CN106018791B - A kind of calcitonin, folic acid, VITAMIN D COMBINATION detection immune chromatography reagent kit and preparation method thereof - Google Patents
A kind of calcitonin, folic acid, VITAMIN D COMBINATION detection immune chromatography reagent kit and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to immuno-chromatographic assay technology field, specifically discloses a kind of calcitonin, folic acid, VITAMIN D COMBINATION detection kit and preparation method thereof.The kit includes plastic plate, glass fibre element film, nitrocellulose filter, sample pad and adsorptive pads, and the mixture of quantum dot-labeled calcitonin antibody, folic acid antibody and the antibody of 25(OH)VD 3 is coated with the glass fibre element film；Detection line 1, detection line 2, detection line 3 and control line are provided with nitrocellulose filter, calcitonin secondary antibody is coated with detection line 1, folic acid secondary antibody is coated with detection line 2, the antigen of 25(OH)VD 3 is coated with detection line 3, sheep anti mouse lgG is coated with control line.The kit, the fluorescent characteristic selected using double antibody/antigen sandwich principle and competition law Cleaning Principle incorporating quantum is realized carries out quick, sensitive joint-detection using the method for fluorescence immune chromatography to calcitonin, folic acid and vitamin D.
Description
Technical field
The present invention relates to immuno-chromatographic assay technology field, and in particular to a kind of calcitonin, folic acid, VITAMIN D COMBINATION inspection
Survey immune chromatography reagent kit and preparation method thereof.
Background technology
Calcitonin is that thyroid parafollicular cell (bright cell or C cells) is produced and secreted, and it is made up of 32 peptides.
Main Physiological Function is to reduce blood calcium, the level of serium inorganic phosphorus.Plasma Ca ion concentration is too high, gastrin (gastrin) and pentapeptide
The rise of gastrin (Pentagastrin) can promote the secretion of calcitonin.Calcitonin have impact on calcium ion and phosphatic metabolism
Process, its function are the effect of antagonism parathormone mostly.In healthy male blood plasma the mean concentration of calcitonin be 0~
14ng/L, the mean concentration of calcitonin is 0~28ng/L in women blood plasma.Clinical research finds to suffer from malignant tumour, such as oat
Cell cancer, lung cancer, cancer of pancreas, uterine cancer, prostate cancer, some ectopic endocrine syndromes, serious osteopathy are kidney trouble, thermophilic
The diseases such as chromium cytoma are to occur in blood plasma that calcitonin raises, and by the concentration of calcitonin in blood plasma to originating from folliculus
The diagnosis of the medullary carcinoma of thyroid gland of parietal cell, judge that operative effect and observation postoperative recurrence etc. are significant.In addition, clinically
Thyroid operation excision, the sufferer of severe hyperthyroidism, it may appear that calcitonin concentration reduces in blood plasma.
Folic acid (folic acid) be also FA, is a kind of water soluble vitamin.Folic acid is by pteridine, p-aminophenyl first
Acid and Pidolidone composition, are also pteroylglutamic acid, it is one kind of B family vitamin.It was once named as after being found：Dimension
Raw plain M, folic acid, R factors etc., nineteen forty-one, because being found that this biotic factor from spinach, are named as leaf
Acid.Folic acid is rich in fresh water fruits and vegetables, meat product, if the folic acid in food can lose 50% through boiling for a long time
~90%.Mainly the folic acid storage capacity in duodenum and proximal jejunum site absorption, human body is 5~20mg to folic acid, folic acid master
To be excreted through urine and excrement, be 2~5ug per daily output.Folic acid is water miscible vitamin, general minimum beyond being grown up
It will not also cause poisoning within 20 times of requirement.In every case the amount of the folic acid exceeded is discharged from urine.Natural folic acid is widely present
It is especially more with comparision contents in yeast, liver and green vegetable in animal-plant kind food.
