CN103364567A - Surfactant protein D nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof - Google Patents

Abstract

The invention discloses a surfactant protein D (SP-D) nano-magnetic particle chemiluminescent immunity quantitative detection kit, which comprises: a surfactant protein D calibrator; a streptavidin coupled nano-magnetic particle suspension solution; a biotin labeled surfactant protein D antibody; a surfactant protein D antibody enzyme conjugate, with the enzyme being horseradish peroxidase with purity RZ of greater than or equal to 3 and activity of greater than or equal to 250U/mL; a surfactant protein D quality control material; a chemiluminescent liquid A and a chemiluminescent liquid B; a 20-time concentrated lotion; and reaction tubes. In addition, the invention also discloses a preparation method of the kit provided in the invention. Compared with the existing kits, the kit disclosed in the invention has the advantages of high sensitivity, wide measurable concentration range, long effective period of reagents, low reagent cost, simple operation, and high automation detection degree.

Description

A kind of Surfactant proteinD nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof

Technical field

The present invention relates to the immunoassay medical domain, concrete, the invention provides a kind of Surfactant proteinD (SP-D) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof.

Background technology

Pulmonary surfactant (pulmonary surfactant, PS) be synthetic by alveolar type II cells and secrete a kind of lipoprotein of complexity, a kind ofly has a surface-active compound by what phosphatide and protein formed, wherein 90% is phosphatide, 8%-9% is surfactant protein (Surfactant protein, SP), SP mainly comprises SP-A, SP-B, SP-C, SP-D, wherein SP-B and SP-C are hydrophobic small molecules albumen, SP-A and SP-D are large hydrophilic molecular albumen, the above two can reduce alveolar surface tension, and both then mainly regulate the lung immunologic function afterwards.SP-D at first finds in respiratory system, that a kind of molecular weight is the glycoprotein of 43KD, studies show that SP-D can strengthen the invasion and attack of resistivity, defence microorganism and the dust of respiratory tract as immunologic active material, not only can directly be combined with silicious dust specifically, and can also strengthen pulmonary macrophage to the phagocytosis of silicious dust, after silicious dust entered lung tissue, the expression of lung tissue SP-D can increase.

Interstitial lung disease (ILD) is a kind ofly to have multi-form and inflammation and fiber degree with alveolus wall and alveolar space and turn to the characteristic pathological change, is the infiltration shadow of extensive distribution as the general name of the diffusivity lung disease of main clinical manifestation take carrying out property expiratory dyspnea and x-ray rabat.Judge that at present ILD mainly relies on the means such as imaging examination, biopsy, routine blood test, the detection of enzyme linked immunological serology, studies show that ILD patient's x-ray inspection of 10% is normal, but proved by pathology is ILD, and patient's this moment serum SP-D level in the situation that x-ray can not detect significantly raises; Biopsy is because the pathology position difference of getting also may cause undetected.

It is simple to operate, cheap that enzymoimmunoassay possesses, precision is high, specificity is good, accuracy is good, be a kind of preferably micro substance detection technique, but susceptibility is relatively low, difference is large between plate, measured value repeatability is relatively poor, but used mark enzyme-to-substrate quantitative measurement narrow range reaches the shortcomings such as Instrument measuring narrow range.

Chemiluminescent immunoassay(CLIA) research is started in the beginning of the eighties, and fast development is applied to the nineties, is current the most responsive skeptophylaxis determination method.

Summary of the invention

The defectives such as the problem to be solved in the present invention provides chemiluminescence immunoassay immue quantitative detection reagent box of surfactant protein and preparation method thereof, has avoided the measured value poor repeatability, and euzymelinked immunosorbent assay (ELISA) sensitivity is low, and sensing range is narrow.

