CN109709329A

CN109709329A - For detecting chronic nasosinusitis with the ELISA kit and preparation method thereof of nasal polyp hypotype

Info

Publication number: CN109709329A
Application number: CN201811437289.8A
Authority: CN
Inventors: 张罗; 王成硕; 闫冰; 王阳
Original assignee: Beijing Institute Of Otorhinolaryngology; Beijing Tongren Hospital
Current assignee: Beijing Institute Of Otorhinolaryngology; Beijing Tongren Hospital
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2019-05-03

Abstract

The present invention proposes a kind of ELISA kit and preparation method thereof for detecting chronic nasosinusitis with nasal polyp hypotype, ELISA Plate including being coated with Xia Keleidengshi crystalline protein CLC antibody, the Xia Keleidengshi crystalline protein CLC antibody that is coated with is mouse anti human CLC monoclonal antibody.The present invention uses mouse anti human CLC monoclonal antibody, it is applied in ELISA kit, to realize detection chronic nasosinusitis with the purpose of nasal polyp hypotype, the kit can quickly identify nasal polyp hypotype, and it is higher compared to more traditional pathological examination method accuracy, this ELISA kit can carry out high-volume, quickly detection to sample simultaneously, save human cost and medical treatment cost.

Description

For detecting chronic nasosinusitis with the ELISA kit and its preparation of nasal polyp hypotype Method

Technical field

The invention belongs to biomedicine technical fields, more particularly to one kind is for detecting chronic nasosinusitis with nasal polyp hypotype ELISA kit and preparation method thereof.

Background technique

Chronic nasosinusitis is with nasal polyp (Chronic rhinosinusitis with nasal polyps, CRSwNP) The chronic inflammation of sinus mucosa, the visible nasal cavity of physical examination or middle nasal meatus polyp are formed.CRSwNP common sympton is stifled nose, runny nose or nose Tears refluence, hyposphresia, the bored swollen sense of face or feeling of stress, duration are more than 12 weeks.Illness rate is about 0.5-4%, CRSwNP It is often accompanied by asthma and allergic rhinitis, has been reported that 7% asthmatic patient suffers from CRSwNP, and the CRSwNP of 26-48% is with heavy breathing Asthma.CRSwNP pathogenesis is also uncertain at present, and mucosal epithelial cells destroy, host immune system is unbalance and pathogenic microorganism enters Invade the main reason for may be its morbidity.The main therapeutic modality of CRSwNP is operation and drug therapy.But studies have shown that even if By the drug or operative treatment of specification, chronic nasosinusitis is still up to 56% with the recurrence rate of nasal polyp, and it is raw to seriously affect patient Bioplasm amount, while bringing high medical to pay, but clinical shortage radical treatment method, thus become nasology research field Emphasis.

Degree according to eosinophils can divide CRSwNP for eosinophil type (Eosinophilic CRSwNP, ECRSwNP) and Non eosinophilic granulocyte type (Noneosinophilic CRSwNP, nonECRSwNP), the two is faced Bed performance, medication and prognosis are different, and the clinical symptoms of eosinophil type are heavier, with nose is stifled and hyposphresia based on, it is more It is associated with asthma, Postoperative recurrent rate is higher.Eosinophils degree and relapse are the closest in human nasal polyps tissues, When the cell percentages are more than 27% in tissue, risk of recurrence is more than 90%.Eosinophil type polyp is to sugared cortical hormone The susceptibility of plain class drug is significantly higher than Non eosinophilic granulocyte type.Non eosinophilic granulocyte type clinical symptoms are generally relatively light, and The probability for merging asthma is smaller, and airway inflammation is lighter, and Postoperative recurrent rate is low compared with eosinophil type, to macrolides medicine Object therapeutic response is good.Western countries are mainly shown as TH2 inflammatory reaction based on eosinophil type, and the acidophilus in China Property granulocyte type and Non eosinophilic granulocyte type ratio account for about half, and Non eosinophilic granulocyte type is mainly shown as TH1/TH17 Based on inflammatory reaction.In conclusion eosinophil type and Non eosinophilic granulocyte type are in immunopathogenesis type, clinical condition There are significantly different for shape, drug therapy reaction and prognosis.Different chronic nasosinusitis treat plan with inflammation/pathological of nasal polyp It is slightly different.So the identification to chronic nasosinusitis with the pathological of nasal polyp is particularly important.

