CN110794148A

CN110794148A - Double-antibody sandwich ELISA quantitative detection kit for porcine inflammasome NLRP3 and application thereof

Info

Publication number: CN110794148A
Application number: CN201911076215.0A
Authority: CN
Inventors: 张艳; 刘海隆; 王文秀; 曹宗喜; 谭树义; 黄丽丽
Original assignee: Shandong Binzhou Animal Science & Veterinary Medicine Academy; Animal Husbandry Veterinary Institute Hainan Academy Of Agricultural Sciences
Current assignee: Shandong Binzhou Animal Science & Veterinary Medicine Academy; Animal Husbandry Veterinary Institute Hainan Academy Of Agricultural Sciences
Priority date: 2019-11-06
Filing date: 2019-11-06
Publication date: 2020-02-14
Anticipated expiration: 2039-11-06
Also published as: CN110794148B

Abstract

The invention discloses a double-antibody sandwich ELISA quantitative detection kit of a porcine inflammasome NLRP3 and application thereof, wherein the double-antibody sandwich ELISA quantitative detection kit is characterized in that a solid phase carrier is coated with a biological preservation number of CCTCC NO: the monoclonal antibody secreted by the hybridoma cell strain 9H5 of C201973 realizes quantitative detection by detecting the antibody, substrate color development and a standard curve, and realizes rapid detection of the porcine inflammation corpuscle NLRP3, and the kit has the advantages of high sensitivity and good stability, and makes up the vacancy of the detection of the porcine inflammation corpuscle NLRP3 in the existing market.

Description

Double-antibody sandwich ELISA quantitative detection kit for porcine inflammasome NLRP3 and application thereof

Technical Field

The invention relates to the technical field of biological engineering, in particular to a double-antibody sandwich ELISA quantitative detection kit for a porcine inflammasome NLRP3, and also relates to application of the double-antibody sandwich ELISA quantitative detection kit.

Background

NLRP3 is also called as inflammatory corpuscle, is a cytokine which is produced by leucocytes and mediates intercellular interaction and plays a regulating role in the activation, proliferation and differentiation of cells, protein plays an important role in the body to resist pathogenic microorganisms and inflammatory response, basic data is provided for further discussing the action mechanism of the inflammatory corpuscle in pig inflammatory diseases, the inflammatory corpuscle is a multi-protein complex which is assembled by natural immune recognition receptors in cytoplasm and is important for the generation of inflammatory response, the research on the inflammatory corpuscle and the pathophysiology thereof becomes one of the hot research problems in the field of inflammation generation and regulation, the interaction of protein with ASC, Caspase-1 and the like forms the inflammatory corpuscle, promotes the maturation and activation of Caspase-1 precursors and promotes the maturation and release of 1L-1 β and IL-18, activates the inflammatory response, various inflammatory diseases frequently occur in the production process of the pig, the important loss caused by the maturation of Caspase-1 precursors is urgently needed to research on the mechanisms of the inflammatory diseases, the molecular regulation of the occurrence of the pig, the inflammatory corpuscles plays an important role in supporting and the development of various inflammatory corpuscles, and the important target of the inflammatory corpuscles as important research on the development of various inflammatory pathogens in pig inflammatory diseases, the research of pig inflammatory corpuscles, and the research of pig inflammatory corpuscles.

The rapid and accurate diagnosis of pig inflammation can provide time reference for the correct treatment and prevention and control of inflammatory diseases caused by viruses, bacteria and the like, and the colloidal gold test strip for accurately diagnosing whether the pig has inflammation or not and the antigen ELISA kit for checking expression are all undisclosed to monoclonal antibodies with stable performance and high antigen titer, and no good monoclonal antibodies aiming at pigs exist at home and abroad, so that no targeted diagnostic reagent exists. Therefore, there is a need to develop a kit that can be easily applied to the preparation of pig body and assembly. However, at present, no other report about the sandwich ELISA quantitative detection method for detecting the porcine double monoclonal antibody exists at home and abroad.

Disclosure of Invention

In view of the above, one of the purposes of the present invention is to provide a double-antibody sandwich ELISA quantitative detection kit for a porcine inflamed corpuscle NLRP3, which is rapid, sensitive and stable, and makes up for the vacancy of detecting the porcine inflamed corpuscle NLRP3 in the existing market; the invention also aims to provide the application of the double-antibody sandwich ELISA quantitative detection kit of the porcine inflammasome NLRP 3.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a double-antibody sandwich ELISA quantitative detection kit of a porcine inflammasome NLRP3 is characterized in that a solid phase carrier of the kit is coated with an antibody with a biological preservation number of CCTCC NO: c201973 hybridoma cell line 9H 5.