For solving the problems of the technologies described above, the technical solution used in the present invention is: Surfactant proteinD (SP-D) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box comprises: the Surfactant proteinD calibration object; Coupling has the nano magnetic microparticle suspending liquid of Streptavidin; Biotin labeled Surfactant proteinD antibody; Surfactant proteinD abzyme bond, used enzyme are horseradish peroxidase, horseradish peroxidase purity RZ 〉=3.0, activity 〉=250U/mL; Surfactant proteinD quality-control product, quality-control product comprise the low value quality-control product of concentration 3ng/mL and the high value quality-control product of 600ng/mL; Chemical luminescence for liquid A liquid and B liquid, A liquid are 5mmol/L, the Tris-HCl damping fluid of pH8.6, and contain final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is the 0.675g/L urea peroxide; 20 times of concentrated washing lotions; Reaction tube.

Further, described nano magnetic particulate is that the surface parcel is with the tri-iron tetroxide of amino or carboxyl reactive group, particle diameter 10-50nm.

Further, the material of described reaction tube is transparent polystyrene, tygon, polypropylene or glass.

The preparation method of kit may further comprise the steps:

(1) preparation of Surfactant proteinD calibration object:

Use the calibration object diluted to each concentration point position 0,2,10,50,200,1000ng/mL the Surfactant proteinD sterling;

Described calibration object dilution is the damping fluid that contains 30% cow's serum;

(2) preparation of Surfactant proteinD quality-control product:

With the calibration object dilution Surfactant proteinD sterling is diluted to 3ng/mL and 600ng/mL, 3ng/mL is as the low value quality-control product, and 600ng/mL is as high value quality-control product;

(3) preparation of nano magnetic particulate-Streptavidin suspending liquid:

A, the preparation of ferriferrous oxide nano magnetic particle

Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl ₃6H ₂O and FeCl ₂4H ₂O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under blanket of nitrogen, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;

The coupling of B, nanometer magnetic bead surface carboxyl

Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: get in the ultrasonic 10%PEG8000 of the being dispersed in solution of nano magnetic particulate of above-mentioned preparation, get magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, move into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under the protection of nitrogen, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add successively afterwards the benzoyl peroxide of magnetic fluid volume 3%, stirring rate is about 500rpm, the styrene of magnetic fluid solution equal volume, the acrylic acid of magnetic fluid liquor capacity 25% keeps stream of nitrogen gas, and all the other conditions remain unchanged, reaction 8-10h, products therefrom leaves standstill, and uses the distilled water cyclic washing, regulates pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use again the distilled water cyclic washing, remove the Fe that does not coat ₃O ₄Magnetic is put into 50 ℃ of lower dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nano magnetic particulate that the surface is associated with carboxyl;

The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:

Get 100mL0.1M MES damping fluid, adding 10mg surface is associated with the nano magnetic particulate of carboxyl, stirring at room 40min, add afterwards the 3.5mg Streptavidin, then add 8mg/mLEDC solution, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, be dissolved to 1L with 0.01M PBS at last and get final product;

(4) preparation of biotin labeled Surfactant proteinD antibody

The preparation of A, Surfactant proteinD antibody

Prepare Surfactant proteinD antibody according to the routine immunization method.

The preparation of B, biotin labeled Surfactant proteinD antibody

Get 0.8mg Surfactant proteinD antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 65ug biotin, add simultaneously dimethyl sulfoxide (DMSO), make dimethyl sulfoxide (DMSO) final mass concentration greater than 5%, slowly vibration, lucifuge reaction 2.5h; In mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01M PBS solution at 2～8 ℃ of lower dialysis 24h, during change liquid 2-4 time;

(5) preparation of Surfactant proteinD abzyme bond

After adopting the improvement sodium periodate oxidation that Surfactant proteinD antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:4500, and adds 11% enzyme stabilizers, be stored in 2～8 ℃;

The preparation of (6) 20 times of concentrated washing lotions

20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;

(7) preparation of chemical luminescence for liquid A liquid and B liquid

A liquid is the 0.7g/L luminol, and 0.165g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid before use 5min mix;

(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃, each one bottle of every kind of reagent;

(9) kit that adopts the method preparation is carried out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.