The judgement of two kinds of hypotypes is lacked non-invasive mainly according to histopathology specimen staining after bronchia mucosal biopsy at present Biological markers are used for antidiastole.After patient obtains polyp sample under nasal endoscopes, the Pathologic specimens such as progress paraffin is fixed Conventional treatment, then carry out haematoxylin Yihong dyeing, followed by the inflammatory cell of ultramicroscopic observation tissue infiltration (main inflammatory cell includes eosinophil, neutrophil leucocyte, lymphocyte, thick liquid cell) infiltrates number, carries out cell point Type.The shortcomings that bronchia mucosal pathological biopsy is as follows: 1. be invasive inspection: increasing the infection risk of patient, is not suitable for being immunized Power lower crowd such as children, the elderly etc.；Nasal bleeding when materials often causes patient frightened and worry.2. being difficult to obtain disease The real-time dynamic-change information of disease: pathological biopsy cannot be carried out to healing stage mucous membrane after surgery.But clinical data shows chronic nose Sinusitis can be lapsed to the pathological classification of nasal polyp with drug therapy, operative treatment etc., can not with the pathological biopsy result before treatment Represent the feature of whole disease durations.3. time-consuming and increase medical treatment cost, general to pathological examination is obtained from tissue samples are obtained 3-4 working day.Due to can not obtain the same day or next day as a result, the additional transportation expenses of generations such as nonlocal patient sees a doctor, hotel expense, Registration fee increases medical treatment cost.4. the inflammatory cell quantity that different pathologists counts has can there are certain human error Can be different, influence the judgement of polyp parting.5. tissue pathological slice relatively limits to, it can only reflect the sample inflammation shape of slice position State cannot reflect the overall picture of tissue, be likely to result in mistaken diagnosis.6. every piece requires Pathologis artificial counting, it is difficult to Batch operation.

It can be seen that provide one kind can independent of bronchia mucosal pathological biopsy and can quick and precisely mass detection it is slow Property rhinosinusitis with nasal polyps hypotype method become those skilled in the art's technical problem urgently to be resolved.

Summary of the invention

The present invention for the above technical issues, proposes a kind of for detecting chronic nasosinusitis with nasal polyp hypotype ELISA kit and preparation method thereof.

It is a kind of for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype, including be coated with Xia Keleidengshi The ELISA Plate of crystalline protein CLC antibody.

Preferably, the Xia Keleidengshi crystalline protein CLC antibody that is coated with is mouse anti human CLC monoclonal antibody.

Preferably, in each micropore of the ELISA Plate Xia Keleidengshi crystalline protein CLC antibody package amount For 0.1~1 μ g.

Preferably, further comprising Xia Keleidengshi crystalline protein CLC standard items and its standard dilutions, TMB Substrate developing solution, detection solution A working solution, detection solution B working solution, stop bath and cleaning solution.

Preferably, the standard items are recombination CLC albumen；

The standard dilution includes polysorbas20, bovine serum albumin(BSA) and phosphate buffered saline solution；

The detection solution A working solution includes the biotin mark that 1~10 μ g/mL of concentration is diluted to using standard dilutions People's CLC antibody of note；

The detection solution B working solution includes the antibiont that 0.5~5 μ g/mL of concentration is diluted to using standard dilutions The horseradish peroxidase that plain antibody combines；

The stop bath is sulfuric acid；

The cleaning solution includes polysorbas20 and phosphate buffered saline solution.

Preferably, the standard items be recombination CLC albumen, can directly outsourcing acquire, outsourcing manufacturer is；The mark Quasi- dilution includes the phosphate buffered saline solution containing 1% polysorbas20 and 10% bovine serum albumin(BSA)；

The detection solution A working solution includes the biotin labeling that 5 μ g/mL of concentration is diluted to using standard dilutions People's CLC antibody；

The detection solution B working solution includes that the antibiotin for using standard dilutions to be diluted to concentration 1.25/mL resists The horseradish peroxidase that body combines；

The stop bath is 2N sulfuric acid；

The cleaning solution includes the phosphate buffered saline solution containing 0.05% polysorbas20.

Preferably, being coated with the preparation method of the ELISA Plate of Xia Keleidengshi crystalline protein CLC antibody, including following step It is rapid: mouse anti human CLC monoclonal antibody being diluted with phosphate buffered saline solution, takes ELISA Plate, the mouse diluted is added to it Anti-human CLC monoclonal antibody, 4 DEG C are abandoned board-washing after supernatant overnight, and confining liquid is added in every hole, abandon board-washing after supernatant again.

A kind of preparation method of the above-mentioned ELISA kit for being used to detect chronic nasosinusitis companion's nasal polyp hypotype, including with Lower step:

Step 1: preparation is coated with Xia Keleidengshi crystalline protein CLC antibody: mouse anti human CLC monoclonal antibody is used Phosphate buffered saline solution dilution；

Step 2: preparation is coated with the ELISA Plate of Xia Keleidengshi crystalline protein CLC antibody: taking ELISA Plate, every hole is added The mouse anti human CLC monoclonal antibody diluted, 4 DEG C are abandoned addition cleaning solution board-washing after supernatant overnight, and confining liquid is added in every hole, then Cleaning solution board-washing is added after secondary abandoning supernatant；

Step 3: doubling dilution standard items: taking standard items, standard dilutions is added to it, concentration, which is prepared, is The standard items of Xng/mL take the standard items that concentration is Xng/mL to carry out doubling dilution, successively doubling dilution concentration are as follows: Xng/mL, 0.5Xng/mL, 0.25Xng/mL, 0.625Xng/mL, 0.3125Xng/mL, 0.15625Xng/mL, 0.078125Xng/mL；

Step 3: sample dilution: taking-up sample to room temperature, and carry out gradient dilution processing；

Step 4: dress sample: the ELISA Plate for being coated with Xia Keleidengshi crystalline protein CLC antibody for taking step 2 to prepare, often Hole is sequentially added into the sample after the dilution of the standard items after the doubling dilution of step 3 and step 4, while setting is added with Cleaning solution board-washing is added in the blank well of standard dilutions after incubation；

