CN105510594A - Procalcitionin (PCT) test kit and test method thereof - Google Patents
Procalcitionin (PCT) test kit and test method thereof Download PDFInfo
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Abstract
The invention discloses a procalcitionin (PCT) test kit, which is composed of a magnetic separation reagent, a reagent R1, a reagent R2, a standard substance, a quality control substance, a calibration substance, a cleaning concentrated solution, a dilution solution and a luminous substrate. The invention also discloses a preparation method of the kit. The kit provided by the invention combines the chemiluminescence technique and immune magnetic particles, and provides a reaction system close to a homogeneous phase. Compared with existing ELISA techniques, the kit and the method provided by the invention have the advantages of higher specificity and sensitivity, short detection time, simple operation mode, and accurate and reliable detection result, and greatly lower the product cost.
Description
Technical field
The present invention relates to the kit and method of testing thereof that measure serum, especially relate to the kit and method of testing thereof that measure Procalcitonin content in serum.
Background technology
Procalcitonin (procalcitionin, detect PCT) be a kind ofly to be made up of 116 amino acid, molecular weight is the glycoprotein of 13kDa, be the precursor of calcitonin (CT), be about 20-24 hour at people's Half-life in vivo, good stability.Under normal condition, PCT generates and is cracked into calcitonin in parafollicular cells of thyroid gland, and human serum PCT content is extremely low, about 2.5pg/ml.When serious bacterial, fungi, parasitic infection and pyemia and MOFE, the level of PCT in blood plasma raises.When autoimmunity, allergy and virus infections, PCT can not raise.The bacteriological infection of local finite, slight infection and chronic inflammation can not cause PCT to raise.PCT as a kind of newly, the experimental index of the disease such as the severe bacterial infections with innovative significance, PCT improves the accuracy of clinical diagnosis, for Intensive Care Therapy, chemicotherapy, take the patient such as immunodepressant or organ transplant and merge adstante febre and provide and important diagnosis, further to check and the clinical foundation for the treatment of, PCT can be widely used in ICU ward, hematology, oncology, paediatrics, premature and newborn intensive care unit, surgery, internal medicine, organ transplant section, emergency department, intervention diagnosis and Experiment on therapy room etc.Chinese patent 200710073309.3 discloses a kind of Procalcitonin test kit and method of testing thereof, the reagent that kit comprises has: separation agent, luminous marker, fluorescein-labelled thing and adopt nano immune magnetic microballon as separation agent, adopt the different Derivative of Luminol of anti-PCT labeling of monoclonal antibody as the method for testing of luminous marker, this PCT kit and method of testing thereof have higher sensitivity and specificity, but its time going out testing result is longer, virtually adds the reagent cost of kit.
Summary of the invention
In view of this, technical matters to be solved by this invention overcomes above-mentioned the deficiencies in the prior art, a kind of high specificity is provided, highly sensitive, the time obtaining testing result is short, mode of operation is easy, the test kit of the Procalcitonin (PCT) that testing result is reliable and stable and method of testing thereof.
For achieving the above object, the invention provides following technical scheme:
A kind of Procalcitonin (PCT) test kit, this kit by Magneto separate reagent, reagent R
1, reagent R
2standard items, quality-control product, calibration object, cleaning concentrate, dilution and luminous substrate composition, wherein: Magneto separate reagent: the nano-magnetic microsphere being marked with the monoclonal antibody of mouse anti human PCT, described in be marked with the nano-magnetic microsphere of the monoclonal antibody of mouse anti human PCT concentration be 132 μ g/ml; Reagent R
1: the PCT antibody containing alkali phosphatase enzyme mark, the PCT antibody concentration of described alkali phosphatase enzyme mark is 0.6 μ g/ml; Reagent R
2: the damping fluid containing ox gamma Globulin matter component; Standard items, quality-control product and calibration object: bovine serum albumin(BSA) (BSA) (the being called for short BSAV) solution containing a certain amount of PCT antigen, the concentration of described standard items is 0 (S0), 0.1 (S1), 1 (S2), 5 (S3), 50 (S4), 100 (S5) ng/ml, the concentration of described quality-control product is 0.6,50ng/ml, the concentration of described calibration object is 1,50ng/ml; Cleaning concentrate: the damping fluid containing Tween-20 and Proclin-300; Dilution: the solution containing bovine serum albumin(BSA) (BSA) (BSAV); Luminous substrate: the derivative I UMIPHOS530 of diamantane, the concentration of the derivative I UMIPHOS530 of described diamantane is 10 μ g/ml.
A kind of Procalcitonin (PCT) test kit, this kit is made up of following volume fraction: Magneto separate reagent 4%-6%, reagent R14%-6%, reagent R24%-6%, standard items 4%-6%, quality-control product 1%-3%, calibration object 1%-3%, cleaning concentrate 20%-30%, dilution 10%-20%, luminous substrate 25%-40%.
Preferably, described kit is made up of following volume fraction: Magneto separate reagent 4%, reagent R
14%, reagent R
24%, standard items 4%, quality-control product 1%, calibration object 3%, cleaning concentrate 30%, dilution 20%, luminous substrate 30%.
Preferably, described kit is made up of following volume fraction: Magneto separate reagent 5.1%, reagent R
15.1%, reagent R
25.1%, standard items 5.1%, quality-control product 1.8%, calibration object 1.8%, cleaning concentrate 25%, dilution 17%, luminous substrate 34%.
Preferably, described kit is made up of following volume fraction: Magneto separate reagent 6%, reagent R
16%, reagent R
26%, standard items 6%, quality-control product 3%, calibration object 1%, cleaning concentrate 20%, dilution 12%, luminous substrate 40%.