Principle of the present invention is, adopt the SP-D in double antibody sandwich method mensuration serum or the blood plasma, in Avidin-nano magnetic microparticle suspending liquid, add biotin-SP-D antibody conjugates, compatible reaction by Avidin and biotin, form magnetic particle-Avidin-Biotin-SP-D antibody complex, add sample and enzyme, by antigen-antibody reaction, form magnetic particle-Avidin-Biotin-SP-D antibody-SP-D antigen-SP-D antibody-HRP compound, with magnetic field compound is adsorbed on the test tube bottom, wash free composition, add the substrate working fluid, under the oxygenant effect, HRP catalysis luminol generates the aminophthalic acid ion that is in excited state, when it returns to ground state, discharge the photon of 425nm, measured the luminous value RLU of each well in the 5th minute.The RLU of sample and sample SP-D concentration are proportionate.SP-D concentration in the sample is according to the Log(X that is set up by calibration object SP-D concentration and corresponding RLU)-Log(Y) mathematical model is carried out quantitatively, thus detect the SP-D content in human serum, the blood plasma.

The Surfactant proteinD nano magnetic particulate chemistry electrochemiluminescent immunoassay quantitative determination reagent kit of this patent invention has the following advantages:

(1) highly sensitive, the sensitivity for analysis of this kit is not higher than 0.8ng/mL.

(2) accuracy is good, and imprecision is not higher than 5% in batch, and imprecision is not higher than 10% between batch.

(3) cost is low, compares with like product on the market, and this kit is functional, and cost is low, has clinical value.

(4) have good stability, this product can be deposited more than 7 days at 37 ℃, can deposit 1 year at 2～8 ℃.

Description of drawings

Fig. 1 is the measurement result comparison diagram of kit measurement Surfactant proteinD of the present invention and Enzyme immunoassay Surfactant proteinD, the Surfactant proteinD value that records for this kit of ordinate wherein, horizontal ordinate is that enzyme is exempted from kit measurement Surfactant proteinD value, two kinds of method correlation coefficient r=0.9839, straight-line equation y=0.9657x+0.1997.

Fig. 2 is the preparation technology of Surfactant proteinD antibody

Embodiment

Embodiment 1: preparation Surfactant proteinD nano magnetic particulate chemistry electrochemiluminescent immunoassay quantitative determination reagent kit

(1) preparation of Surfactant proteinD calibration object:

Use the calibration object diluted to each concentration point position 0,2,10,50,200,1000ng/mL Surfactant proteinD sterling (available from US BIO company);

Described calibration object dilution is the damping fluid that contains 30% cow's serum;

(2) preparation of Surfactant proteinD quality-control product:

With the calibration object dilution Surfactant proteinD sterling is diluted to 3ng/mL and 600ng/mL, 3ng/mL is as the low value quality-control product, and 600ng/mL is as high value quality-control product;

(3) preparation of nano magnetic particulate-Streptavidin suspending liquid:

A, the preparation of ferriferrous oxide nano magnetic particle

Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl ₃6H ₂O and FeCl ₂4H ₂O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under blanket of nitrogen, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;

The coupling of B, nanometer magnetic bead surface carboxyl

Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: get in the ultrasonic 10%PEG8000 of the being dispersed in solution of nano magnetic particulate of above-mentioned preparation, get magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, move into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under the protection of nitrogen, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add successively afterwards the benzoyl peroxide of magnetic fluid volume 3%, stirring rate is about 500rpm, the styrene of magnetic fluid solution equal volume, the acrylic acid of magnetic fluid liquor capacity 25% keeps stream of nitrogen gas, and all the other conditions remain unchanged, reaction 8-10h, products therefrom leaves standstill, and uses the distilled water cyclic washing, regulates pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use again the distilled water cyclic washing, remove the Fe that does not coat ₃O ₄Magnetic is put into 50 ℃ of lower dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nano magnetic particulate that the surface is associated with carboxyl;

The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:

Get 100mL0.1M MES damping fluid, adding 10mg surface is associated with the nano magnetic particulate of carboxyl, stirring at room 40min, add afterwards the 3.5mg Streptavidin, then add 8mg/mLEDC solution, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, be dissolved to 1L with 0.01M PBS at last and get final product;

(4) preparation of biotin labeled Surfactant proteinD antibody

The preparation of A, Surfactant proteinD antibody

Technological process according to Fig. 2 prepares Surfactant proteinD antibody.