Step 5: preparation detection solution A working solution: take people's CLC antibody of biotin labeling dilute using standard dilutions It is added after being interpreted into 1~10 μ g/mL to each hole of ELISA Plate, cleaning solution board-washing is added after overlay film and incubation；

Step 6: the anti-of 0.5~5 μ g/mL of concentration preparation detection solution B working solution: is diluted to using standard dilutions It is added after the horseradish peroxidase that biotin antibody combines to each hole of ELISA Plate, cleaning solution is added after overlay film and incubation Board-washing；

Step 7: colour developing: TBM substrate developing solution is prepared after taking TMB that 0.75% hydrogen peroxide of equal volume is added and adds Enter to each hole of ELISA Plate, after observation becomes brown color added with the hole for the standard items that concentration is Xng/mL, ELISA Plate Each hole be added stop bath, it is to be added when to have concentration be that the hole of standard items of Xng/mL becomes blue from yellow, using enzyme The detection of instrument 450nm Detection wavelength, 550nm-570nm reference wavelength colorimetric are marked, production standard curve calculates sample concentration value.

In the step 2, it is anti-that the mouse anti human CLC monoclonal that 100 μ L have diluted is added in each hole of the ELISA Plate Body；The amount that cleaning solution is added in every hole is 300 μ L；The amount that confining liquid is added in every hole is 250 μ L；

In the step 2, the standard items are taken, standard dilutions is added, the standard that concentration is 20ng/mL is prepared Product, according to this doubling dilution concentration are as follows: 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL and The doubling dilution standard items of 0.312ng/mL.

Preferably, the standard dilutions include that the phosphoric acid containing 1% polysorbas20 and 10% bovine serum albumin(BSA) is slow Rush salting liquid；The confining liquid includes the phosphate buffered saline solution containing 2% polysorbas20 and 2% bovine serum albumin(BSA).

Preferably, taking people's CLC antibody of biotin labeling to be diluted to 5 μ using standard dilutions in the step 5 After g/mL, 100 μ L is taken to be added to each hole of ELISA Plate, overlay film and in 37 DEG C be incubated for 1 hour, board-washing 3 times；

In the step 6, combined using the anti-biotin antibodies that standard dilutions are diluted to 1.25 μ g/mL of concentration After horseradish peroxidase, 100 μ L is taken to be added to each hole of ELISA Plate, overlay film and in 37 DEG C be incubated for 0.5 hour, board-washing 5 It is secondary；

In the step 7, the amount that TBM substrate developing solution is added in every hole is 90 μ L；The amount that stop bath is added in every hole is 50 μL。

In order to achieve the above object, the technical solution adopted by the present invention are as follows:

Compared with prior art, the advantages and positive effects of the present invention are:

1, the present invention provides a kind of for detecting ELISA kit and its preparation side of the chronic nasosinusitis with nasal polyp hypotype Method is applied in ELISA kit using mouse anti human CLC monoclonal antibody, to realize detection chronic nasosinusitis with breath The purpose of meat hypotype, the kit can quickly identify nasal polyp hypotype, and compare more traditional pathological examination method Accuracy is higher, this ELISA kit can carry out high-volume, quickly detection to sample simultaneously, has saved human cost and just Cure cost.

2, the present invention provides a kind of ELISA kit for detecting chronic nasosinusitis with nasal polyp hypotype, with polymerase Chain reaction technology (PCR) is compared, and there are following advantages: the regulation of human body has a transcriptional control, post-transcriptional control, translational control, Post-translational control, and three regulations after using PCR detection transcriptional control that can not reflect, and protein level can reflect final expression Amount, and the pharmacodynamic assessment based on protein level that is detected as of protein level provides possibility.The present invention provides a kind of for examining Chronic nasosinusitis is surveyed with the ELISA kit of nasal polyp hypotype by using mouse anti human CLC monoclonal antibody, improves detection The accuracy of chronic nasosinusitis companion's nasal polyp hypotype, and achieved the effect that quick detection.

3, the present invention provide it is a kind of can be comprehensive with the ELISA kit of nasal polyp hypotype for detecting chronic nasosinusitis Response organization's Pathologic Characteristics solve the influence of human error in the prior art, avoid histotomy and reacted tissue local Feature causes the drawbacks of mistaken diagnosis.Clinic is examined by the nasal polyp neuraminidase that ELISA kit carries out fast, accurately and comprehensively Treat it is most important, with as early as possible according to the inflammation hypotype of nasal polyp carry out for change treatment, effectively guidance be directed to chronic nasosinusitis companion Reaction, the judging prognosis effect of drug therapy are accurately estimated in the determination of the medical treatment regime and modus operandi of Nasal Polyps Patients.

Detailed description of the invention

Some specific embodiments of the present invention is described in detail by way of example and not limitation with reference to the accompanying drawings hereinafter. Identical appended drawing reference denotes same or similar part or part in attached drawing.It should be appreciated by those skilled in the art that these What attached drawing was not necessarily drawn to scale.In attached drawing:

Fig. 1 is the canonical plotting of effect detection of embodiment of the present invention test result.

Specific embodiment

The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects It encloses.

