CN103364567B - A kind of surfactant protein D nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of Surfactant proteinD (SP-D) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box, described kit comprises: Surfactant proteinD calibration object; Coupling has the nano magnetic microparticle suspending liquid of Streptavidin; Biotin labeled Surfactant proteinD antibody; Surfactant proteinD abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; Surfactant proteinD quality-control product; Chemical luminescence for liquid A liquid and B liquid; 20 times of concentrated washing lotions; Reaction tube.The invention also discloses the preparation method of kit of the present invention in addition.Kit of the present invention is highly sensitive compared with available reagent box, can measure that concentration range is wide, the reagent term of validity is long, reagent cost is low, simple to operate, detect automaticity advantages of higher.

Description

A kind of surfactant protein D nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof

Technical field

The present invention relates to field of immunoassay medicine, 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 synthesized by alveolar type II cells and secrete a kind of lipoprotein of complexity, the one be made up of phosphatide and protein has surface-active compound, 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 is hydrophobic small molecules albumen, SP-A and SP-D is large hydrophilic molecular albumen, the above two can reduce alveolar surface tension, and rear both then mainly regulate lung immunologic function.First SP-D finds in respiratory system, the glycoprotein of to be a kind of molecular weight be 43KD, research shows that SP-D can strengthen the resistivity of respiratory tract as immunologic active material, defend the invasion and attack of microorganism and dust, not only can directly be combined with silicious dust specifically, and the phagocytosis of pulmonary macrophage to silicious dust can also be strengthened, after silicious dust enters lung tissue, the expression of lung tissue SP-D can increase.

Interstitial lung disease (ILD) a kind ofly has the inflammation of multi-form and degree and fiber with alveolus wall and alveolar space and turn to characteristic pathological and change, the general name of the Diffuse-type gastric carcinoma being main clinical manifestation in the infiltration shadow extensively distributed with Progressive symmetric erythrokeratodermia expiratory dyspnea and x-ray rabat.Current judgement ILD mainly relies on the means such as imaging examination, biopsy, routine blood test, enzyme linked immunological Serologic detection, research show 10% the inspection of ILD patient's x-ray normal, but proved by pathology is ILD, now patient's serum SP-D level when x-ray can not detect significantly raises; Biopsy also may cause undetected due to got pathology location difference.

Enzymoimmunoassay possesses simple to operate, cheap, precision is high, specificity is good, accuracy is good, be a kind of minimal feeding technology preferably, but susceptibility is relatively low, between plate, difference is large, measured value repeatability is poor, and mark enzyme-to-substrate used can quantitative measurement narrow range, and the shortcoming 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 microimmuno-assays the most responsive.

Summary of the invention

The problem to be solved in the present invention chemiluminescence immunoassay immue quantitative detection reagent box being to provide surfactant protein and preparation method thereof, avoids measured value poor repeatability, the defects such as 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, comprising: Surfactant proteinD calibration object; Coupling has the nano magnetic microparticle suspending liquid of Streptavidin; Biotin labeled Surfactant proteinD antibody; Surfactant proteinD abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; Surfactant proteinD quality-control product, quality-control product comprises the low value quality-control product of concentration 3ng/mL and the high level quality-control product of 600ng/mL; Chemical luminescence for liquid A liquid and B liquid, A liquid is the Tris-HCl damping fluid of 5mmol/L, pH8.6, and containing final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is 0.675g/L urea peroxide; 20 times of concentrated washing lotions; Reaction tube.

Further, described nano magnetic particulate is the tri-iron tetroxide that surface wraps up with 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, comprises the following steps:

(1) preparation of Surfactant proteinD calibration object:

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

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

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

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

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

Prepared by A, ferriferrous oxide nano magnetic particle

Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) by FeCl ₃6H ₂o and FeCl ₂4H ₂o joins in distilled water with mol ratio 2:1, dissolved with vigorous agitation; 2) add 0.5M ammoniacal liquor under nitrogen atmosphere in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3), after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;

The coupling of B, nanometer magnetic bead surface carboxyl groups

Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10%PEG8000 solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, moves 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 DEG C, constant temperature stirs 30min, add the benzoyl peroxide of magnetic fluid volume 3% afterwards successively, stirring rate is about 500rpm, the styrene of magnetic fluid solution same volume, the acrylic acid of magnetic fluid liquor capacity 25%, keep stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, products therefrom leaves standstill, and uses distilled water cyclic washing, then regulates pH=1 with hydrochloric acid, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe ₃o ₄magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that 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, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mLEDC solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally dissolved to 1L with 0.01M PBS;

(4) preparation of biotin labeled Surfactant proteinD antibody

The preparation of A, Surfactant proteinD antibody

Conveniently immunization method prepares Surfactant proteinD antibody.