A kind of preparation method of calcium element former (PCT) test kit, comprises the steps:
The first step: the preparation process of Magneto separate reagent
One, magnetic bead buffer solution formulation operations code:
(1) take Tris4.58g and NaCl6.81g in 1L container, take 0.96gTween-20 and add after suitable quantity of water makes it dissolve completely in 20ml container, pour in said vesse;
(2) Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water with pipettor, pour in above-mentioned 1L container; Then in 1L container, add 800ml purified water, fully stir, reagent is dissolved completely;
(3) PH measurement is regulated to measure its pH value; Adjust PH with 4MHCl or 4MNaOH, measure its PH and namely meet the requirements between 7.95-8.05;
(4) taking BSAV (bovine serum albumin(BSA) (BSA)) 3g pours in above-mentioned 1L container;
(5) be finally settled to 1000ml, survey pH value, namely scope meets the requirements between 7.95-8.05, with 0.2um frit and get final product; Label is posted in 2-8 DEG C of refrigeration house storage after having filtered;
Two, the preparation of Magneto separate reagent:
(1) be dissolved in 50ulDMSO by 1.0mgDSS (disuccinimidyl suberate), namely concentration is 20mg/mL; Get 2mgPCT antibody be dissolved in the 0.1mol/LPB damping fluid of PH9.5 to cumulative volume be 1ml;
(2) input amount of DSS is calculated, according to following formulae discovery: (antibody mass/16000) × 10 × 368/C
dSS), wherein C
dSSrefer to the substance withdrawl syndrome mol/L of DSS;
(3) join in the antibody-solutions of step 1 with the DSS of liquid-transfering gun absorption respective volume, put room temperature 90min;
(4) join in Centricon-10 concentration tube by step 3 antibody-solutions, then putting into and under the centrifugal force of 3000g, concentrating about 30min in sigma2-16k high speed freezing centrifuge is 0.5ml to volume;
(5) get 0.5ml magnetic bead, described bead diameter, between 0.01-5.0 μm, adds in 5ml reaction cup, puts into test tube rack special, draws supernatant through magnet adsorption after 2 minutes;
(6) add 1.5mlPH9.50.1mol/LPB at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; Antibody-solutions step 4 obtained joins in magnetic bead, and room temperature reaction 4 hours after mixing, keeps mixing state;
(7) add the Tris solution 37 DEG C 15 minutes of 0.3ml1mol/L, wherein the application of sample amount of Tris is that 1mg antibody adds 0.15mlTris;
(8) add 1.5mlPH7.20.1mol/LPB at every turn and clean the magnetic bead marked, mix 30 seconds, added, remove supernatant, repetitive operation 3 times;
(9) with 100ml magnetic bead conserving liquid, magnetic bead is proceeded to 125ml vial, be the PCT Magneto separate reagent of 0.05%;
(10) Magneto separate reagent magnetic bead buffer solution step 9 obtained mixes according to the ratio of 1:1, obtains Magneto separate reagent in kit of the present invention;
Second step: reagent R
1preparation process
One, reagent R
1diluent preparing working specification:
(1) Tris6.06g, NaCl13.0g, Zncl is got
20.05g, Proclin-3000.2ml and MgCl
20.05g is in flask; Then in flask, add 800ml purified water, fully stir, reagent is dissolved completely;
(2) adjust PH with 4MHCl or 4MNaOH, measure and make PH within the scope of 7.35-7.45;
(3) taking BSAV3g pours in above-mentioned flask;
(4) be finally settled to 1000ml, survey pH value, namely scope meets the requirements between 7.35-7.45, uses 0.2um frit; After having filtered, post label in 2-8 DEG C of refrigeration house storage;
Two, reagent R
1preparation (the PCT monoclonal antibody of alkaline phosphatase (ALP) coupling)
(1) get 10mgALP to add in 5ml physiological saline, join in Centricon-10 concentration tube, centrifugal about 20 minutes of 3000rpm, is concentrated into 1 milliliter;
(2) in upper liquid, add the 0.1MNaIO that 0.2ml newly joins
4solution, under room temperature, lucifuge stirs 20 minutes;
(3) loaded in bag filter by above-mentioned solution, dialyse with the sodium-acetate buffer of 1mMPH4.4,4 DEG C are spent the night;
(4) add 20 μ l0.2MPH9.5 carbonate buffer solutions, make the PH of above hydroformylation ALP be elevated to 9.0 ~ 9.5, then add 2.5mgIgG antibody immediately, in 1ml0.01M carbonate buffer solution, room temperature lucifuge stirs 2 hours gently;
(5) 4mg/mlNaBH that 0.1ml newly joins is added
4liquid, mixing, then put 4 DEG C 2 hours;
(6) load in bag filter by above-mentioned liquid, to 0.15MPH7.4PBS dialysis, 4 DEG C are spent the night;
(7) under agitation dropwise add equal-volume saturated ammonium sulfate, put 4 DEG C 1 hour;
(8) 3000rpm centrifugal half an hour, supernatant is abandoned.Sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of a small amount of 0.15MPH7.4;
(9) load in bag filter by above-mentioned solution, dialyse about 5 hours to the PB buffer saline of 0.15MPH7.4, after removing ammonium ion, 10000rpm removes precipitation in centrifugal 30 minutes, and supernatant is enzyme conjugates, adds the 1MMgcl of volume 1/100
2solution 4 DEG C preservation.The alkaline phosphatase (ALP) collected mixes with the volume ratio of 1:1000 with above-mentioned enzyme reaction thing dilution with the conjugate of PCT monoclonal antibody, obtains seminal plasma fructose detection kit R of the present invention
1;
3rd step: reagent R
2formulation operations process
(1) Tris (trishydroxymethylaminomethane, molecular formula: (HOCH2) 3CNH2) 1.56g and NaCl4.23g is taken in 1L beaker; Proclin-300 measured after 0.2ml dissolves completely in the beaker of a small amount of purified water with pipettor, pour in above-mentioned 1L container;
(2) measure 800ml purified water in beaker with graduated cylinder, fully stir, until dissolve completely; Adjust PH with 4MHCL or 4MNaOH, measure its scope between 7.35-7.45;
(3) ox gamma Globulin (IgG) 0.9g is taken in the beaker of 800ml purified water;
(4) last constant volume 1000ml, after dissolving completely, survey pH value, namely scope meets the requirements between 7.35-7.45, uses 0.2um frit; After having filtered, post label in 2-8 DEG C of refrigeration house storage;
4th step: standard items, the preparation of quality-control product and calibration object:
(1) Procalcitonin (PCT) quantitative determination reagent kit PCT standard items raw material is mixed with concentration point is 0.5,1,5,20,100ng/ml; The concentration point of quality-control product preparation is 0.6,50ng/ml.The concentration point of calibration object preparation is 1,50ng/ml;
(2), after fully mixing, label is posted in 2-8 DEG C of refrigeration house storage;
5th step: cleaning concentrate formulation operations code:
(1) Tris12.54g and NaCl325.6g is taken in 1L container;
(2) take 5gTween-20 adds after 20ml water makes it dissolve completely in 100ml container, pours in said vesse;
(3) Proclin-300 measured after 0.2ml dissolves completely in the beaker filling 10ml purified water with pipettor, pour in above-mentioned 1L container;
(4) measure 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolve completely;
(5) adjust PH with 4MHCL or 4MNaOH, measure its scope between 7.35-7.45;
(6) last constant volume 1000ml, survey pH value, namely scope meets the requirements between 7.35-7.45, with 0.2um frit and get final product after dissolving completely; After having filtered, post label in 2-8 DEG C of refrigeration house storage;
6th step: diluent preparing process:
(1) NaCl9.0g and BSAV60g is taken in the container of 1L;
(2) with pipettor, Proclin-300 is measured 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
(3) last constant volume 1000ml, after dissolving completely, uses 0.2um frit, posts label in 2-8 DEG C of refrigeration house storage;
7th step: luminous substrate formulation operations code:
(1) Tris2.35g, NaCl6.41g, Na is taken
2sO
30.002g and Proclin-3000.2ml is in 1L beaker;
(2) measure 800ml purified water in beaker with graduated cylinder, fully stir, until dissolve completely; Adjust PH with 4MHCl or 4MNaOH, measure its scope between 7.95-8.05;
(3) be finally settled to 1000ml, survey pH value, namely scope meets the requirements between 7.95-8.05, uses 0.2um frit; After having filtered, add 250mlIUMIPHOS530, after mixing, post label in 2-8 DEG C of refrigeration house storage.