The preparation of B, biotin labeled Surfactant proteinD antibody

Get 0.8mg Surfactant proteinD antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 65ug biotin, add simultaneously dimethyl sulfoxide (DMSO), make dimethyl sulfoxide (DMSO) final mass concentration greater than 5%, slowly vibration, lucifuge reaction 2.5h; In mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01M PBS solution at 2～8 ℃ of lower dialysis 24h, during change liquid 2-4 time;

(5) preparation of Surfactant proteinD abzyme bond

After adopting the improvement sodium periodate oxidation that Surfactant proteinD antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:4500, and adds 11% enzyme stabilizers, be stored in 2～8 ℃;

Improvement sodium periodate oxidizing process step comprises:

The A:HRP activation

1) configuration 10mg/mL HRP solution;

2) configuration 12.8mg/mL sodium periodate NaIO ₄Solution;

3) with above-mentioned 1 and 2 obtain solutions 1:1 mixing by volume, 4 ℃ of lucifuges reaction 30min;

4) configuration concentration is the glycol water of 20uL/mL, mix with equal volume with mentioned solution 3, and reacting at normal temperature without light 20min, activation is namely finished, and puts-20 ℃ and preserves (holding time is no more than 3 months).

B, SP-D labeling of monoclonal antibody

1) raw material to be marked is packed in the bag filter, with the carbonate buffer solution of 0.05M pH9.6, dialysis 30min;

2) with the HRP of mark raw material and activation in mass ratio 1:2 mix, during 4 ℃ of dialysis 24h(, change liquid 2-3 time with the 0.05M carbonate buffer solution afterwards);

3) configuration concentration is the NaBH of 2mg/mL ₄Aqueous solution adds the NaBH that 80uL prepares by 1mgHRP ₄The ratio of aqueous solution is mixed, and in 4 ℃ of lucifuge reaction 2h;

4) marking fluid of above-mentioned steps 3 being finished in 4 ℃ of dialysis 24h, adds equal-volume glycerine ,-20 ℃ of preservations with 0.01M PBS.

Comprise 10mL/L2M NaOH in the enzyme dilution, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000(is available from Sigma company), 1.05g/L Triton X-100(is available from Sigma company), the 2.5mL/L gentamicin sulphate, 1mL/L is carmine, and (famille rose is powder solid, being mixed with concentration 40mg/mL uses later on), 2g/L Tween-20(is available from Sigma company), 1mL/L ProClin300(is available from Sigma company);

The preparation of (6) 20 times of concentrated washing lotions

20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;

(7) preparation of chemical luminescence for liquid A liquid and B liquid

A liquid is the 0.7g/L luminol, and 0.165g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid before use 5min mix;

(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃, each one bottle of every kind of reagent;

(9) kit that adopts the method preparation is carried out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.

Embodiment 2: the inspection of kit of the present invention

(1) physical examination: liquid component should be clarified, without precipitation or floccus; Other components should be without packages in damaged condition.

(2) linearity of dose-response curve: use the double-log Model fitting, dose-response curve correlation coefficient r absolute value in the 0-1000ng/mL concentration range is not less than 0.9900.

(3) sensitivity for analysis: the kit sensitivity for analysis is not higher than 0.8ng/mL.

(4) precision: the high value of 10 hole replicate determinations and low value quality-control product, the mean concentration of calculating measurement result

With standard deviation (SD), imprecision in batch $(\mathrm{CV}\%)=\mathrm{SD}/\stackrel{\‾}{X}\×100\%;$ Use 3 batches of products to carry out 3 tests, calculate the mean concentration of measurement result

With standard deviation (SD), imprecision between batch

The result should meet batch interior imprecision (CV%) should not be higher than 5%; Imprecision (CV%) should not be higher than 10% between batch.