In the invention, the coating concentration of the coating antibody is 2-5 mug/mL.

In the invention, the kit also comprises a detection antibody, wherein the detection antibody is a polyclonal antibody for recognizing the porcine inflammasome NLRP3, and the detection antibody is labeled with horseradish peroxidase HRP.

Preferably, the concentration of the detection antibody is 10-15 mug/mL. The diluent for detecting the antibody is phosphate buffer solution, the phosphate buffer solution contains calf serum with the volume percentage concentration of 5%, polyethylene glycol (PEG) with the mass percentage concentration of 1.5-3% and Tween20 (Tween20) with the volume percentage concentration of 0.2-0.5%. And (3) replacing rabbit polyclonal antibody with the detection antibody.

In the invention, the kit also comprises a confining liquid, a color developing agent, a stop solution, a washing solution and a standard substance.

In the invention, the confining liquid is a phosphate buffer containing calf serum with a volume fraction of 5%, Tween20 with a mass fraction of 0.1% and a preservative with a mass fraction of 0.01%, and the preservative is procline 300.

In the invention, the color developing agent is 3, 3 ', 5' -tetramethyl benzidine (TMB) single-component color developing solution or TMB-H₂O₂A bi-component color developing liquid.

In the invention, the washing solution is PBS containing 0.1% of Tween20 by mass fraction.

The standard substance is NLRP3 recombinant protein or NLRP3 protein.

2. The kit is applied to quantitative detection of the porcine inflammasome NLRP 3.

The invention has the beneficial effects that: the invention discloses a double-antibody sandwich ELISA quantitative detection kit of a porcine inflammasome NLRP3, which takes the biological preservation number of CCTCC NO: the antibody secreted by the hybridoma cell strain 9H5 of C201973 is an envelope antibody detection antigen, and the antibody has the characteristics of good specificity and high sensitivity, can specifically recognize NLRP3, and has the characteristics of high sensitivity and specificity after being prepared into a kit; also has good stability and reproducibility, shortened operation time, and reduced cost.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows SDS-PAGE analysis of NLRP3 recombinant protein (M: protein Marker; 1: pET32 a-inducible strain; 2: pET32a-NLRP 3-uninduced strain; 3: pET32a-NLRP 3-inducible strain; 4: supernatant; 5: precipitation; 6: purified protein).

FIG. 2 shows the indirect immunofluorescence assay NLRP3 monoclonal antibody levels (A-G show the results of indirect immunofluorescence assays on porcine PK cells using mAbs 9H5, 10G4, 13A11, 13E1, 13E3, 13E11, and 15E2, respectively, and H is control).

FIG. 3 is a standard curve of NLRP3 protein double antibody sandwich ELISA detection method.

FIG. 4 is the content determination of NLRP3 protein in pig lung tissue.

Biological preservation

The hybridoma cell strain 9H5 is delivered to China center for type culture Collection for preservation, and the preservation number is CCTCCNO: c201973, the address is located at Wuhan university in Wuhan, China, the preservation date is 4 months and 28 days in 2019, and the cell line is classified and named as a mouse bone marrow hybridoma cell line 9H 5.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1 preparation of porcine NLRP3 truncated recombinant protein

The preparation process of the truncated recombinant protein of the pig NLRP3 comprises the following steps:

(1) obtaining pig NLRP3 target fragment

And performing RT-PCR amplification reaction by using the extracted total RNA of the pig liver as a template and using a one-step RT-PCR amplification kit.

The one-step RT-PCR reaction system is as follows: mu.L of 2X 1Step B. mu.ffer, 1. mu.L of PrimeScript 1StepEnzyme Mix, 0.5. mu.L each of the upstream and downstream primers P1, P2(20 pmol/. mu.L), 5. mu.L of RNA (0.02. mu.g/. mu.L), and water to 50. mu.L.