The preparation of B, biotin labeled Surfactant proteinD antibody

Get 0.8mg Surfactant proteinD antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 65ug biotin, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) final mass concentration be greater than 5%, slowly vibrate, lucifuge reaction 2.5h; 250uL1M ammonium chloride solution is added, reacting at normal temperature without light 30-60min in above-mentioned solution; To dialyse at 2 ~ 8 DEG C 24h by 0.01M PBS solution, period changes liquid 2-4 time;

(5) preparation of Surfactant proteinD abzyme bond

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

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

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

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

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

(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C, each one bottle of often kind of reagent;

(9) to adopting the kit prepared of the method to carry out physical examination, the measured value of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product and stability are measured.

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

The Surfactant proteinD nano magnetic particulate chemistry electrochemiluminescent immunoassay quantitative determination reagent kit of 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 in batch, imprecision is not higher than 5%, and between batch, imprecision is not higher than 10%.

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

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

Accompanying drawing explanation

Fig. 1 is the measurement result comparison diagram of kit measurement Surfactant proteinD of the present invention and Enzyme immunoassay Surfactant proteinD, wherein ordinate is the Surfactant proteinD value that this kit records, horizontal ordinate is that enzyme exempts from kit mensuration 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: prepare Surfactant proteinD nano magnetic particulate chemistry electrochemiluminescent immunoassay quantitative determination reagent kit

(1) preparation of Surfactant proteinD calibration object:

By Surfactant proteinD sterling (purchased from US BIO company) calibration object diluted extremely each concentration point position 0,2,10,50,200,1000ng/mL;

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

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

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

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

Prepared by A, ferriferrous oxide nano magnetic particle

Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) by FeCl ₃6H ₂o and FeCl ₂4H ₂o joins in distilled water with mol ratio 2:1, dissolved with vigorous agitation; 2) add 0.5M ammoniacal liquor under nitrogen atmosphere in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3), after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;

The coupling of B, nanometer magnetic bead surface carboxyl groups

Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10%PEG8000 solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, moves 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 DEG C, constant temperature stirs 30min, add the benzoyl peroxide of magnetic fluid volume 3% afterwards successively, stirring rate is about 500rpm, the styrene of magnetic fluid solution same volume, the acrylic acid of magnetic fluid liquor capacity 25%, keep stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, products therefrom leaves standstill, and uses distilled water cyclic washing, then regulates pH=1 with hydrochloric acid, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe ₃o ₄magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that 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, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mLEDC solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally dissolved to 1L with 0.01M PBS;

(4) preparation of biotin labeled Surfactant proteinD antibody

The preparation of A, Surfactant proteinD antibody

Surfactant proteinD antibody is prepared according to the technological process of Fig. 2.

The preparation of B, biotin labeled Surfactant proteinD antibody

Get 0.8mg Surfactant proteinD antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 65ug biotin, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) final mass concentration be greater than 5%, slowly vibrate, lucifuge reaction 2.5h; 250uL1M ammonium chloride solution is added, reacting at normal temperature without light 30-60min in above-mentioned solution; To dialyse at 2 ~ 8 DEG C 24h by 0.01M PBS solution, period changes liquid 2-4 time;

(5) preparation of Surfactant proteinD abzyme bond

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

Improvement sodium periodate oxidizing process step comprises:

A:HRP activates

1) 10mg/mL HRP solution is configured;

2) 12.8mg/mL sodium periodate NaIO is configured ₄solution;

3) by the 1:1 mixing by volume of above-mentioned 1 and 2 obtain solutions, 4 DEG C of lucifuge reaction 30min;

4) configuration concentration is the glycol water of 20uL/mL, mixes, reacting at normal temperature without light 20min with above-mentioned solution 3 with same volume, and namely activation completes, and puts-20 DEG C of preservations (holding time is no more than 3 months).

B, SP-D labeling of monoclonal antibody

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

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

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

4) marking fluid 0.01M PBS above-mentioned steps 3 completed, in 4 DEG C of dialysis 24h, adds equal-volume glycerine ,-20 DEG C of preservations.

Enzyme dilution comprises 10mL/L2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000(available from Sigma), 1.05g/L Triton X-100(available from Sigma), 2.5mL/L gentamicin sulphate, (famille rose is powder solid to 1mL/L famille rose, be mixed with concentration 40mg/mL to use later), 2g/L Tween-20(available from Sigma), 1mL/L ProClin300(available from Sigma);

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

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

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

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

(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C, each one bottle of often kind of reagent;

(9) to adopting the kit prepared of the method to carry out physical examination, the measured value of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product and stability 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 without packages in damaged condition.

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

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

(4) precision: 10 hole replicate determination high level and low value quality-control product, calculates the mean concentration of 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 result should meet batch interior imprecision (CV%) should not higher than 5%; Between batch, imprecision (CV%) should not higher than 10%.

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

(6) stability: place 7 days for 37 DEG C, measured value should meet above-mentioned requirements.

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

(1) kit to be checked is balanced 30 minutes under room temperature (18 ~ 25 DEG C).

(2) washing lotion is prepared: washing lotion will be concentrated by 1:20 dilution (1mL washing lotion adds 19mL distilled water) with distilled water.If concentrated washing lotion has crystallization, concentrated washing lotion can be placed in room temperature or 37 DEG C, dilute again after dissolving to be crystallized.