The invention also discloses the method for testing of a kind of Procalcitonin (PCT), comprise the following steps:
(1), before using, kit internal calibration product (choosing) need be used to carry out curvature correction, and carry out quality control with quality-control product.Test result can carry out the detection of sample within the scope of Quality Control;
(2) add bottom PCT standard items, quality-control product, sample to be measured to corresponding test tube;
(3) reagent adding R
1to in each test tube;
(4) reagent adding R
2to in each test tube;
(5) Magneto separate reagent is added in each test tube;
(6) use covered rearing with plastic film test tube, multitube vortex mixer gently tube shaken frame, after 30 seconds, puts 37 DEG C of water-baths 15 minutes;
(7) test tube frame linking is put on magnetic separator, guarantees that often propping up test tube all contacts with separator surface, precipitates 2 minutes.Supernatant poured out by the separation vessel that reverses slowly, and the test tube of reversing is placed on filter paper together with separation vessel, firmly bounces separator bottom to remove all drops be bonded on tube wall;
(8) after cleaning concentrate purified water dilutes 7 times, add the cleaning fluid after dilution in each test tube, to put on multitube vortex mixer vibration mixing 30s gently, application of sample dynamics should be avoided excessive and cause magnetic bead to spill during application of sample, mixing is wanted thoroughly;
(9) repeat step 6,7,6 one times;
(10) add in substrate solution to test tube and mix 3 seconds, detect with ready luminometer rapidly;
(11) as run into high level HOOK sample, suggestion clinician selects suitable extension rate dilution to dilute sample according to all the other test indexs.
Beneficial effect of the present invention is:
(1) chemiluminescence combines with immune magnetic particle by kit of the present invention, provide a kind of reaction system close to homogeneous phase, compared with prior art, going out the few 10-20 minute of more traditional euzymelinked immunosorbent assay (ELISA) on the result time, the consumption of antibody reduces more than 20% simultaneously, while enhancing product performance, greatly reduce cost of products.
(2) the invention discloses a kind of new special agent R
2, make course of reaction more reliable and more stable, there is higher detection sensitivity and specificity, and reach preferably performance parameter.
(3) the Magneto separate reagent in kit, reagent R
1, reagent R
2, standard items, quality-control product, calibration object, the proportioning of cleaning concentrate, dilution and luminous substrate is all the optimization formulas under reaction system, provides powerful guarantee to the use term of validity of this kit and detection perform.
(4) degree of accuracy of kit of the present invention, sensitivity and stability are all better than market like product, and with low cost, simple to operate, have a extensive future.
Embodiment
Below in conjunction with embodiment, the preferred embodiments of the present invention are described in detail.
Embodiment 1:
A kind of Procalcitonin (PCT) test kit of the present invention, this kit by Magneto separate reagent, reagent R
1, reagent R
2standard items, quality-control product, calibration object, cleaning concentrate, dilution and luminous substrate composition, wherein: Magneto separate reagent: the nano-magnetic microsphere being marked with the monoclonal antibody of mouse anti human PCT, described in be marked with the nano-magnetic microsphere of the monoclonal antibody of mouse anti human PCT concentration be 132 μ g/ml; Reagent R
1: the PCT antibody containing alkali phosphatase enzyme mark, the PCT antibody concentration of described alkali phosphatase enzyme mark is 0.6 μ g/ml; Reagent R
2: the damping fluid containing ox gamma Globulin matter component; Standard items, quality-control product and calibration object: bovine serum albumin(BSA) component five (BSAV) solution containing a certain amount of PCT antigen, the concentration of described standard items is 0 (S0), 0.1 (S1), 1 (S2), 5 (S3), 50 (S4), 100 (S5) ng/ml, the concentration of described quality-control product is 0.6,50ng/ml, the concentration of described calibration object is 1,50ng/ml; Cleaning concentrate: the damping fluid containing Tween-20 and Proclin-300; Dilution: the solution containing bovine serum albumin(BSA) component five (BSAV); Luminous substrate: the derivative I UMIPHOS530 of diamantane, the concentration of the derivative I UMIPHOS530 of described diamantane is 10 μ g/ml;
A kind of Procalcitonin (PCT) test kit of the present embodiment, is made up of following volume fraction: Magneto separate reagent 4%, reagent R
14%, reagent R
24%, standard items 4%, quality-control product 1%, calibration object 3%, cleaning concentrate 30%, dilution 20%, luminous substrate 30%.