The one-step RT-PCR reaction conditions are that 50 ℃ is 60min, 95 ℃ is 5min, 94 ℃ is 45s, 56 ℃ is 45s, 72 ℃ is 1min, 30 cycles are carried out, 72 ℃ is 10min, RT-PCR amplification products are subjected to agarose gel electrophoresis, a gel recovery kit is utilized to recover and purify a target gene fragment, the purified product is taken to be connected with a pMD18-T vector overnight at 4 ℃, the connection product is converted into DH5 α competent cells, a positive recombinant plasmid pMD18-T-NLRP3 is obtained through PCR and BamH I/Xho I double enzyme digestion identification, the plasmid is sent to a company Limited in the Biotechnology (Shanghai) for sequence determination, antigen epitope analysis is carried out, fragments rich in antigen epitopes are intercepted, a specific primer is designed for PCR amplification, a pig NLRP3 fragment consisting of 288 amino acid residues is obtained, the molecular weight is about 50KDa, the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence of the coded amino acid is shown as SEQ ID NO. 2.

(2) Construction of recombinant expression plasmid pET32a-NLRP3 of NLRP3 truncated fragment

The nucleotide sequence shown in SEQ ID NO.2 is connected into an expression vector pET-32a through BamH I and Xho I to obtain a recombinant expression vector pET32a-NLRP3, and after sequencing verification, an expression vector with correct target gene sequence is selected for protein expression.

(3) The recombinant protein is expressed by the following specific expression method:

converting a pET32a-NLRP3 recombinant plasmid with correct sequencing identification into a BL21(DE3) competent cell, selecting a single colony for overnight enrichment, inoculating the single colony into 300mL of LB liquid culture medium according to a ratio of 1:100 the next day, culturing at 37 ℃ until the A600 is about 0.6-0.7, adding IPTG until the final concentration is 0.1mmol/L, continuously culturing at 25 ℃ for 8h, centrifuging at 5000 r/min, collecting thalli, washing with PBS for 2 times, ultrasonically cracking the thalli cells for 5s at an interval of 10s for 30 min. Then, the mixture is centrifuged at 12000r/min for 10min, and the supernatant and the precipitate are respectively collected and analyzed by SDS-PAGE to identify whether the target protein is expressed and the expression form of the protein.

Transforming BL21(DE3) competent cells with the pET32a-NLRP3 recombinant plasmid with correct sequencing identification, picking single colonies, and respectively inoculating the single colonies into 3mL LB culture medium containing kan resistance; culturing to OD600 of 0.4-0.6, with a ratio of 1: inoculating 100 proportion into 300mL LB liquid culture medium, culturing at 37 deg.C to A₆₀₀About 0.6-0.7, adding IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 0.1mmol/L, carrying out induced expression for 8h at 25 ℃, centrifuging the bacterial liquid at 5000 r/min, collecting the bacterial cells, washing the bacterial cells for 2 times by PBS (phosphate buffer solution), carrying out ultrasonic lysis on the bacterial cells for 5s at an interval of 10s, and carrying out lysis for 30 min. Then, the cells were centrifuged at 12000r/min for 10min, and the expression was examined by SDS-PAGE on the supernatant and the precipitate, respectively.

(4) The recombinant protein is purified by the following specific method:

purifying the supernatant, and purifying the recombinant protein expressed in a soluble form according to the use instruction of the Ni NTA P [ mu ] configuration System because the protein expressed in the invention is soluble protein, wherein the purification method is as follows: 1mL of 50% Ni-NTAHis Bind resin suspension was added to 4mL of 1 XNi-NTA binding buffer and gently mixed. After the resin settled naturally, 4mL of the supernatant was aspirated off with a pipette tip. 4mL of the prepared lysate was added, mixed by gentle shaking and combined for 60 minutes at 4 ℃. Adding the lysis solution Ni-NTAHis Bind resin mixture into a hollow chromatographic column with the lower end closed. Removing the lower closed cover, and collecting effluent. After the protein is purified, the purification effect is detected by SDS-PAGE electrophoresis, and the concentration of the purified protein is determined by an ultraviolet spectrophotometer. The purified protein was determined to have a concentration of 1.28mg/mL by UV spectrophotometer, and the result of SDS-PAGE of the purified protein is shown in FIG. 1. The results show that the purified protein is of high purity.

Example 2 animal immunization

Experimental animals and immunization methods: 5 Balb/C mice with the age of 5-8 weeks are selected, NLRP3 recombinant protein prepared in example 1 is used as antigen, back multi-point injection is carried out, 100 mu g of antigen/experimental mouse is injected, 50 mu g of antigen/experimental mouse is injected in a boosting immunization mode, Freund's complete adjuvant is used in the first injection and is mixed with the antigen in the same volume, the Freund's incomplete adjuvant is used in the boosting immunization injection and is mixed with the antigen in the same volume, and the specific immunization time and the immunization period are shown in Table 1.