The embodiment of the invention provides a kind of for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype, packet Include the ELISA Plate for being coated with Xia Keleidengshi crystalline protein CLC antibody.

The above-mentioned gene I/D for being coated with Xia Keleidengshi crystalline protein CLC antibody is 1178, DNA sequence dna such as SEQIDNO: Shown in 1, gene NM is 001828.5.

In an alternative embodiment, the Xia Keleidengshi crystalline protein CLC antibody that is coated with is mouse anti human CLC Dan Ke Grand antibody.

In an alternative embodiment, the Xia Keleidengshi crystalline protein CLC antibody in each micropore of the ELISA Plate Package amount be 0.1~1 μ g.Wherein in order to further increase the sensibility of ELISA kit, each of preferably described ELISA Plate The package amount of the Xia Keleidengshi crystalline protein CLC antibody is 0.5 μ g in micropore.

In an alternative embodiment, further comprises Xia Keleidengshi crystalline protein CLC standard items and its standard items are dilute Release liquid, tmb substrate developing solution, detection solution A working solution, detection solution B working solution, stop bath and cleaning solution.It is preferred that described Standard items are recombination CLC albumen, and wherein recombinant C LC albumen is commercially available to be directly paid, and purchase manufacturer can be；The standard is dilute Releasing liquid includes polysorbas20, bovine serum albumin(BSA) and phosphate buffered saline solution；The detection solution A working solution includes using standard items Diluted at the biotin labeling of 1~10 μ g/mL of concentration people's CLC antibody；The detection solution B working solution includes using Standard dilutions are diluted to the horseradish peroxidase that the anti-biotin antibodies of 0.5~5 μ g/mL of concentration combine；The termination Solution is sulfuric acid；The cleaning solution includes polysorbas20 and phosphate buffered saline solution.In order to further make the ELISA reagent obtained Box, detection is sensitiveer, efficient, and testing result is more accurate, and more preferably, the standard dilution includes spitting containing 1% The phosphate buffered saline solution of temperature 20 and 10% bovine serum albumin(BSA)；The detection solution A working solution includes being diluted using standard items Liquid is diluted to people's CLC antibody of the biotin labeling of 5 μ g/mL of concentration；The detection solution B working solution includes dilute using standard items It releases liquid and is diluted to the horseradish peroxidase that the anti-biotin antibodies of 1.25 μ g/mL of concentration combine；The stop bath is 2N sulphur Acid；The cleaning solution includes the phosphate buffered saline solution containing 0.05% polysorbas20.

In an alternative embodiment, it is coated with the preparation method of the ELISA Plate of Xia Keleidengshi crystalline protein CLC antibody, is wrapped It includes following steps: mouse anti human CLC monoclonal antibody being diluted with phosphate buffered saline solution, takes ELISA Plate, dilution is added to it Good mouse anti human CLC monoclonal antibody, 4 DEG C are abandoned board-washing after supernatant overnight, and confining liquid is added in every hole, are washed after abandoning supernatant again Plate.

In an alternative embodiment, the standard dilutions include containing 1% polysorbas20 and 10% bovine serum albumin(BSA) Phosphate buffered saline solution；The confining liquid includes that the phosphate-buffered salt containing 2% polysorbas20 and 2% bovine serum albumin(BSA) is molten Liquid.

In an alternative embodiment, in the step 5, take people's CLC antibody of biotin labeling using standard dilutions After being diluted to 5 μ g/mL, 100 μ L is taken to be added to each hole of ELISA Plate, overlay film and in 37 DEG C be incubated for 1 hour, board-washing 3 times；

It is introduced provided by the embodiment of the present invention in detail to become apparent from for detecting chronic nasosinusitis with nasal polyp Asia ELISA kit of type and preparation method thereof is described below in conjunction with specific embodiment.

Embodiment 1:

It is a kind of for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype, including following components:

It is coated with the ELISA Plate of Xia Keleidengshi crystalline protein CLC antibody, it is described to be coated with Xia Keleidengshi crystalline protein CLC antibody is mouse anti human CLC monoclonal antibody, the Xia Keleidengshi crystalline protein in each micropore of the ELISA Plate The package amount of CLC antibody is 0.5 μ g；

Standard dilution: the phosphate buffered saline solution containing 1% polysorbas20 and 10% bovine serum albumin(BSA)；

Detection solution A working solution: the people CLC for being diluted to the biotin labeling of 5 μ g/mL of concentration using standard dilutions is anti- Body；

Detection solution B working solution: it is combined using the anti-biotin antibodies that standard dilutions are diluted to concentration 1.25/mL Horseradish peroxidase；

Stop bath: 2N sulfuric acid；

Cleaning solution: the phosphate buffered saline solution containing 0.05% polysorbas20.

Embodiment 2:

Step 1: preparation is coated with Xia Keleidengshi crystalline protein CLC antibody:

Step 1: animal immune: taking people's recombinant C LC albumen 1:1 that Freund's complete adjuvant and freund 's incomplete adjuvant is added, and resists Former and adjuvant mixes in equal volume, is ground into the chyle shape of Water-In-Oil.

(1) initial immunity, only, the subcutaneous multi-point injection 1.5mL of Freund Freund's complete adjuvant is spaced 3 weeks 50 μ g/ of recombinant protein.