(5) measured value of quality-control product: the quality-control product of the high value in replicate determination 10 holes and low value, with Log (X)-Log (Y) Model fitting, the quality-control product measured value should be in allowed band, and low value quality-control product measured value is at 2.4-3.6ng/mL, and high value quality-control product measured value is at 480-720ng/mL.

(6) stability: placed 7 days for 37 ℃, measured value should meet above-mentioned requirements.

Embodiment 3: the using method of kit of the present invention

(1) kit to be checked was descended balance 30 minutes in room temperature (18～25 ℃).

(2) preparation washing lotion: will concentrate washing lotion by 1:20 dilution (the 1mL washing lotion adds 19mL distilled water) with distilled water.If concentrated washing lotion has crystallization, can place room temperature or 37 ℃ with concentrating washing lotion, dilute again after the dissolving to be crystallized.

(3) preparation luminescent solution: use and got an amount of luminescent solution A in front 5 minutes and mix with luminescent solution B equal-volume.

(4) reaction tube is numbered, in test tube, add successively 25uL calibration object or serum specimen, 50uL magnetic-particle-Streptavidin suspending liquid, 50uL biotin-surfactant protein antibody conjugates, 100uL surfactant protein abzyme bond, 37 ℃ of lower oscillating reactions 30min, test tube rack placed separate 5min on the magnetic separator, then pour out supernatant, add the 500uL washing lotion, fully behind the mixing, on magnetic separator, separate, pour out washing lotion, repeat 3 times, in each pipe, add Chemoluminescent substrate 100uL, fully mixing is secretly put 5min, measures the luminous value (RLU) of each pipe at the tube-type chemical light-emitting appearance, take the Log value of calibration object concentration as horizontal ordinate, take the Log of luminous value as ordinate, the drawing standard curve can calculate the concentration of surfactant protein according to the luminous value of serum specimen.

Embodiment 4: the evaluation of methodology result of this kit

Sensing range: scope is 0-1000ng/mL, measures after should diluting first greater than the sample of 1000ng/mL for concentration again.

Sensitivity: 0.8ng/mL.

Precision: less than 5%.

Accuracy: the mean value of the recovery is in 0.90～1.10 scope.

The quality-control product measured value: all in allowed band, low value quality-control product measured value is at 2.4-3.6ng/mL for the measured value of low value quality-control product QcL and high value quality-control product QcH, and high value quality-control product measured value is at 480-720ng/mL.

Stability: each reagent component in the kit in 37 ℃ of lower placement 7d, is had good stability.

Embodiment 5: the clinical comparison experiment of this kit

The kit of this patent invention has carried out clinical examination, total sample number 280 examples of this clinical testing, first with after the inspection-free test agent box test of Surfactant proteinD enzyme, measure with the kit (chemiluminescence) of this patent invention again, the result shows, straight-line equation is y=0.9657x+0.1997, correlation coefficient r=0.9839.As seen the kit of this method preparation and enzyme are exempted from measured value preferably consistance.With the SPSS13.0 statistical analysis software related coefficient is carried out t check (inspection level α=0.05), P＜0.001, the related intimate degree of the surfactant protein value of two kinds of method mensuration is conspicuousnesses, as seen the surfactant protein value of two kinds of method mensuration is closely related, the diagnosis capability that kit is described is stronger, can promote clinical practice.