TABLE 1 immune cycle

Course of the experiment	Date
		Prime immune	2018.07.15
First boost immunization	2018.07.23
		Second boost immunization	2018.07.31
Third booster immunization	2018.08.08
		Fusion	2018.08.16
Screening	2018.08.24
		Sub-first screening	2018.09.01
Screening for Asia two	2018.09.09
		Ascites (ascites)	2018.09.24

And (3) antiserum detection:

taking a small amount of blood from tail veins of immunized mice to prepare antiserum, detecting the titer of the antiserum by adopting an indirect ELISA method, wherein the reaction conditions are as follows:

antigen coating: coating for 2h at 37 ℃; and (3) sealing: sealing for 1h at 37 ℃; serum antibody incubation: incubating at 37 ℃ for 1 h; secondary antibody (HRP-labeled goat anti-mouse IgG): the secondary antibody is prepared from the following components in percentage by weight: 10000 dilution, 37 ℃ binding for 30min, and finally 37 ℃ color development for 10min, the results are shown in Table 2.

TABLE 2 antibody titers determined at different immunization times and different concentrations

The result shows that the antigen coating concentration is 5K, the effect is the best, the titer of the hyperimmune serum is higher, and the specificity of the antibody is better.

3 mice immunized with recombinant protein were tested using the best conditions for screening. The results showed that 3 mice responded well to the recombinant protein, and it was decided to use M0131-4 mice for subsequent experiments and screening with the recombinant protein.

Example 3 cell fusion and subcloning

(1) Myeloma cell preparation: one week prior to fusion, SP2/0 cells were revived and cultured normally to log phase.

(2) Preparation of splenocytes: mice to be fused were selected, sacrificed on the day of fusion by cervical dislocation, spleens were removed, splenocytes collected and counted in a standard procedure.

(3) Cell fusion: myeloma cells and spleen cells were mixed at a ratio of 1:3 to 1:10, cell fusion was performed according to the standard protocol, followed by culturing in HAT DMEM complete medium, hybridoma cells were observed 3 days after fusion, 1/2HAT complete medium was changed 7 days, 1/2HT medium was changed 8 days, and screening was performed about 10 days after fusion.

Cell fusion results: after fusion, HAT selective medium is used for culture, and observation is carried out under a microscope, a plurality of growing hybridoma cells are observed, and the success of the fusion operation is proved.

(4) Fusion screening: cell supernatants were aspirated at 100. mu.L/well for indirect ELISA detection. And judging the positive holes according to the ELISA result, picking and checking the positive holes detected by the whole plate by using a single-channel pipettor, and performing secondary recheck to further confirm the positive holes.

(5) Subcloning: two rounds of subcloning were performed on the rescreened positive well cells (since the positive well cell line obtained from the first subcloning was not stable yet and could contain multiple hybridoma cells, it was generally accepted that the hybridoma cells were a single cell line after the second subcloning and were determined to be positive).

Subcloning cells in the positive hole for the first time, adding an HT DMEM culture medium into the multiple holes for culture, observing under a microscope for about 7 days, detecting the hole with clone growth by indirect ELISA, and taking the hole with a high OD value as the positive hole; and (3) selecting the cells of the positive holes for secondary subcloning, detecting the stable and positive hybridoma cell strains as the cells for finally preparing the monoclonal antibody, and performing expanded culture.

(6) Monoclonal antibody subtype identification

The subtype of each supernatant was determined separately using the monoclonal antibody subtype identification kit from Southern Biotech, USA. Preparing a lath coated with immunogen protein, collecting 600 mu l of supernatant of each clone, and respectively dripping the supernatant into enzyme-labeled wells of 6 corresponding proteins, wherein the volume of each well is 100 mu l; incubating at 37 ℃ for 1h, washing PBST for three times, adding the diluted antibodies of the typing secondary antibodies, namely anti-IgM, IgA, IgG1, IgG2a, IgG2b and IgG3 into 6 holes, incubating at 37 ℃ for 1h, washing PBST for three times, and developing TMB; the identified secondary antibody subtype corresponding to the signal reaction hole is the subtype of the antibody.

After two rounds of subcloning and retesting, the following positive cell lines and subtypes were determined: 9H5, 10G4, 13a11, 13E1, 13E3, 13E11, 15E 2.

(8) And finally determining the number and subtype of the positive cell strain, and determining that the positive cell strain is as follows: 9H 5; sending the culture to China center for type culture collection for collection, wherein the collection number is CCTCC NO: C201973.

example 4 ascites preparation and antibody purification

(1) Preparing ascites: the positive cells are subjected to amplification culture and injected into the abdominal cavity of a Balb/C mouse (sensitized by Freund's incomplete adjuvant), and ascites is generated when the abdominal bulge of the mouse is seen in 7-10 days generally. When the mouse has obvious ascites, the ascites is extracted in time.