(2) it is immunized for second, dosage routes are same as above, Freund Freund's incomplete adjuvant, are spaced 3 weeks.

(3) third time is immune, and dosage is same as above, and adjuvant is not added, and is injected intraperitoneally, its potency is surveyed in blood sampling after 7 days, detects immune effect Fruit is spaced 3 weeks.

(4) booster immunization, 50 μ g of dosage, intraperitoneal injection.

After (5) 3 days, spleen is taken to merge；

Step 2: cell fusion:

(1) preparation of feeder cells

After cell fusion in selective incubation, since a large amount of myeloma cells and splenocyte die off, at this time The hybridoma of single or a small number of dispersions is not easy to survive mostly, it is often necessary to and other living cells are added and are allowed to breed, this quilt The living cells of addition is known as feeder cells；

The preparation of Turnover of Mouse Peritoneal Macrophages:

1. choosing the BALB/c mouse of 6-10 week old；

2. being soaked in 75% alcohol, 3min is sterilized, injects 6mL culture solution to peritonaeum suction pipe, punching is sucked out in repeated flushing Washing lotion；

3. being put into 10mL centrifuge tube, 1200rpm is centrifuged 5min；

4. being suspended with the culture solution of 20% calf serum, adjustment cell number is 1*10⁵96 orifice plates, 100 μ L/ are added in/mL Hole is put into 37 degree of incubator cultures；

(2) preparation of myeloma cell

1. myeloma cell is expanded and is cultivated 48-36 hours before fusion

2. on the fusion same day, cell gently being blown down from bottle wall with connector bend dropping tube, is collected in 50mL centrifuge tube or fusion pipe.

3. 1000r/min is centrifuged 5-10 minutes, discard supernatant.

4. 30mL incomplete culture medium is added, centrifuge washing is primary.Then Cell resuspension is not exclusively cultivated in 10mL Base mixes.

5. myeloma cell's suspension is taken, it is spare after adding 0.4% phenol indigo plant dye liquor to make viable count.

(3) preparation of splenocyte

Immunized BALB/c mouse is taken, positive when extracing eyeball blood sampling, and separating serum as antibody test is right According to serum.It simultaneously by mouse, is soaked in 75% alcohol 5 minutes, is placed in that have filled 10mL endless in taking out spleen on culture dish In the plate of full culture medium, surrounding connective tissue is washed and peelled off, in horizontalization ware on stainless steel mesh, is ground with syringe needle core At counting after cell suspension, splenocyte is made to enter the incomplete culture medium in plate, for several times with suction pipe piping and druming, is made unicellular outstanding Liquid, every mouse 1 × 10⁸-2.5×10⁸A splenocyte；

(4) cell fusion

1. by 1 × 10⁸Splenocyte and 1 × 10⁷Myeloma cell SP2/0 is mixed in a 50mL fusion pipe, is added endless Full culture medium is mixed well to 30mL；

2. 1000r/min is centrifuged 5-10 minutes, supernatant is exhausted as far as possible；

3. tapping bottom of fusion pipe on palm, keep sedimentation cell loosely uniform；

4. 50%PEG (polyethylene glycol) 1mL of preheating is added in 30s with 1mL suction pipe, side edged is gently mixed；

5. sucking suction pipe stands 1min；

6. the endless full nutrient solution of preheating is added, PEG effect is terminated, is separately added into 1mL, 2mL, 3mL in continuous every 2min, 4mL, 5mL, 10mL；

7. 800rpm, 5 minutes；It discards supernatant；

8. 5mL complete medium is added, gently pressure-vaccum sedimentation cell, makes it suspend and mix, and then adds complete culture Base is to 40-50mL；96 porocyte culture plates are dispensed, then culture plate is set 37 DEG C, 5%CO by every 100 μ L of hole₂Training in incubator It supports；

9. adding Selective agar medium after 6h, every 50 μ L of hole partly changed liquid with Selective agar medium after 3 days；

10. supernatant is sucked out for antibody when its length to 1/10 or more hole floor space in often observation Growth of Hybridoma Cell situation Detection；

(5) selection of hybridoma

1. antigen is diluted to 10 μ g/mL with coating buffer；

It is added in ELISA Plate hole 2. being measured with 100 holes μ L/, sets 4 DEG C overnight or 37 DEG C adsorb 2 hours；

3. discarding the liquid in hole, while being washed 3 times, 3 minutes every time, is patted dry with cleaning solution；

4. every hole adds 37 DEG C of 100 μ L confining liquid to close 1 hour；

5. cleaning solution is washed 3 times；

6. every hole adds 100 μ L Hybridoma Cell Culture supernatant to be checked, while setting up positive, negative control and blank control； 37 DEG C are incubated for 1 hour；Washing, pats dry.