Clinical research shows that folic acid is worked in the form of tetrahydrofolic acid in vivo, has following physiological function：
1) coenzyme as one carbon unit transfer enzyme system in internal biochemical reaction, plays a part of one carbon unit carrier；
2) synthesis of purine and thymidine in nucleic acid synthesis, further synthetic DNA and RNA are participated in as coenzyme；
3) participate in amino acid metabolism, participate in two carbon amino acid and three carbon amino acid and mutually convert, glycine and serine,
The carrier of one carbon unit is served as in mutual conversion process between histidine and glutamic acid, homocysteine and methionine；
4) synthesis of hemoglobin and methyl compound such as adrenaline, choline, creatine etc. is participated in：Human body lacks folic acid can
Cause the exception of red blood cell, the increase of immature cell, anaemia and white blood cell are reduced；
5) metabolism of albumen is helped, and generation and the maturation of red blood cell are collectively promoted with vitamin B12, is manufacture red blood cell
Indispensable material；
6) also serve as the promotion proliferation factor of cheese lactic acid bacteria and other microorganisms and work；
7) it is the indispensable nutrient of embryo growth and development.Pregnant woman lacks folic acid and is likely to result in occurring during fetal birth
Under-weight, harelip, heart defect etc..If pregnancy first 3 months in lack folic acid, can cause fetal neural tube development lack
Fall into, and cause deformity.Therefore, prepare the women of pregnancy, can begin to take 100 micrograms before pregnancy daily to 300 microgram leaves
Acid.
Vitamin D (vitamin D) is sterol analog derivative, has anti-rachitic effect, also known as antirachitic vitamin.Mesh
Before think that vitamin D is also a kind of steroid hormone, most important member is VD2 (calciferols in vitamin D family member
Alcohol) and VD3 (Vitamin D3).Vitamin D is derivative of the different provitamin Ds after ultraviolet irradiation, and plant is without dimension
Raw plain D, but provitamin D all exists in animal and plant body.Vitamin D is a kind of liposoluble vitamin, there is five kinds of compounds,
Closer to healthy relation is calciferol and vitamine D3, and they have following three points characteristic：It is present in part Natural Food
In thing；Human body subcutaneous depot has the 7-DHC from cholesterol generation, after by ultraviolet irradiation, can be changed into vitamin
D3；Appropriate sunbath is sufficient for needs of the human body to vitamin D.
Vitamin D deficiency can cause juvenile's rickets and the osteomalacia of adult.Rickets is mainly in infant, main table
It is now the change of neuropsychic symptom and bone.Hidrosis, sleep terror fright at night, irritability are shown as on neuropsychic symptom.The change of bone
The factors such as the degree with age, growth rate and vitamin D deficiency are relevant.It may occur in which Lu osteomalacia, beading of ribs etc..Osteomalacia
Disease is more common in the women of pregnant fecund and weak and sickly old man occurred in adult.Most common symptom is ostalgia, myasthenia
With bone tenderness.Vitamin D is mainly used in forming and maintains the strong of bone, and it is used to prevent and treat the rickets of children with adult's
Rickets, arthralgia etc., the people with osteoporosis can effectively improve calcium by adding suitable vitamin D and magnesium
The trap of ion.In addition, vitamin D is also used for the probability for reducing colon cancer, breast cancer and prostate cancer, to immune
System also has humidification.
Research shows that the precursor (raw material of generation vitamin D) of vitamin D is present in skin, can be sent out when direct sunlight
Raw reaction is converted into vitamine D3, and D3 molecules, which are transported to liver and are converted into 25 lists of another form of vitamin D, to be taken off
Oxygen cholesterol, the effectiveness of this form are bigger.Then 25 monodeoxy cholesterol be transported to again kidneys and there by
1,25 dihydroxy courage calcification (steroid) alcohol are converted into, this form is the maximally effective state of vitamin D.Then vitamin D will and first
Glandular hormone and calcitonin act synergistically to balance the content of calcium ion and phosphorus in blood by shape, particularly strengthen human body to calcium
The absorbability of ion.Vitamin D should not be used to the too high patient of blood calcium, or the higher people of calcium ion content in blood.