(2) Ascites purification: and purifying the ascites of the cells, wherein the purity of the purified antibody is more than 90%. The purification method comprises the following steps: purifying by ammonium caprylate and DEAE ion column method, centrifuging ascites, sucking out light yellow liquid to calculate volume, diluting with 4 times volume of 60mM acetic acid buffer solution (pH4.0) at 1:3, dropwise adding caprylic acid (final concentration is 25 μ l/ml diluted ascites), stirring at room temperature for 30min, and standing at 4 deg.C for more than 2 hr to precipitate completely. 10000r/min, 4 ℃, 20min, collecting the supernatant, adding 1/10 volume 10 × PBS (0.1 MpH7.4). Adding 0.277g solid ammonium sulfate (0.291 g/ml 45% saturated ammonium sulfate at 0 deg.C) per ml of the above mixture, and standing for at least 60 min; 10000r/min, 4 ℃, 20min, discarding the supernatant, dissolving the precipitate in a small amount of PBS, dialyzing the PBS, and dialyzing overnight at 4 ℃.

(3) Concentration determination of purified antibody: hybridoma cell CCTCC No: the ascites fluid prepared from C201973 was purified to obtain the NLRP3 monoclonal antibody 9H5, which was measured at a concentration of 1.78mg/ml using a Smart Spec plus nucleic acid protein analyzer manufactured by BIO-RAD.

(4) Titer identification of purified antibody: mu.g of the protein prokaryotically expressed from the truncated fragment of NLRP3 was dissolved in 10ml of 0.05M carbonate-coated buffer pH9.6 and added to 96-well plates at 100. mu.L per well overnight at 4 ℃. Washing the plate three times with PBS (containing 0.05% (V/V) Tween-20), blocking with 10mM PBS containing 1% BSA blocking solution 150. mu.l/well at 37 ℃ for 2h, washing the plate three times with PBS (containing 0.05% (V/V) Tween-20), adding 100. mu.l purified antibody per well, incubating at 37 ℃ for 1h, washing the plate three times with PBS (containing 0.05% (V/V) Tween-20), adding horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody as a secondary antibody, incubating at 37 ℃ for 30min, washing the plate three times with PBS (containing 0.05% (V/V) Tween-20), adding 100. mu.l TMB per well, developing color, adding 2M H after incubating at 37 ℃ for 15min₂SO₄The solution stops the reaction, and the microplate reader detects the absorbance value at 450 nm.

(5) Detecting the specificity of the purified antibody by an indirect immunofluorescence method:

digesting 1 bottle of porcine retinal epithelial cells with 0.25% pancreatin, inoculating to a 96-well plate, inoculating to 1 × 10⁷CCU/ml mycoplasma hyopneumoniae, 2% DMEM medium to continue to culture for 2 days; 2 days after inoculation of mycoplasma, cell supernatant is discarded, PBS is added to each hole gently for washing for 3 times, and cells are prevented from being suspended; the liquid in the wells of the cells was removed as much as possible, and 100. mu.L of absolute ethanol was added to each well and fixed at 4 ℃ for 2 hours.

Removing the fixing solution, drying, adding ascites 1 of 7 monoclonal antibodies of 9H5, 10G4, 13A11, 13E1, 13E3, 13E11 and 15E 2: 100, cell supernatants were added as controls and incubated at 37 ℃ for 1 hour.

Wash 3 times with PBS, gently tap the plate to spin the liquid, add anti-mouse fluorescent antibody 1 from Sigma: incubate at 100, 37 ℃ for 1 hour.

Wash 3 times with PBS, gently tap the plate to drain the liquid, and observe under a fluorescent microscope, see figure 2. The result shows that the 9H5 monoclonal antibody has better reactogenicity.