7. enzyme mark secondary antibody, every 100 μ L of hole, 37 DEG C are incubated for 1 hour, and washing pats dry；

8. plus substrate solution, every hole add the substrate of Fresh using 100 μ L of liquid, 37 DEG C are kept for 20 minutes；

9. with 2mol/L H₂SO₄Reaction is terminated, OD value is read on enzyme linked immunological reading apparatus；

10. result judgement: being the positive with P/N≤2.1 or P≤N+3SD.If negative control hole is colourless or close to colourless, sun Property control wells clearly develop the color, then can directly detect by an unaided eye result；

(6) cloning (limiting dilution assay) of hybridoma

1. preparing mouse boosting cell is feeder cells；

2. preparing hybridoma suspension to be cloned, every milliliter is diluted to containing 5,10 with the HT culture medium containing 20% serum The dilution different with 20 3 kinds of cells；

3. being added 5 × 10 by every milliliter⁴-1×10⁵The ratio of cell is separately added into abdomen in above-mentioned hybridoma suspension Chamber macrophage；

4. every kind of hybridoma dispenses one piece of 96 orifice plate, every hole amount is 100 μ L；

5. 37 DEG C, 5%CO₂Culture 6 days, macroscopic clone occur can be detected antibody；It is seen under inverted microscope It examines, marks the hole of only single clonal growth, supernatant is taken to make antibody test；

6. taking the cell expansion culture in antibody test positive hole, and freeze；

Step 3: the Ig class of monoclonal antibody and the identification of subclass:

1. 50 holes μ L/, 4 DEG C overnight with the antigen coat ELISA Plate of 10 μ g/mL concentration；

2. be added monoclonal antibody sample to be checked after washing, 100 holes μ L/, 37 DEG C 1 hour；If negative, Positive control wells；

3. after washing, the anti-mouse class of HRP (horseradish peroxidase) label and the antibody reagent of subclass Ig, 100 μ are added The hole L/, 37 DEG C are protected from light colour developing 20 minutes；With 2mol/L H₂SO₄After terminating reaction, the hypotype of antibody is judged according to color；

Step 4: the production and purifying of monoclonal antibody

(1) monoclonal antibody is produced in animal body

1. grow up BALB/c mouse intraperitoneal inoculation norphytane or atoleine, every mouse 0.3-0.5mL；

2. intraperitoneal inoculation PBS or the diluted hybridoma of serum free medium, every mouse 5 × 10 after 7-10 days⁵/ 0.2mL；

3. mouse ascites production after 5 days, is observed, if abdomen obviously expands, when touching, skin has in interval daily Tension can acquire ascites.Usual every mouse can adopt 3mL ascites；

4. ascites is centrifuged (2000r/min 5 minutes), cell component and other sediments are removed, supernatant is collected, is surveyed Determine antibody titer, dispense, -70 DEG C freeze it is spare；

(2) purifying (octanoic acid-ammonium sulfate precipitation method) of monoclonal antibody

1. 4 DEG C of 12000rpm of ascites are centrifuged 15min, impurity is removed；

2. 1 part of ascites is taken to add 2 parts of 0.06mol/L PH5.0 acetate buffer solutions, add 33 μ L octanoic acids by every milliliter of dilution ascites Ratio, be stirred at room temperature down and octanoic acid be added dropwise, mixed at room temperature 30min；

3. 4 DEG C stand 2 hours, takes out 12000g and be centrifuged 30 minutes, abandon precipitating；

4. supernatant is filtered through nylon mesh, 4 DEG C of dialysis 6h in the 0.01MPH7.4PBS of 50 times of volumes；

5. isometric saturated ammonium sulfate solution is added in the supernatant after dialysis；

6. 4 DEG C of standing 1h or more, 10000g are centrifuged 30 minutes, supernatant is abandoned；

7. precipitating is dissolved in appropriate PBS (NaCl containing 137mmol/L, 2.6mol/L KCl, 0.2mmol/L EDTA), in Dialysed overnight in the PBS of 50-100 times of body；

8. taking after sample suitably dilutes after a small amount of dialysis, with UV spectrophotometer measuring protein content, SDS-PAGE, WB Detect antibody purity；

Step 2: prepare sample: serum or blood plasma (EDTA or anticoagulant heparin): taking 1000g to be centrifuged 15 minutes in 4 DEG C, Supernatant is taken, is frozen in -80 DEG C of refrigerators, it is spare；Other body fluid (tissue homogenate, phlegm, nasal secretion, various arthral fluids Deng): it takes 1000g to be centrifuged 15 minutes in 4 DEG C, takes supernatant, freeze in -80 DEG C of refrigerators, it is spare；

Step 3: preparation is coated with the ELISA Plate of Xia Keleidengshi crystalline protein CLC antibody: taking 96 hole elisa Plates, every hole The mouse anti human CLC monoclonal antibody that 100 μ L have diluted is added, 4 DEG C overnight；30 times of dilutions of washing lotion of concentration are made with distilled water Standby to obtain cleaning solution, every hole addition 300 μ L washing standing discards after 2 minutes after abandoning supernatant, and drains on blotting paper, so again 2 times repeatedly, 250 μ L confining liquids are added in every hole, repeat above-mentioned board-washing step after abandoning supernatant again；The confining liquid includes containing The phosphate buffered saline solution of 2% polysorbas20 and 2% bovine serum albumin(BSA)；