(3) luminescent solution is prepared: use first 5 minutes and get appropriate luminescent solution A and mix with luminescent solution B equal-volume.

(4) reaction tube is numbered, 25uL calibration object or serum specimen is added successively in test tube, 50uL magnetic-particle-Streptavidin suspending liquid, 50uL biotin-surfactant protein antibody conjugates, 100uL surfactant protein abzyme bond, oscillating reactions 30min at 37 DEG C, test tube rack is placed on magnetic separator and is separated 5min, then supernatant is poured out, add 500uL washing lotion, after abundant mixing, be separated on magnetic separator, pour out washing lotion, repeat 3 times, Chemoluminescent substrate 100uL is added in each pipe, abundant mixing, secretly put 5min, tube-type chemical light-emitting appearance measures the luminous value (RLU) of each pipe, with the Log value of calibration object concentration for horizontal ordinate, with the Log of luminous value for ordinate, drawing standard curve, the concentration of surfactant protein can be calculated 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 the sample being greater than 1000ng/mL for concentration should first dilute again.

Sensitivity: 0.8ng/mL.

Precision: be less than 5%.

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

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

Stability: reagent component each in kit is placed 7d at 37 DEG C, has good stability.

Embodiment 5: the clinical comparison experiment of this kit

The kit of invention has carried out clinical examination, total sample number 280 example of this clinical testing, after first exempting from detection kit test with Surfactant proteinD enzyme, the kit of invention (chemiluminescence) is used to measure again, result shows, straight-line equation is y=0.9657x+0.1997, correlation coefficient r=0.9839.Kit prepared by visible this method and enzyme exempt from measured value good consistance.With SPSS13.0 statistical analysis software, t inspection (inspection level α=0.05) is carried out to related coefficient, P<0.001, the related intimate degree of the surfactant protein value of two kinds of method mensuration is conspicuousnesses, the surfactant protein value that visible two kinds of methods measure is closely related, illustrate that the diagnosis capability of kit is comparatively strong, can clinical practice be promoted.

Claims (1)

1. a surfactant protein D nano-magnetic particle chemiluminescent immunity quantitative detection kit, is characterized in that, described kit comprises:

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

2) coupling has the nano magnetic microparticle suspending liquid of Streptavidin, and described nano magnetic particulate is the tri-iron tetroxide that surface wraps up with amino or carboxyl-reactive group, particle diameter 10-50nm;

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

4) Surfactant proteinD abzyme bond, described antibody is polyclonal antibody, and enzyme used 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 level quality-control product of 600ng/mL;

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

7) 20 times of concentrated washing lotions;

8) reaction tube, the material of described reaction tube is transparent polystyrene, transparent polyethylene, transparent polypropylene or clear glass;

It is characterized in that, preparation method comprises the following steps:

(1) preparation of Surfactant proteinD calibration object:

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

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

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

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

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

Prepared by A, ferriferrous oxide nano magnetic particle

Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) by FeCl ₃6H ₂o and FeCl ₂4H ₂o joins in distilled water with mol ratio 2:1, dissolved with vigorous agitation; 2) add 0.5M ammoniacal liquor under nitrogen atmosphere in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3), after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;

The coupling of B, nanometer magnetic bead surface carboxyl groups

Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10%PEG8000 solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, moves 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 DEG C, constant temperature stirs 30min, add the benzoyl peroxide of magnetic fluid volume 3% afterwards successively, stirring rate is about 500rpm, the styrene of magnetic fluid solution same volume, the acrylic acid of magnetic fluid liquor capacity 25%, keep stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, products therefrom leaves standstill, and uses distilled water cyclic washing, then regulates pH=1 with hydrochloric acid, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe ₃o ₄magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that 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, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mLEDC solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally settled to 1L with 0.01M PBS;

(4) preparation of biotin labeled Surfactant proteinD antibody

Get 0.8mg Surfactant proteinD antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 65 μ g biotins, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) final mass concentration be greater than 5%, slowly vibrate, lucifuge reaction 2.5h; 250 μ L1M ammonium chloride solutions are added, reacting at normal temperature without light 30-60min in above-mentioned solution; To dialyse at 2 ~ 8 DEG C 24h by 0.01M PBS solution, period changes liquid 2-4 time;

(5) preparation of Surfactant proteinD abzyme bond

After adopting improvement sodium periodate oxidation that Surfactant proteinD antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:4500 with enzyme dilution, and added 11% enzyme stabilizers, be stored in 2 ~ 8 DEG C; Enzyme dilution comprises 10mL/L 2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000,1.05g/L TritonX-100,2.5mL/L gentamicin sulphate, 1mL/L is carmine, and famille rose is powder solid, is mixed with concentration 40mg/mL and uses later, 2g/L Tween-20,1mL/L ProClin300;

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

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

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

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

(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C, each one bottle of often kind of reagent;

(9) to adopting the kit prepared of the method to carry out physical examination, the measured value of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product and stability are measured.