Example 5 establishment of NLRP3 truncated protein double-antibody sandwich ELISA detection method

An establishment method of an NLRP3 truncated protein double-antibody sandwich ELISA detection method comprises the following steps:

(1) preparation of HRP enzyme-labeled antibody mAb2

Dissolving HRP enzyme into ultrapure water to make the final concentration of the HRP enzyme be 10mg/ml, taking 1ml, taking 500ml again, taking 10mg/ml HRP enzyme, reacting for 1 hour at 4 ℃, adding 500 mul 1% ethylene glycol, uniformly mixing, reacting for 1 hour at 4 ℃, adding 5mg mAb2 into 250 mul of the final product, uniformly mixing, dialyzing for 16 hours in 0.05M carbonate buffer solution with pH9.6, changing the solution once in the process, adding 30 mul 5mg/ml sodium borohydride to stop the reaction, centrifuging for 2 hours at 4 ℃, desalting by using a G25 desalting column to obtain the final enzyme-labeled HRP-mAb2 marker, adding 50% glycerol, and standing for-20 ℃ for storage. mAb2 is a polyclonal antibody NLRP3, prepared by immunizing mice with a NLRP3 truncated protein.

(2) Coating antibody and labeled antibody concentration determination

Determining the optimal reaction concentration of the coated antibody and the labeled antibody by chessboard titration

Coating: diluting NLRP3 monoclonal antibody mAb1 with 0.05M carbonate buffer solution with pH value of 9.6, adding into 96-well enzyme label plate with each well being 100 μ l, coating amount being 50 μ g, 100 μ g, 200 μ g, 400 μ g, and standing overnight at 4 deg.C;

and (3) sealing: PBST (PBST) washing liquid is washed for 3 times and dried, blocking liquid (0.02M PBS + 5% skimmed milk powder + 0.5% Tween20) is added, and incubation is carried out for 1 hour;

sample incubation: PBST wash liquor is washed for 3 times and dried, 100 mul of NLRP3 truncated protein with the concentration of 0.1 mug/ml is added into each hole to be used as a positive sample, PBS is used as a negative control, and the positive sample is incubated for 1 hour at 37 ℃;

and (3) secondary antibody incubation: PBST wash was dried 3 times, and 100. mu.l of HRP-mAb2 was added to each well at 1:20, 1: 40,1:80,1:160, incubation at 37 ℃ for 1 hour;

color development: preparing TMB two-component color developing solution A and B at a ratio of 1:100, adding into enzyme labeling plate, incubating at 37 deg.C in dark for 15min with each well of 100 μ l, adding 1M H₂SO₄The reaction was terminated.

Solution A: 12.6g of sodium acetate, 1.6g of citric acid and 0.3g of carbamide peroxide are added to the mixture to be constant volume to 500 ml;

and B, liquid B: EDTA-2Na 0.2g + citric acid 0.95g +50ml glycerol + tetramethylbenzidine 0.2g (dissolved in 1ml DMSO) to 500 ml.

Reading: readings were taken with a microplate reader at OD450 nm.

The results are shown in tables 3 and 4:

TABLE 3 Absorbance values for different coated and labeled antibody concentrations

TABLE 4 Absorbance values for different coated and labeled antibody concentrations

The result shows that the optimal mAb1 antibody coating amount is 200 mug/hole, and the optimal coating concentration is 2-5 mug/mL; the optimal dilution of the labeled antibody is 1:80, and the optimal reaction concentration is 10-15 mu g/mL.

Example 6 determination of detection range of NLRP3 truncated protein double-antibody sandwich ELISA detection kit

The specific method for drawing the standard curve of the NLRP3 truncated protein double-antibody sandwich ELISA detection kit and determining the detection range comprises the following steps:

coating: diluting NLRP3 monoclonal antibody mAb1 with 0.05M carbonate buffer solution with the pH value of 9.6 respectively, adding the diluted solution into a 96-hole enzyme label plate, wherein each hole is 100 mu l, the coating concentration is 2-5 mu g/mL, and standing overnight at 4 ℃;

sample incubation: PBST wash liquor is washed for 3 times and dried, NLRP3 truncated protein of 0.1 mu g/ml is diluted in a multiple ratio for 11 concentrations, PBS is used as a negative control, and the PBST wash liquor is incubated for 1 hour at 37 ℃;

and (3) secondary antibody incubation: washing the PBST washing solution for 3 times, drying, adding 100 mu l of HRP-mAb2 into each hole, wherein the concentration is 10-15 mu g/mL, and incubating for 1 hour at 37 ℃;

color development: the TMB two-component developing solution A and B are prepared according to the proportion of 1:100, added into an enzyme label plate with each hole being 100 mul,incubating at 37 deg.C in the dark for 15min, adding 1M H₂SO₄The reaction was terminated.

Reading: readings were taken with a microplate reader at OD450 nm.