Step 4: standard items are prepared: takes standard items recombinant C LC protein dry powder to be placed in room temperature 30min, is diluted with standard items After liquid 1mL dissolution mixes repeatedly, 10 minutes are stood, the standard items that dissolved standard items are configured to concentration 20ng/mL are labeled as Standard items 1 are taking 6 EP pipe label standard items 2~7 respectively, and 500 μ L standard dilutions are added, then from standard items 1 It takes out in 500 μ L liquid addition 2 pipe of standard items and mixes in pipe, then take 500 μ L liquid to be added to standard items 3 from 2 pipe of standard items Guan Zhong, operate according to this to standard items 7 manage, finally acquire concentration be according to this 20ng/mL, 10ng/mL, 5ng/mL, The doubling dilution standard items of 2.5ng/mL, 1.25ng/mL, 0.625ng/mL and 0.312ng/mL；

Step 5: sample sample dilution: is taken out to room temperature, after serum or 2 times of the standard dilutions dilution of blood plasma sample Detection；

Step 6: dress sample: the ELISA Plate for being coated with Xia Keleidengshi crystalline protein CLC antibody for taking step 2 to prepare, often Hole is sequentially added into the sample after the dilution of the standard items after the doubling dilution of 100 μ L step 3 and step 4, is arranged simultaneously Blank well added with standard dilutions, encloses lid, 37 DEG C be incubated for 2 hours after repeat board-washing 3 times；

Step 5: preparation detection solution A working solution: take people's CLC antibody of biotin labeling dilute using standard dilutions After being interpreted into 5 μ g/mL, 100 μ L is taken to be added to each hole of ELISA Plate, overlay film and in 37 DEG C be incubated for 1 hour, board-washing 3 times；

Step 6: the antibiosis of 1.25 μ g/mL of concentration preparation detection solution B working solution: is diluted to using standard dilutions Object element antibody combine horseradish peroxidase after, take 100 μ L to be added to each hole of ELISA Plate, overlay film and in 37 DEG C be incubated for 0.5 hour, board-washing 5 times；

Step 7: colour developing: being prepared TBM substrate developing solution after taking TMB solution that 0.75% hydrogen peroxide of equal volume is added, 90 μ LTBM substrate developing solutions are added in each hole of the ELISA Plate, room temperature is protected from light incubation 15-30 minutes, when observation is added with Concentration is after the hole of the standard items of Xng/mL becomes brown color, and the stop bath 2N sulfuric acid of 50 μ L is added in each hole of ELISA Plate, It is to be added to be detected using microplate reader 450nm Detection wavelength when to have concentration be that the hole of standard items of Xng/mL becomes blue from yellow, 550nm-570nm reference wavelength colorimetric calculates sample concentration value with 4-pl pattern making standard curve.

Embodiment 3:

Effect detection test:

Test method: enzyme linked immunosorbent assay (ELISA), each original OD value (absorbance value) of the first behavior of cell, Second behavior subtracts OD value (absorbance value) after blank control, the corresponding actual concentrations of third behavior OD value.As shown in Figure 1, the 1st Column sample is various concentration standard items, and 2-6 column sample is test secretion sample, can obtain each secretion based on data Actual sample concentration.

Experiment conclusion: if 1 data of table are it is found that ELISA kit provided by the invention can be used for detecting chronic nasosinusitis companion Nasal polyp hypotype, sample 3B (3 be column B be it is capable, similarly hereinafter), 6B, 3C, 3D, 4D, 6D, 3E, 5E, 3F, 5F, 6F, 3G, 4G, 5G, 6G, CLC is not detected in 3H, 4H, 5H, is thought of as the nasal polyp of no eosinophils, other sample standard deviations have different degrees of Eosinophils, foundation quartile section (25%, 50%, 75% confidence interval is respectively 0,0.09,2.2), The concentration of sample 2A, 4A, 6A, 2B, 2C, 5C, 6C, 5D, 4E are above 75% confidence interval, are high eosinophils Nasal polyp, other samples are the nasal polyp of low eosinophils.

Wherein the standard curve of 1 data acquisition of table is as shown in Figure 1, have the reliability of this experiment known to Fig. 1, R²Value is greater than 0.95 is that test passes through, which is 0.998, the results showed that ELISA kit can accurately reflect very much dilution by OD value Multiple.Use method for enzyme linked immunosorbent assay (ELISA).

1 effect detection experimental result of table

Sequence table

<110>it raises

Wang Chengshuo

Yan Bing

Wang Yang

Beijing Otorhinolaryngology Inst.

Capital University Of Medical Sciences Affiliated Beijing Tongren Hospital

<120>for detecting chronic nasosinusitis with the ELISA kit and preparation method thereof of nasal polyp hypotype

<130> CC18K10247CCN

<141> 2018-11-27

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 649

<212> DNA

<213> human

<400> 1

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Claims

1. a kind of for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype, it is characterised in that: including being coated with the summer The ELISA Plate of Ke Leidengshi crystalline protein CLC antibody.

2. according to claim 1 for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype, feature exists In: the Xia Keleidengshi crystalline protein CLC antibody that is coated with is mouse anti human CLC monoclonal antibody.

3. according to claim 1 for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype, feature exists In: the package amount of the Xia Keleidengshi crystalline protein CLC antibody is 0.1~1 μ g in each micropore of the ELISA Plate.