Claims (4)

1. Surfactant proteinD nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box is characterized in that described kit comprises:

1) Surfactant proteinD calibration object, concentration are 0,2,10,50,200,1000ng/mL;

2) coupling has the nano magnetic microparticle suspending liquid of Streptavidin;

3) biotin labeled Surfactant proteinD antibody, described antibody is monoclonal antibody;

4) Surfactant proteinD abzyme bond, described antibody are polyclonal antibody, and used enzyme is horseradish peroxidase, horseradish peroxidase purity RZ 〉=3.0, activity 〉=250U/mL;

5) Surfactant proteinD quality-control product: quality-control product comprises the low value quality-control product of concentration 3ng/mL and the high value quality-control product of 600ng/mL;

6) chemical luminescence for liquid A liquid and B liquid; A liquid is 5mmol/L, the Tris-HCl damping fluid of pH8.6, and contain final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is the 0.675g/L urea peroxide;

7) 20 times of concentrated washing lotions;

8) reaction tube.

2. Surfactant proteinD nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box according to claim 1 is characterized in that, described nano magnetic particulate is that the surface parcel is with the tri-iron tetroxide of amino or carboxyl reactive group, particle diameter 10-50nm.

3. Surfactant proteinD nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box according to claim 1 is characterized in that the material of described reaction tube is transparent polystyrene, tygon, polypropylene or glass.

4. a method for preparing the described kit of the arbitrary claim of described claim 1-3 is characterized in that, may further comprise the steps:

(1) preparation of Surfactant proteinD calibration object:

Use the calibration object diluted to each concentration point position 0,2,10,50,200,1000ng/mL the Surfactant proteinD sterling;

Described calibration object dilution is the damping fluid that contains 30% cow's serum;

(2) preparation of Surfactant proteinD quality-control product:

With the calibration object dilution Surfactant proteinD sterling is diluted to 3ng/mL and 600ng/mL, 3ng/mL is as the low value quality-control product, and 600ng/mL is as high value quality-control product;

(3) preparation of nano magnetic particulate-Streptavidin suspending liquid:

A, the preparation of ferriferrous oxide nano magnetic particle

Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl ₃6H ₂O and FeCl ₂4H ₂O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under blanket of nitrogen, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;

The coupling of B, nanometer magnetic bead surface carboxyl

Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: get in the ultrasonic 10%PEG8000 of the being dispersed in solution of nano magnetic particulate of above-mentioned preparation, get magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, move into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under the protection of nitrogen, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add successively afterwards the benzoyl peroxide of magnetic fluid volume 3%, stirring rate is about 500rpm, the styrene of magnetic fluid solution equal volume, the acrylic acid of magnetic fluid liquor capacity 25% keeps stream of nitrogen gas, and all the other conditions remain unchanged, reaction 8-10h, products therefrom leaves standstill, and uses the distilled water cyclic washing, regulates pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use again the distilled water cyclic washing, remove the Fe that does not coat ₃O ₄Magnetic is put into 50 ℃ of lower dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nano magnetic particulate that the surface is associated with carboxyl;

The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:

Get 100mL0.1M MES damping fluid, adding 10mg surface is associated with the nano magnetic particulate of carboxyl, stirring at room 40min, add afterwards the 3.5mg Streptavidin, then add 8mg/mLEDC solution, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, be dissolved to 1L with 0.01M PBS at last and get final product;

(4) preparation of biotin labeled Surfactant proteinD antibody

Get 0.8mg Surfactant proteinD antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 65ug biotin, add simultaneously dimethyl sulfoxide (DMSO), make dimethyl sulfoxide (DMSO) final mass concentration greater than 5%, slowly vibration, lucifuge reaction 2.5h; In mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 30-60min; With 0.01M PBS solution at 2～8 ℃ of lower dialysis 24h, during change liquid 2-4 time;

(5) preparation of Surfactant proteinD abzyme bond

After adopting the improvement sodium periodate oxidation that Surfactant proteinD antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:4500, and adds 11% enzyme stabilizers, be stored in 2～8 ℃;

The preparation of (6) 20 times of concentrated washing lotions

20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;

(7) preparation of chemical luminescence for liquid A liquid and B liquid

A liquid is the 0.7g/L luminol, and 0.165g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid before use 5min mix;

(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃, each one bottle of every kind of reagent;

(9) kit that adopts the method preparation is carried out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.

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