The results are shown in Table 5:

TABLE 5 absorbance at different concentrations

Standard fit curves are plotted fig. 3, with the standard curve fit equation being 0.0303x +0.0661 for y and 0.9949 for R2. The minimum detectable concentration of the ELISA detection kit is determined to be 0.1ng/ml, and the maximum detectable concentration is 0.1 mu g/ml.

Example 7 application of double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit for NLRP3 truncated protein in detection of porcine lung tissue samples

Selecting 40 healthy piglets of 5-15 days old with negative detection of mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus, circovirus-2 and porcine infectious pleuropneumonia, randomly dividing into two groups, wherein one group is a virus attacking group and is inoculated with the mycoplasma hyopneumoniae, and the other group is inoculated with sterilized PBS as a control by the same method. And collecting the pig lung tissue samples for liquid nitrogen preservation on 7 th, 14 th, 21 th, 28 th and 35 th days after immunization. 0.1mg of the cryopreserved pig lung tissue sample is weighed, 900 mu L of PBS is added to be fully ground, the mixture is centrifuged at 8000rpm for 10min, and the supernatant is taken and stored at-20 ℃ to be used as the sample to be detected.

sample incubation: PBST washing liquid is washed for 3 times, a lung tissue sample to be detected is added, PBS is used as a negative control for each sample, and incubation is carried out for 1 hour at 37 ℃;

color development: the TMB two-component color developing solution A and B are prepared according to the proportion of 1:100, added into an enzyme label plate, each hole is 100 mu l, incubated for 15min at 37 ℃ in a dark place, and added with 1M H2SO4 to terminate the reaction.

Reading: the A values were read with a microplate reader at OD450 nm.

The results are shown in Table 6:

TABLE 6 results of the detection of porcine lung tissue samples by the double antibody sandwich ELISA detection kit

Sampling time (sky)	7	14	21	28	35
						Mean value of A values of control group	0.8767	1.0352	0.9842	0.9337	1.0645
Mean value of A values of challenge groups	1.0824	1.1662	1.2491	1.4446	1.2919

The standard curve prepared in example 6 was compared with the a value of the sample to be tested, and the dilution factor was multiplied to calculate the NLRP3 protein content of the sample to be tested. As shown in the results obtained in figure 4, the expression level of the challenge group NLRP3 is obviously higher than that of the healthy group, and the expression level is increased along with the increase of the infection days, and the expression level of the healthy group NLRP3 is basically stable until the highest level is reached on the 28 th day.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> institute of zootechnics of academy of agricultural sciences in Hainan province

Shandong province Binzhou animal husbandry veterinary research institute

<120> double-antibody sandwich ELISA quantitative detection kit of pig inflammasome NLRP3 and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>288

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Met Ser Met Ala Ser Val Arg Cys Lys Leu Ala Arg Tyr Leu Glu Asp

1 5 10 15

Leu Glu Asp Val Asp Phe Lys Lys Phe Lys Met His Leu Glu Asp Tyr

20 25 30

Pro Ser Gln Lys Gly Cys Ile Ser Leu Pro Arg Gly Gln Thr Glu Lys

35 40 45

Ala Asp His Val Asp Leu Ala Thr Leu Met Ile Asp Phe Asn Gly Glu

50 55 60

Glu Lys Ala Trp Ala Met Ala Thr Trp Ile Phe Ala Ala Ile Asn Arg

65 70 75 80

Arg Asp Leu Tyr Glu Lys Ala Lys Arg Asp Glu Pro Glu Trp Asp Asn

85 90 95

Ala Asn Leu Ser Val Ile Ser Gln Glu Glu Ser Leu Glu Glu Glu Trp

100 105 110

Met Gly Leu Leu Gly Tyr Leu Ser Arg Ile Ser Ile Cys Lys Lys Lys

115 120 125

Lys Asp Tyr Cys Lys Lys Tyr Arg Lys His Val Arg Ser Arg Phe Gln

130 135 140

Cys Ile Lys Asp Arg Asn AlaArg Leu Gly Glu Ser Val Asn Leu Asn

145 150 155 160

Lys Arg Phe Thr Arg Leu Arg Leu Ile Lys Glu His Arg Ser Gln Gln

165 170 175

Glu Arg Glu His Glu Leu Leu Ala Ile Gly Arg Thr Ser Ala Lys Met

180 185 190

Gln Asp Gly Pro Val Ser Ser Leu Asn Leu Glu Leu Leu Phe Asp Pro

195 200 205

Glu Asp Gln His Ser Glu Pro Val His Thr Val Val Phe Gln Gly Ala

210 215 220

Ala Gly Ile Gly Lys Thr Ile Leu Ala Arg Lys Ile Met Leu Asp Trp

225 230 235 240

Ala Ser Glu Lys Leu Tyr Gln Glu Lys Phe Asp Tyr Leu Phe Tyr Ile

245 250 255

His Cys Arg Glu Val Ser Leu Gly Thr Arg Arg Ser Leu Gly Asp Leu

260 265 270

Ile Ala Ser Cys Cys Pro Gly Pro Asn Pro Pro Ile Gly Lys Ile Val

275 280 285

<210>2

<211>864

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atgagcatgg caagcgtccg ctgcaagctg gctcgttacc tggaggacct agaagatgta 60