4. according to claim 1 for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype, feature exists In: further comprise Xia Keleidengshi crystalline protein CLC standard items and its standard dilutions, tmb substrate developing solution, detection Solution A working solution, detection solution B working solution, stop bath and cleaning solution.

5. according to claim 4 for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype, feature exists In: the standard items are recombination CLC albumen；

The detection solution A working solution includes the biotin labeling that 1~10 μ g/mL of concentration is diluted to using standard dilutions People's CLC antibody；

The detection solution B working solution includes that the antibiotin for using standard dilutions to be diluted to 0.5~5 μ g/mL of concentration resists The horseradish peroxidase that body combines；

The stop bath is sulfuric acid；

6. according to claim 1 for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype, feature exists In: it is coated with the preparation method of the ELISA Plate of Xia Keleidengshi crystalline protein CLC antibody, comprising the following steps: by mouse anti human CLC monoclonal antibody is diluted with phosphate buffered saline solution, takes ELISA Plate, and the mouse anti human CLC monoclonal diluted is added to it Antibody, 4 DEG C are abandoned board-washing after supernatant overnight, and confining liquid is added in every hole, abandon board-washing after supernatant again.

7. a kind of claim 1~6 is described in any item for detecting chronic nasosinusitis with the ELISA kit of nasal polyp hypotype Preparation method, it is characterised in that: the following steps are included:

Step 1: preparation is coated with Xia Keleidengshi crystalline protein CLC antibody: by mouse anti human CLC monoclonal antibody phosphoric acid Buffer salt solution dilution；

Step 2: preparation is coated with the ELISA Plate of Xia Keleidengshi crystalline protein CLC antibody: taking ELISA Plate, dilution is added in every hole Good mouse anti human CLC monoclonal antibody, 4 DEG C are abandoned addition cleaning solution board-washing after supernatant overnight, and every hole is added confining liquid, abandons again Cleaning solution board-washing is added after supernatant；

Step 3: doubling dilution standard items: taking standard items, and standard dilutions are added to it, and it is Xng/mL that concentration, which is prepared, Standard items, take the standard items that concentration is Xng/mL to carry out doubling dilution, successively doubling dilution concentration are as follows: Xng/mL, 0.5Xng/ ML, 0.25Xng/mL, 0.625Xng/mL, 0.3125Xng/mL, 0.15625Xng/mL, 0.078125Xng/mL；

Step 4: dress sample: the ELISA Plate for being coated with Xia Keleidengshi crystalline protein CLC antibody for taking step 2 to prepare, every hole point Standard items after not sequentially adding the doubling dilution of step 3 and the sample after the dilution of step 4, while the standard of being added with is set Cleaning solution board-washing is added in the blank well of product dilution after incubation；

Step 5: preparation detection solution A working solution: people's CLC antibody of biotin labeling is taken to be diluted to 1 using standard dilutions It is added after~10 μ g/mL to each hole of ELISA Plate, cleaning solution board-washing is added after overlay film and incubation；

Step 6: the antibiont of 0.5~5 μ g/mL of concentration preparation detection solution B working solution: is diluted to using standard dilutions It is added after the horseradish peroxidase that plain antibody combines to each hole of ELISA Plate, cleaning solution is added after overlay film and incubation and washes Plate；

Step 7: colour developing: TBM substrate developing solution is prepared after taking TMB that 0.75% hydrogen peroxide of equal volume is added and is added extremely In each hole of ELISA Plate, after observation becomes brown color added with the hole of standard items that concentration is Xng/mL, ELISA Plate it is every A hole is added stop bath, to be added when to have concentration be that the hole of the standard items of Xng/mL becomes blue from yellow, using microplate reader The detection of 450nm Detection wavelength, 550nm-570nm reference wavelength colorimetric, production standard curve calculate sample concentration value.

8. according to claim 7 for detecting chronic nasosinusitis with the preparation side of the ELISA kit of nasal polyp hypotype Method, it is characterised in that: in the step 2, it is mono- that the mouse anti human CLC that 100 μ L have diluted is added in each hole of the ELISA Plate Clonal antibody；The amount that cleaning solution is added in every hole is 300 μ L；The amount that confining liquid is added in every hole is 250 μ L；

In the step 2, the standard items are taken, standard dilutions is added, the standard items that concentration is 20ng/mL is prepared, Doubling dilution concentration according to this are as follows: 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL and The doubling dilution standard items of 0.312ng/mL.

9. according to claim 7 for detecting chronic nasosinusitis with the preparation side of the ELISA kit of nasal polyp hypotype Method, it is characterised in that: in the step 5, people's CLC antibody of biotin labeling is taken to be diluted to 5 μ g/ using standard dilutions After mL, 100 μ L is taken to be added to each hole of ELISA Plate, overlay film and in 37 DEG C be incubated for 1 hour, board-washing 3 times；

In the step 6, the horseradish of the anti-biotin antibodies combination of 1.25 μ g/mL of concentration is diluted to using standard dilutions After peroxidase, 100 μ L is taken to be added to each hole of ELISA Plate, overlay film and in 37 DEG C be incubated for 0.5 hour, board-washing 5 times；

In the step 7, the amount that TBM substrate developing solution is added in every hole is 90 μ L；The amount that stop bath is added in every hole is 50 μ L.