The above-mentioned Procalcitonin of the present invention (PCT) measures the preparation method of kit, and its concrete steps are as follows:
The first step: the preparation process of Magneto separate reagent
One, magnetic bead buffer solution formulation operations code: fill a prescription in table 1, to prepare 1L:
(1) take Tris4.58g and NaCl6.81g in 1L container, take 0.96gTween-20 and add after suitable quantity of water makes it dissolve completely in 20ml container, pour in said vesse;
(2) Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water with pipettor, pour in above-mentioned 1L container; Then in 1L container, add 800ml purified water, fully stir, reagent is dissolved completely;
(3) PH measurement is regulated to measure its pH value; Adjust PH with 4MHCl or 4MNaOH, measure its PH and namely meet the requirements between 7.95-8.05;
(4) taking BSAV (bovine serum albumin(BSA) (BSA)) 3g pours in above-mentioned 1L container;
(5) be finally settled to 1000ml, survey pH value, namely scope meets the requirements between 7.95-8.05, with 0.2um frit and get final product; Label is posted in 2-8 DEG C of refrigeration house storage after having filtered;
Magnetic bead buffer solution (table 1)
Two, the preparation of Magneto separate reagent
(1) be dissolved in 50ulDMSO by 1.0mgDSS (disuccinimidyl suberate), namely concentration is 20mg/mL; Get 2mgPCT antibody be dissolved in the 0.1mol/LPB damping fluid of PH9.5 to cumulative volume be 1ml;
(2) input amount of DSS is calculated, according to following formulae discovery: (antibody mass/16000) × 10 × 368/C
dSS), wherein C
dSSrefer to the substance withdrawl syndrome mol/L of DSS;
(3) join in the antibody-solutions of step 1 with the DSS of liquid-transfering gun absorption respective volume, put room temperature 90min;
(4) join in Centricon-10 concentration tube by step 3 antibody-solutions, then putting into and under the centrifugal force of 3000g, concentrating about 30min in sigma2-16k high speed freezing centrifuge is 0.5ml to volume;
(5) get 0.5ml magnetic bead, described bead diameter, between 0.01-5.0 μm, adds in 5ml reaction cup, puts into test tube rack special, draws supernatant through magnet adsorption after 2 minutes;
(6) add 1.5mlPH9.50.1mol/LPB at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; Antibody-solutions step 4 obtained joins in magnetic bead, and room temperature reaction 4 hours after mixing, keeps mixing state;
(7) add the Tris solution 37 DEG C 15 minutes of 0.3ml1mol/L, wherein the application of sample amount of Tris is that 1mg antibody adds 0.15mlTris;
(8) add 1.5mlPH7.20.1mol/LPB at every turn and clean the magnetic bead marked, mix 30 seconds, added, remove supernatant, repetitive operation 3 times;
(9) with 100ml magnetic bead conserving liquid, magnetic bead is proceeded to 125ml vial, be the PCT Magneto separate reagent of 0.05%;
(10) Magneto separate reagent magnetic bead buffer solution step 9 obtained mixes according to the ratio of 1:1, obtains separation agent in kit of the present invention;
Second step: reagent R
1preparation process
One, reagent R
1diluent preparing working specification: fill a prescription in table 2, to prepare 1L:
(1) Tris6.06g, NaCl13.0g, Zncl is got
20.05g, Proclin-3000.2ml and MgCl
20.05g is in flask; Then in flask, add 800ml purified water, fully stir, reagent is dissolved completely;
(2) adjust PH with 4MHCl or 4MNaOH, measure and make PH within the scope of 7.35-7.45;
(3) taking BSAV3g pours in above-mentioned flask;
(4) be finally settled to 1000ml, survey pH value, namely scope meets the requirements between 7.35-7.45, uses 0.2um frit; After having filtered, post label in 2-8 DEG C of refrigeration house storage;
Reagent R
1dilution (table 2)
Two, reagent R
1preparation (the mouse anti human PCT monoclonal antibody that alkaline phosphatase (ALP) marks)
(1) get 10mgALP to add in 5ml physiological saline, join in Centricon-10 concentration tube, centrifugal about 20 minutes of 3000rpm, is concentrated into 1 milliliter;
(2) in upper liquid, add the 0.1MNaIO that 0.2ml newly joins
4solution, under room temperature, lucifuge stirs 20 minutes;
(3) loaded in bag filter by above-mentioned solution, dialyse with the sodium-acetate buffer of 1mMPH4.4,4 DEG C are spent the night;
(4) add 20 μ l0.2MPH9.5 carbonate buffer solutions, make the PH of above hydroformylation ALP be elevated to 9.0 ~ 9.5, then add 2.5mgIgG antibody immediately, in 1ml0.01M carbonate buffer solution, room temperature lucifuge stirs 2 hours gently;
(5) 4mg/mlNaBH that 0.1ml newly joins is added
4liquid, mixing, then put 4 DEG C 2 hours;
(6) load in bag filter by above-mentioned liquid, to 0.15MPH7.4PBS dialysis, 4 DEG C are spent the night;
(7) under agitation dropwise add equal-volume saturated ammonium sulfate, put 4 DEG C 1 hour;
(8) 3000rpm centrifugal half an hour, abandon supernatant, sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of a small amount of 0.15MPH7.4;
(9) load in bag filter by above-mentioned solution, dialyse about 5 hours to the PB buffer saline of 0.15MPH7.4, after removing ammonium ion, 10000rpm removes precipitation in centrifugal 30 minutes, and supernatant is enzyme conjugates, adds the 1MMgcl of volume 1/100
2solution 4 DEG C preservation, the alkaline phosphatase (ALP) collected mixes with the volume ratio of 1:1000 with above-mentioned enzyme reaction thing dilution with the conjugate of PCT monoclonal antibody, obtains seminal plasma fructose detection kit R of the present invention
1;
3rd step: reagent R
2formulation operations code: formula see (table 3), to prepare 1L:
(1) Tris (trishydroxymethylaminomethane, molecular formula: (HOCH2) 3CNH2) 1.56g and NaCl4.23g is taken in 1L beaker; Proclin-300 measured after 0.2ml dissolves completely in the beaker of a small amount of purified water with pipettor, pour in above-mentioned 1L container;
(2) measure 800ml purified water in beaker with graduated cylinder, fully stir, until dissolve completely; Adjust PH with 4MHCL or 4MNaOH, measure its scope between 7.35-7.45;
(3) ox gamma Globulin IgG0.9g is taken in the beaker of 800ml purified water;
(4) last constant volume 1000ml, after dissolving completely, survey pH value, namely scope meets the requirements between 7.35-7.45, uses 0.2um frit; After having filtered, post label in 2-8 DEG C of refrigeration house storage;
Reagent R
2preparation (table 3)
4th step: the preparation of standard items and quality-control product:
(1) peak: maximum concentration point is X, impact point concentration is A, B, C, D, E, F, when preparing the solution of V volume, need add the volume of raw material for being respectively: (table 4)
| Concentration | Add standard dilutions volume | Add X volume |
| A | V-A*V/X | A*V/X |
| B | V-B*V/X | B*V/X |
| C | V-C*V/X | C*V/X |
| D | V-D*V/X | D*V/X |
| E | V-E*V/X | E*V/X |
| F | V-F*V/X | F*V/X |
(2) Procalcitonin (PCT) quantitative determination reagent kit PCT standard items raw material is mixed with concentration point is 0,0.1,1,5,50,100ng/ml; The concentration point of quality-control product preparation is 0.6,50ng/ml.The concentration point of calibration object preparation is 1,50ng/ml
(3), after dissolving completely, label is posted in 2-8 DEG C of refrigeration house storage;
5th step: cleaning concentrate formulation operations code: formula is shown in (table 5), to prepare 1L:
(1) Tris12.54g and NaCl325.6g is taken in 1L container;
(2) take 5gTween-20 adds after 20ml water makes it dissolve completely in 100ml container, pours in said vesse;
(3) Proclin-300 measured after 0.2ml dissolves completely in the beaker filling 10ml purified water with pipettor, pour in above-mentioned 1L container;
(4) measure 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolve completely;
(5) adjust PH with 4MHCL or 4MNaOH, measure its scope between 7.35-7.45;
(6) last constant volume 1000ml, survey pH value, namely scope meets the requirements between 7.35-7.45, with 0.2um frit and get final product after dissolving completely; After having filtered, post label in 2-8 DEG C of refrigeration house storage;
The preparation (table 5) of cleaning concentrate
6th step: dilution formula is shown in (table 6), to prepare 1L:
(1) NaCl9.0g and BSAV60g is taken in the container of 1L;
(2) with pipettor, Proclin-300 is measured 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
(3) last constant volume 1000ml, after dissolving completely, uses 0.2um frit, posts label in 2-8 DEG C of refrigeration house storage;
The preparation (table 6) of dilution
7th step: luminous substrate formulation operations code: formula is shown in (table 7), to prepare 1L:
(1) Tris2.35g, NaCl6.41g, Na is taken
2sO
30.002g and Proclin-3000.2ml is in 1L beaker;
(2) measure 800ml purified water in beaker with graduated cylinder, fully stir, until dissolve completely; Adjust PH with 4MHCl or 4MNaOH, measure its scope between 7.95-8.05;
(3) be finally settled to 1000ml, survey pH value, namely scope meets the requirements between 7.95-8.05, uses 0.2um frit; After having filtered, add 250mlIUMIPHOS530, after mixing, post label in 2-8 DEG C of refrigeration house storage;
The preparation (table 7) of luminous substrate
The method of testing (for 100 μ l) of a kind of Procalcitonin (PCT) of the present invention, comprises the following steps:
(1) before using, 3 μ l calibration objects (choosing) in kit need be used to carry out curvature correction, and carry out quality control with X μ l (X<1 μ l) quality-control product, test result can carry out the detection of sample within the scope of Quality Control.