gattttaaga aattcaagat gcacttagaa gactatcctt ctcagaaggg ctgcatctct 120

ctccctcggg gccagacaga aaaagcagat catgtggatc tagccactct gatgatagat 180

ttcaatggag aggagaaggc atgggccatg gccacatgga tttttgctgc gatcaacagg 240

agagaccttt atgagaaagc taagagggat gagccagaat gggacaatgc aaatctttct 300

gtgataagcc aggaagaaag ccttgaagag gaatggatgg gtttactggg gtacctttcc 360

agaatctcta tttgtaagaa aaagaaagat tattgtaaga agtacagaaa gcacgtgaga 420

agcagattcc agtgcatcaa agacaggaat gcacgtctgg gtgagagtgt gaacctcaac 480

aaacgcttca ccaggctgcg tctcatcaag gaacaccgga gtcagcagga gagggagcat 540

gagctccttg ccattggcag gacctcagcc aagatgcaag atggccccgt gagttccctg 600

aacttggaat tgctgtttga tcctgaggac caacactctg agcctgtgca cacggtagta 660

ttccagggag cagcaggcat tgggaaaaca atactggcca ggaagatcat gttggactgg 720

gcatcagaga aactttacca ggaaaaattt gactatttgt tttacattca ctgtcgggag 780

gtgagcctag gaactcggag gagcctggga gacctgatcg ccagctgctg ccctggccca 840

aacccaccca taggcaagat tgtg 864

Claims

1. A double-antibody sandwich ELISA quantitative detection kit of a porcine inflammasome NLRP3 is characterized in that: the solid phase carrier of the kit is coated with an antibody with a biological preservation number of CCTCC NO: c201973 hybridoma cell line 9H 5.

2. The double antibody sandwich ELISA quantitative detection kit of porcine inflammasome NLRP3 as claimed in claim 1, wherein: the coating concentration of the coating antibody is 2-5 mug/mL.

3. The double antibody sandwich ELISA quantitative detection kit of porcine inflammasome NLRP3 as claimed in claim 1, wherein: the kit also comprises a detection antibody, wherein the detection antibody is a polyclonal antibody for recognizing the porcine inflammasome NLRP3, and the detection antibody is labeled with horseradish peroxidase (HRP).

4. The double antibody sandwich ELISA quantitative detection kit of porcine inflammasome NLRP3 as claimed in claim 1, wherein: the concentration of the detection antibody is 10-15 mug/mL.

5. The double antibody sandwich ELISA quantitative detection kit of porcine inflammasome NLRP3 as claimed in claim 1, wherein: the kit also comprises a confining liquid, a color developing agent, a stop solution, a washing liquid and a standard substance.

6. The double antibody sandwich ELISA quantitative detection kit of porcine inflammasome NLRP3 as claimed in claim 1, wherein: the confining liquid is a phosphate buffer solution containing calf serum with the volume fraction of 5%, Tween20 with the mass fraction of 0.1% and a preservative with the mass fraction of 0.01%, and the preservative is procline 300.

7. The double antibody sandwich ELISA quantitative detection kit of porcine inflammasome NLRP3 as claimed in claim 1, wherein: the color developing agent is 3, 3 ', 5' -tetramethyl benzidine single-component color developing liquid or 3, 3 ', 5' -tetramethyl benzidine-H₂O₂A bi-component color developing liquid.

8. The double antibody sandwich ELISA quantitative detection kit of porcine inflammasome NLRP3 as claimed in claim 1, wherein: the washing solution was PBS containing 0.1% mass fraction Tween 20.

9. The double antibody sandwich ELISA quantitative detection kit of porcine inflammasome NLRP3 as claimed in claim 1, wherein: the standard substance is NLRP3 recombinant protein or NLRP3 protein.

10. Use of the kit of any one of claims 1 to 8 for the quantitative detection of porcine inflammasome NLRP 3.