensity unit of the calibrator as an abscissa, preparing a PSP calibrator concentration-fluorescence intensity unit correlation curve to obtain an equation, y =26.267 x-24.27, R²=0.9885, see fig. 5, from which PSP concentration standard card is obtained as a basis for quantitative analysis of PSP concentration contained in sample

。

And inputting the standard card into an immunofluorescence analyzer, establishing an automatic operation system, and automatically calculating the PSP concentration in the sample to be detected by the immunofluorescence analyzer through corresponding analysis software.

Detection of PSP concentration in samples Using PSP immunochromatographic test paper cards

Adding 5ul of serum to a sample adding hole part of a PSP chromatography test paper card, adding 100 chromatographic solutions into a sample hole, carrying out membrane chromatography reaction, and measuring the PSP concentration by using an immunofluorescence analyzer automatic detection system after 5-10 minutes, wherein the result is as follows:

。

<110> Changchun Heng-Xiao Biotechnology Limited liability company

<120> method for preparing pancreatic stone protein antibody and establishing and detecting PSP

<160>4

<210>1

<211>144

<213>PSP

<400>1

QEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA 60

FVASLIKESGTDDFNVWIGLHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYCVSLTS 120

STGFQKWKDVPCEDKFSFVCKFKN

<210>1

<211>33

<213>PSP

<400>1

CQNMNSGNLVSVLTQAEGAFVASLIKESGTDDF

<210>1

<211>36

<213>PSP

<400>1

LHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYC

<210>1

<211>137

<213>PSP

<400>1

MKHHHHHHQLHAGAHQEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA

Claims

1. A method for producing a pancreatic stone protein antibody, comprising: constructing a label-PSP recombinant protein vector by using a gene recombination technology, expressing a label-PSP recombinant protein by using bacteria, yeast, insect cells, animal or human cells, and purifying the label-PSP recombinant protein to be used as a PSP recombinant protein immunogen; PSP protein amino acid sequence:

QEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA 60

FVASLIKESGTDDFNVWIGLHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYCVSLTS 120

STGFQKWKDVPCEDKFSFVCKFKN。

2. the antibody for producing pancreatic stone protein according to claim 1, characterized in that:

synthesis of PSP epitope peptide-N, amino acid sequence

SEQ ID NO.1：CQNMNSGNLVSVLTQAEGAFVASLIKESGTDDF；

Synthesis of PSP epitope peptide-C, amino acid sequence

SEQ ID NO.2：LHDPKKNRRWHWSSGSLVSYKSWGIGAPSSVNPGYC。

3. The antibody for producing pancreatic stone protein according to claim 2, characterized in that: PSP epitope peptide-N or C coupled carrier protein including Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Blue Carrier Protein (BCP) and the like is prepared into PSP epitope peptide-carrier protein immunogen.

4. The antibody for producing pancreatic stone protein according to claim 1, characterized in that: PSP recombinant protein immunogen, immune animals including mice, rats, rabbits, goats, sheep, alpacas, donkeys and horses, and PSP monoclonal antibody or polyclonal antibody is produced.

5. The antibody for producing pancreatic stone protein according to claim 3, characterized in that: PSP epitope peptide-carrier protein immunogen, immune animals including mouse, rat, rabbit, goat, sheep, alpaca, donkey, horse, producing PSP monoclonal antibody or polyclonal antibody.

6. The antibody for producing pancreatic stone protein according to claim 1, characterized in that: the PSP recombinant protein is used for protein renaturation and is used as a calibrator for a PSP detection method.

7. The antibody for producing pancreatic stone protein according to claim 4, 5 and 6, characterized in that: PSP monoclonal antibody or polyclonal antibody, and PSP calibrator, and establishing PSP detecting method including ELISA, chemiluminescence method, immunofluorescence method, immunoturbidimetry, immunochromatography, immunoagglutination, etc.

8. The antibody for producing pancreatic stone protein according to claim 4, 5 and 6, characterized in that: the PSP in-vitro diagnostic kit is established by a PSP monoclonal antibody or a polyclonal antibody and a PSP calibrator, wherein one PSP in-vitro diagnostic kit is a rapid quantitative detection PSP immunochromatographic test strip, and the test strip comprises a sample pad, a binding pad, a nitrocellulose membrane and absorbent paper which are sequentially connected and fixed on a PVC (polyvinyl chloride) bottom plate.

9. The antibody for producing pancreatic stone protein according to claim 1, characterized in that: adsorbing the PSP antibody labeled fluorescent microspheres by the binding pad; and a detection line (T line) formed by a PSP antibody and a quality control line (C line) formed by an anti-IgG antibody are fixed on the nitrocellulose membrane.

10. The antibody for producing pancreatic stone protein according to claim 1, characterized in that: the preparation process of the PSP recombinant protein comprises the following steps:

His-PSP recombinant protein amino acid sequence:

MKHHHHHHQLHAGAHQEAQTELPQARISCPEGTNAYRSYCYYFNEDRETWVDADLYCQNMNSGNLVSVLTQAEGA