(2) add bottom 4 μ lPCT standard items, (1-X) μ l quality-control product, sample to be measured to corresponding test tube;
(3) 4 μ l reagent R are added
1to in each test tube;
(4) 4 μ l reagent R are added
2to in each test tube;
(5) 4 μ l Magneto separate reagent are added in each test tube;
(6) use covered rearing with plastic film test tube, multitube vortex mixer, gently after tube shaken frame 30s, puts 37 DEG C of water-baths 15 minutes;
(7) test tube frame linking is put on magnetic separator, guarantee that often propping up test tube all contacts with separator surface, precipitate 2 minutes, supernatant poured out by the separation vessel that reverses slowly, the test tube of reversing is placed on filter paper together with separation vessel, firmly bounces separator bottom to remove all drops be bonded on tube wall;
(8) after 30 μ l cleaning concentrate purified water being diluted 7 times, add 200 μ l dilute after cleaning fluid in each test tube, to put on multitube vortex mixer vibration mixing 30s gently; Application of sample dynamics should be avoided during application of sample excessive and cause magnetic bead to spill, mixing is wanted thoroughly;
(9) repeat step 6,7,6 one times;
(10) add in 30 μ l substrate solutions to test tube and mix 3 seconds, detect with ready luminometer rapidly;
(11) as run into high level HOOK sample, suggestion clinician selects suitable extension rate 20 μ l dilutions to dilute sample according to all the other test indexs.
Embodiment 2:
A kind of Procalcitonin (PCT) test kit of the present invention, this kit by Magneto separate reagent, reagent R
1, reagent R
2standard items, quality-control product, calibration object, cleaning concentrate, dilution and luminous substrate composition, wherein: Magneto separate reagent: the nano-magnetic microsphere being marked with the monoclonal antibody of mouse anti human PCT, described in be marked with the nano-magnetic microsphere of the monoclonal antibody of mouse anti human PCT concentration be 132 μ g/ml; Reagent R1: the PCT antibody containing alkali phosphatase enzyme mark, the PCT antibody concentration of described alkali phosphatase enzyme mark is 0.6 μ g/ml; Reagent R2: the damping fluid containing ox gamma Globulin matter component; Standard items, quality-control product and calibration object: bovine serum albumin(BSA) component five (BSAV) solution containing a certain amount of PCT antigen, the concentration of described standard items is 0 (S0), 0.1 (S1), 1 (S2), 5 (S3), 50 (S4), 100 (S5) ng/ml, the concentration of described quality-control product is 0.6,50ng/ml, the concentration of described calibration object is 1,50ng/ml; Cleaning concentrate: the damping fluid containing Tween-20 and Proclin-300; Dilution: the solution containing bovine serum albumin(BSA) component five (BSAV); Luminous substrate: the derivative I UMIPHOS530 of diamantane, the concentration of the derivative I UMIPHOS530 of described diamantane is 10 μ g/ml;
A kind of Procalcitonin (PCT) test kit of the present embodiment, is made up of following volume fraction: Magneto separate reagent 5.1%, reagent R
15.1%, reagent R
25.1%, standard items 5.1%, quality-control product 1.8%, calibration object 1.8%, cleaning concentrate 25%, dilution 17%, luminous substrate 34%.
The method of testing (for 100 μ l) of a kind of Procalcitonin (PCT) of the present invention, comprises the following steps:
(1) before using, 1.8 μ l calibration objects (choosing) in kit need be used to carry out curvature correction, and carry out quality control with X μ l (X<1.8 μ l) quality-control product, test result can carry out the detection of sample within the scope of Quality Control.
(2) add bottom 5.1 μ lPCT standard items, (1-X) μ l quality-control product, sample to be measured to corresponding test tube;
(3) 5.1 μ l reagent R are added
1to in each test tube;
(4) 5.1 μ l reagent R are added
2to in each test tube;
(5) 5.1 μ l Magneto separate reagent are added in each test tube;
(6) use covered rearing with plastic film test tube, multitube vortex mixer, gently after tube shaken frame 30s, puts 37 DEG C of water-baths 15 minutes;
(7) test tube frame linking is put on magnetic separator, guarantee that often propping up test tube all contacts with separator surface, precipitate 2 minutes, supernatant poured out by the separation vessel that reverses slowly, the test tube of reversing is placed on filter paper together with separation vessel, firmly bounces separator bottom to remove all drops be bonded on tube wall;
(8) after 25 μ l cleaning concentrate purified water being diluted 7 times, add 200 μ l dilute after cleaning fluid in each test tube, to put on multitube vortex mixer vibration mixing 30s gently; Application of sample dynamics should be avoided during application of sample excessive and cause magnetic bead to spill, mixing is wanted thoroughly;
(9) repeat step 6,7,6 one times;
(10) add in 34 μ l substrate solutions to test tube and mix 3 seconds, detect with ready luminometer rapidly;
(11) as run into high level HOOK sample, suggestion clinician selects suitable extension rate 17 μ l dilutions to dilute sample according to all the other test indexs.
The compound method of each component of kit described in the present embodiment adopts is identical with embodiment 1.
Embodiment 3:
A kind of Procalcitonin (PCT) test kit of the present invention, this kit by Magneto separate reagent, reagent R
1, reagent R
2standard items, quality-control product, calibration object, cleaning concentrate, dilution and luminous substrate composition, wherein: Magneto separate reagent: the nano-magnetic microsphere being marked with the monoclonal antibody of mouse anti human PCT, described in be marked with the nano-magnetic microsphere of the monoclonal antibody of mouse anti human PCT concentration be 132 μ g/ml; Reagent R
1: the PCT antibody containing alkali phosphatase enzyme mark, the PCT antibody concentration of described alkali phosphatase enzyme mark is 0.6 μ g/ml; Reagent R
2: the damping fluid containing ox gamma Globulin matter component; Standard items, quality-control product and calibration object: bovine serum albumin(BSA) component five (BSAV) solution containing a certain amount of PCT antigen, the concentration of described standard items is 0 (S0), 0.1 (S1), 1 (S2), 5 (S3), 50 (S4), 100 (S5) ng/ml, the concentration of described quality-control product is 0.6,50ng/ml, the concentration of described calibration object is 1,50ng/ml; Cleaning concentrate: the damping fluid containing Tween-20 and Proclin-300; Dilution: the solution containing bovine serum albumin(BSA) component five (BSAV); Luminous substrate: the derivative I UMIPHOS530 of diamantane, the concentration of the derivative I UMIPHOS530 of described diamantane is 10 μ g/ml;
A kind of Procalcitonin (PCT) test kit of the present embodiment, is made up of following volume fraction: Magneto separate reagent 6%, reagent R
16%, reagent R
26%, standard items 6%, quality-control product 3%, calibration object 1%, cleaning concentrate 20%, dilution 12%, luminous substrate 40%.
The method of testing (for 100 μ l) of a kind of Procalcitonin (PCT) of the present invention, comprises the following steps:
(1) before using, 1 μ l calibration object (choosing) in kit need be used to carry out curvature correction, and carry out quality control with X μ l (X<3 μ l) quality-control product, test result can carry out the detection of sample within the scope of Quality Control.
(2) add bottom 6 μ lPCT standard items, (1-X) μ l quality-control product, sample to be measured to corresponding test tube;
(3) 6 μ l reagent R are added
1to in each test tube;
(4) 6 μ l reagent R are added
2to in each test tube;
(5) 6 μ l Magneto separate reagent are added in each test tube;
(6) use covered rearing with plastic film test tube, multitube vortex mixer, gently after tube shaken frame 30s, puts 37 DEG C of water-baths 15 minutes;
(7) test tube frame linking is put on magnetic separator, guarantee that often propping up test tube all contacts with separator surface, precipitate 2 minutes, supernatant poured out by the separation vessel that reverses slowly, the test tube of reversing is placed on filter paper together with separation vessel, firmly bounces separator bottom to remove all drops be bonded on tube wall;
(8) after 20 μ l cleaning concentrate purified water being diluted 7 times, add 200 μ l dilute after cleaning fluid in each test tube, to put on multitube vortex mixer vibration mixing 30s gently; Application of sample dynamics should be avoided during application of sample excessive and cause magnetic bead to spill, mixing is wanted thoroughly;
(9) repeat step 6,7,6 one times;
(10) add in 40 μ l substrate solutions to test tube and mix 3 seconds, detect with ready luminometer rapidly;
(11) as run into high level HOOK sample, suggestion clinician selects suitable extension rate 12 μ l dilutions to dilute sample according to all the other test indexs.
The compound method of each component of kit described in the present embodiment adopts is identical with embodiment 1.
Clinical testing:
1, data are detected
Sample picks up from the normal health check-up of 1000 example, blood donor.Sample physical examination result is all without liver, brain, kidney, disease of digestive tract, and without blood transfusion and major operation history in half a year, women is not in the gestational period and lactation.Measured value is carried out statistical analysis, draws normal serum term of reference: <0.05ng/ml
Notebook data is only for reference, and different regions, Different Individual and employing distinct methods detect, and measured PCT level also can be different, advises that the range of normal value of oneself is set up in each laboratory.The PCT value that can not only draw with this method makes diagnosis, only as intermediate data reference role, in conjunction with other Data Analysis Results clinical, should comprise concrete condition and the treatment situation of patient.PCT concentration value in the sample obtained by additive method and this kit measurement result do not have direct comparability.Exceed the sample of kit measurement scope, system cannot provide definite numerical value.As for measuring its definite result, measure again after suggestion dilution.The testing result of this kit only supplies clinical reference, can not separately as making a definite diagnosis or the foundation of Excluded cases, and for reaching diagnostic purpose, this testing result will be combined with clinical examination, medical history and other inspection.This product can be used for the mensuration of serum sample, and the reliability for PCT concentration determination in other body fluid samples is fully confirmed.
2, kit performance index of the present invention
Sensitivity for analysis is defined as: to the mensuration of 20 zero standard product, gets its mean deviation of 2 times, and its concentration corresponding on typical curve is sensitivity for analysis; Sensitivity for analysis: 0.03ng/ml; Accuracy: variation within batch CV%≤10.0%; Batch variation CV%≤15.0%; Linear coefficient: r >=0.9900; The range of linearity: 0.03-100ng/ml:
With the cross reaction situation of main analog:
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (7)
1. a Procalcitonin test kit, this kit by Magneto separate reagent, reagent R
1, reagent R
2standard items, quality-control product, calibration object, cleaning concentrate, dilution and luminous substrate composition, wherein: Magneto separate reagent: the nano-magnetic microsphere being marked with the monoclonal antibody of mouse anti human PCT, described in be marked with the nano-magnetic microsphere of the monoclonal antibody of mouse anti human PCT concentration be 132 μ g/ml; Reagent R
1: the PCT antibody containing alkali phosphatase enzyme mark, the PCT antibody concentration of described alkali phosphatase enzyme mark is 0.6 μ g/ml; Reagent R
2: the damping fluid containing ox gamma Globulin matter component; Standard items, quality-control product and calibration object: bovine serum albumin(BSA) (BSA) (the being called for short BSAV) solution containing a certain amount of PCT antigen, the concentration of described standard items is 0 (S0), 0.1 (S1), 1 (S2), 5 (S3), 50 (S4), 100 (S5) ng/ml, the concentration of described quality-control product is 0.6,50ng/ml, the concentration of described calibration object is 1,50ng/ml; Cleaning concentrate: the damping fluid containing Tween-20 and Proclin-300; Dilution: the solution containing bovine serum albumin(BSA) (BSA) (being called for short BSAV); Luminous substrate: the derivative I UMIPHOS530 of diamantane, the concentration of the derivative I UMIPHOS530 of described diamantane is 10 μ g/ml.
2. a kind of Procalcitonin test kit according to claim 1, is characterized in that: described kit is made up of following volume fraction: Magneto separate reagent 4%-6%, reagent R
14%-6%, reagent R
24%-6%, standard items 4%-6%, quality-control product 1%-3%, calibration object 1%-3%, cleaning concentrate 20%-30%, dilution 10%-20%, luminous substrate 25%-40%.
3. a kind of Procalcitonin test kit according to claim 2, is characterized in that: described kit is made up of following volume fraction: Magneto separate reagent 4%, reagent R
14%, reagent R
24%, standard items 4%, quality-control product 1%, calibration object 3%, cleaning concentrate 30%, dilution 20%, luminous substrate 30%.
4. a kind of Procalcitonin test kit according to claim 2, is characterized in that: described kit is made up of following volume fraction: Magneto separate reagent 5.1%, reagent R
15.1%, reagent R
25.1%, standard items 5.1%, quality-control product 1.8%, calibration object 1.8%, cleaning concentrate 25%, dilution 17%, luminous substrate 34%.
5. a kind of Procalcitonin test kit according to claim 2, is characterized in that: described kit is made up of following volume fraction: Magneto separate reagent 6%, reagent R
16%, reagent R
26%, standard items 6%, quality-control product 3%, calibration object 1%, cleaning concentrate 20%, dilution 12%, luminous substrate 40%.
6., according to the arbitrary described a kind of Procalcitonin test kit of claim 1-5, it is characterized in that: in described kit, each component obtains according to following preparation method:
The first step: the preparation process of Magneto separate reagent
One, magnetic bead buffer solution formulation operations code:
(1) take Tris4.58g and NaCl6.81g in 1L container, take 0.96gTween-20 and add after suitable quantity of water makes it dissolve completely in 20ml container, pour in said vesse;
(2) Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water with pipettor, pour in above-mentioned 1L container; Then in 1L container, add 800ml purified water, fully stir, reagent is dissolved completely;
(3) PH measurement is regulated to measure its pH value; Adjust PH with 4MHCl or 4MNaOH, measure its PH and namely meet the requirements between 7.95-8.05;
(4) taking BSAV (bovine serum albumin(BSA) (BSA)) 3g pours in above-mentioned 1L container;
(5) be finally settled to 1000ml, survey pH value, namely scope meets the requirements between 7.95-8.05, with 0.2um frit and get final product; Label is posted in 2-8 DEG C of refrigeration house storage after having filtered;
Two, the preparation of Magneto separate reagent:
(1) be dissolved in 50ulDMSO by 1.0mgDSS (disuccinimidyl suberate), namely concentration is 20mg/mL; Get 2mgPCT antibody be dissolved in the 0.1mol/LPB damping fluid of PH9.5 to cumulative volume be 1ml;
(2) input amount of DSS is calculated, according to following formulae discovery: (antibody mass/16000) × 10 × 368/C
dSS), wherein C
dSSrefer to the substance withdrawl syndrome mol/L of DSS;
(3) join in the antibody-solutions of step 1 with the DSS of liquid-transfering gun absorption respective volume, put room temperature 90min;
(4) join in Centricon-10 concentration tube by step 3 antibody-solutions, then putting into and under the centrifugal force of 3000g, concentrating about 30min in sigma2-16k high speed freezing centrifuge is 0.5ml to volume;
(5) get 0.5ml magnetic bead, described bead diameter, between 0.9-1.5 μm, adds in 5ml reaction cup, puts into test tube rack special, draws supernatant through magnet adsorption after 2 minutes;
(6) add 1.5mlPH9.50.1mol/LPB at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; Antibody-solutions step 4 obtained joins in magnetic bead, and room temperature reaction 4 hours after mixing, keeps mixing state;
(7) add the Tris solution 37 DEG C 15 minutes of 0.3ml1mol/L, wherein the application of sample amount of Tris is that 1mg antibody adds 0.15mlTris;
(8) add 1.5mlPH7.20.1mol/LPB at every turn and clean the magnetic bead marked, mix 30 seconds, added, remove supernatant, repetitive operation 3 times;
(9) with 100ml magnetic bead conserving liquid, magnetic bead is proceeded to 125ml vial, be the PCT Magneto separate reagent of 0.05%;
(10) Magneto separate reagent magnetic bead buffer solution step 9 obtained mixes according to the ratio of 1:2, obtains Magneto separate reagent in kit of the present invention;
Second step: reagent R
1preparation process
One, reagent R
1diluent preparing working specification:
(1) Tris6.06g, NaCl13.0g, Zncl is got
20.05g, Proclin-3000.2ml and MgCl
20.05g is in flask; Then in flask, add 800ml purified water, fully stir, reagent is dissolved completely;
(2) adjust PH with 4MHCl or 4MNaOH, measure and make PH within the scope of 7.35-7.45;
(3) taking BSAV3g pours in above-mentioned flask;
(4) be finally settled to 1000ml, survey pH value, namely scope meets the requirements between 7.35-7.45, uses 0.2um frit; After having filtered, post label in 2-8 DEG C of refrigeration house storage;
Two, reagent R
1the preparation coupling of PCT monoclonal antibody (alkaline phosphatase (ALP) with)
(1) get 10mgALP to add in 5ml physiological saline, join in Centricon-10 concentration tube, centrifugal about 20 minutes of 3000rpm, is concentrated into 1 milliliter;
(2) in upper liquid, add the 0.1MNaIO that 0.2ml newly joins
4solution, under room temperature, lucifuge stirs 20 minutes;
(3) loaded in bag filter by above-mentioned solution, dialyse with the sodium-acetate buffer of 1mMPH4.4,4 DEG C are spent the night;
(4) add 20 μ l0.2MPH9.5 carbonate buffer solutions, make the PH of above hydroformylation ALP be elevated to 9.0 ~ 9.5, then add 2.5mgIgG antibody immediately, in 1ml0.01M carbonate buffer solution, room temperature lucifuge stirs 2 hours gently;
(5) 4mg/mlNaBH that 0.1ml newly joins is added
4liquid, mixing, then put 4 DEG C 2 hours;
(6) load in bag filter by above-mentioned liquid, to 0.15MPH7.4PBS dialysis, 4 DEG C are spent the night;
(7) under agitation dropwise add equal-volume saturated ammonium sulfate, put 4 DEG C 1 hour;
(8) 3000rpm centrifugal half an hour, abandon supernatant, sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of a small amount of 0.15MPH7.4;
(9) load in bag filter by above-mentioned solution, dialyse about 5 hours to the PB buffer saline of 0.15MPH7.4, after removing ammonium ion, 10000rpm removes precipitation in centrifugal 30 minutes, and supernatant is enzyme conjugates, adds the 1MMgcl of volume 1/100
2solution 4 DEG C preservation; The alkaline phosphatase (ALP) collected mixes with the volume ratio of 1:1000 with above-mentioned enzyme reaction thing dilution with the conjugate of PCT monoclonal antibody, obtains seminal plasma fructose detection kit R of the present invention
1;
3rd step: reagent R
2formulation operations process
(1) Tris (trishydroxymethylaminomethane, molecular formula: (HOCH2) 3CNH2) 1.56g and NaCl4.23g is taken in 1L beaker; Proclin-300 measured after 0.2ml dissolves completely in the beaker of a small amount of purified water with pipettor, pour in above-mentioned 1L container;
(2) measure 800ml purified water in beaker with graduated cylinder, fully stir, until dissolve completely; Adjust PH with 4MHCL or 4MNaOH, measure its scope between 7.35-7.45;
(3) ox gamma Globulin (IgG) 0.9g is taken in the beaker of 800ml purified water;
(4) last constant volume 1000ml, after dissolving completely, survey pH value, namely scope meets the requirements between 7.35-7.45, uses 0.2um frit; After having filtered, post label in 2-8 DEG C of refrigeration house storage;
4th step: the preparation of standard items, quality-control product and calibration object:
(1) Procalcitonin (PCT) quantitative determination reagent kit PCT standard items raw material is mixed with concentration point is 0.5,1,5,20,100ng/ml; The concentration point of quality-control product preparation is 0.6,20ng/ml; The concentration point of calibration object is 1,50ng/ml;
(2), after fully mixing, label is posted in 2-8 DEG C of refrigeration house storage;
5th step: cleaning concentrate formulation operations code:
(1) Tris12.54g and NaCl325.6g is taken in 1L container;
(2) take 5gTween-20 adds after 20ml water makes it dissolve completely in 100ml container, pours in said vesse;
(3) Proclin-300 measured after 0.2ml dissolves completely in the beaker filling 10ml purified water with pipettor, pour in above-mentioned 1L container;
(4) measure 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolve completely;
(5) adjust PH with 4MHCL or 4MNaOH, measure its scope between 7.35-7.45;
(6) last constant volume 1000ml, survey pH value, namely scope meets the requirements between 7.35-7.45, with 0.2um frit and get final product after dissolving completely; After having filtered, post label in 2-8 DEG C of refrigeration house storage;
6th step: diluent preparing process:
(1) NaCl9.0g and BSAV60g is taken in the container of 1L;
(2) with pipettor, Proclin-300 is measured 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
(3) last constant volume 1000ml, after dissolving completely, uses 0.2um frit, posts label in 2-8 DEG C of refrigeration house storage;
7th step: luminous substrate formulation operations process
(1) Tris2.35g, NaCl6.41g, Na is taken
2sO
30.002g and Proclin-3000.2ml is in 1L beaker;
(2) measure 800ml purified water in beaker with graduated cylinder, fully stir, until dissolve completely; Adjust PH with 4MHCl or 4MNaOH, measure its scope between 7.95-8.05;
(3) be finally settled to 1000ml, survey pH value, namely scope meets the requirements between 7.95-8.05, uses 0.2um frit; After having filtered, add 250mlIUMIPHOS530, after mixing, post label in 2-8 DEG C of refrigeration house storage.
7. a method of testing for Procalcitonin, comprises the following steps:
(1), before using, kit internal calibration product (choosing) need be used to carry out curvature correction, and carry out quality control with quality-control product, test result can carry out the detection of sample within the scope of Quality Control;
(2) add bottom PCT standard items, quality-control product, sample to be measured to corresponding test tube;
(3) reagent adding R
1to in each test tube;
(4) reagent adding R
2to in each test tube;
(5) Magneto separate reagent is added in each test tube;
(6) use covered rearing with plastic film test tube, multitube vortex mixer gently tube shaken frame, after 30 seconds, puts 37 DEG C of water-baths 15 minutes;
(7) test tube frame linking is put on magnetic separator, guarantee that often propping up test tube all contacts with separator surface, precipitate 2 minutes, supernatant poured out by the separation vessel that reverses slowly, the test tube of reversing is placed on filter paper together with separation vessel, firmly bounces separator bottom to remove all drops be bonded on tube wall;
(8) after cleaning concentrate purified water dilutes 7 times, add the cleaning fluid after dilution in each test tube, to put on multitube vortex mixer vibration mixing 30s gently; Application of sample dynamics should be avoided during application of sample excessive and cause magnetic bead to spill, mixing is wanted thoroughly;
(9) repeat step 6,7,6 one times;
(10) add in substrate solution to test tube and mix 3 seconds, detect with ready luminometer rapidly;
(11) as run into high level HOOK sample, suggestion clinician selects suitable extension rate dilution to dilute sample according to all the other test indexs.
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| CN105823893A (en) * | 2016-05-17 | 2016-08-03 | 北京豪迈生物工程有限公司 | IV type collagen test kit and test method thereof |
| CN106324254A (en) * | 2016-08-12 | 2017-01-11 | 泰州泽成生物技术有限公司 | Anti-insulin antibody detection kit and detection method thereof |
| CN106501503A (en) * | 2016-09-26 | 2017-03-15 | 王键 | Hyaluronic acid（HA）Test kit and preparation method thereof |
| CN106501504A (en) * | 2016-09-26 | 2017-03-15 | 王键 | Laminin，LN test kit and preparation method thereof |
| CN109374884A (en) * | 2018-12-24 | 2019-02-22 | 四川沃文特生物技术有限公司 | A kind of PCT concentration detection kit and preparation method thereof |
| CN111896754A (en) * | 2020-08-04 | 2020-11-06 | 四川沃文特生物技术有限公司 | PCT calibrator buffer solution |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105823893A (en) * | 2016-05-17 | 2016-08-03 | 北京豪迈生物工程有限公司 | IV type collagen test kit and test method thereof |
| CN106324254A (en) * | 2016-08-12 | 2017-01-11 | 泰州泽成生物技术有限公司 | Anti-insulin antibody detection kit and detection method thereof |
| CN106501503A (en) * | 2016-09-26 | 2017-03-15 | 王键 | Hyaluronic acid（HA）Test kit and preparation method thereof |
| CN106501504A (en) * | 2016-09-26 | 2017-03-15 | 王键 | Laminin，LN test kit and preparation method thereof |
| CN109374884A (en) * | 2018-12-24 | 2019-02-22 | 四川沃文特生物技术有限公司 | A kind of PCT concentration detection kit and preparation method thereof |
| CN109374884B (en) * | 2018-12-24 | 2021-10-22 | 四川沃文特生物技术有限公司 | PCT concentration detection kit and preparation method thereof |
| CN111896754A (en) * | 2020-08-04 | 2020-11-06 | 四川沃文特生物技术有限公司 | PCT calibrator buffer solution |
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Application publication date: 20160420 |