Also must carefully it be used for the patient with kidney stone and artery sclerosis in addition, because vitamin D may cause them
Disease of parathyroid glands, weaken renal function even cause heart disease.Effect of the corticosteroid to vitamin D has counteracting to make
With cholestyramine and mineral oil can suppress absorption of the human body to vitamin D, so should avoid eating together.Dilantin sodium and sleeping
Medicine may increase the effect of vitamin D and reduce blood pressure.D be deficient in vitamin except that can cause the rickets of juvenile and be grown up soft
Outside osteopathy, other classical symptoms also include muscular atrophy, dysentery diarrhoea, insomnia, anxiety etc..Live in earth south poles
People and the people of some long-term office works itself can not often synthesize enough vitamin Ds, and also some diseases also influence dimension life
Plain D absorption, such as Crohn's disease (Crohn's disease) etc..
Therefore, a kind of detection method and means realization is researched and developed to examine the joint of calcitonin, folic acid and vitamin D
Survey, contribute to the health status of total evaluation pregnant woman, including the function such as bone conditions, anaemia and calcium uptake function, it is quick accurate
True diagnosis is advantageous in time carry out pregnant woman overall effective treatment.
The content of the invention
The defects of in order to overcome prior art, it is an object of the invention to provide a kind of high sensitivity, the degree of accuracy is high, operation is simple
Just, the low calcitonin of cost, folic acid, VITAMIN D COMBINATION detection kit, are realized to calcitonin, folic acid and vitamin D antibody
Joint-detection while three kinds of compositions.
The present invention also aims to provide the preparation side of a kind of calcitonin, folic acid, VITAMIN D COMBINATION detection kit
Method.
In order to realize the above object the technical solution adopted in the present invention is：
A kind of calcitonin, folic acid, VITAMIN D COMBINATION detection kit, including plastic plate, the glass that is arranged on plastic plate
Glass cellulose membrane, the nitrocellulose filter being arranged on glass fibre element film, one end is provided with sample on the nitrocellulose filter
Pad, the corresponding other end are provided with adsorptive pads, quantum dot-labeled calcitonin antibody, quantum are coated with the glass fibre element film
The folic acid antibody of point mark and the mixture of the quantum dot-labeled antibody of 25(OH)VD 3；Set on the nitrocellulose filter
Detection line 1, detection line 2, detection line 3 and control line are equipped with, calcitonin secondary antibody, the detection line 2 are coated with the detection line 1
On be coated with folic acid secondary antibody, the antigen of 25(OH)VD 3 is coated with the detection line 3, goat-anti is coated with the control line
Mouse lgG.
Coated quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody and quantum on the glass fibre
Quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody and amount in the mixture of the vitamin D antibody of point mark
The mass ratio of the antibody of 25(OH)VD 3 of son point mark is 1: 1: 1.
The quantum dot is cadmium selenide or cadmiumsulfide quantum dot.
Between the detection line 1 and detection line 2 that are set on the nitrocellulose filter, between detection line 2 and detection line 3, inspection
Interval between survey line 1 and control line is not less than 5mm.
Above-mentioned calcitonin, folic acid, the preparation method of VITAMIN D COMBINATION detection kit, including following operating procedure：
1) preparation of raw material：
A：Prepare glass fibre element film：By quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody, quantum
The antibody of 25(OH)VD 3 mixing of point mark is coated on glass fibre element film, is dried, standby；
B：Prepare nitrocellulose filter：Nitrocellulose filter is taken, interval marks off detection line 1, detection line 2, the and of detection line 3
Control line, calcitonin secondary antibody, folic acid secondary antibody, the antigen of 25(OH)VD 3, sheep anti mouse lgG are then coated on nitric acid fibre respectively
Tie up on detection line 1, detection line 2, detection line 3 and the control line of plain film, the nitrocellulose filter after coating is put into closing afterwards
Close, dry in liquid, it is standby；
2) assemble：Glass fibre element mould prepared by step 1) is placed on plastic plate, the glass then prepared in step 1)
Nitrocellulose filter prepared by step 1) is placed on cellulose membrane, sample pad is then placed in one end of nitrocellulose, it is corresponding
The other end place adsorptive pads, dry, that is, complete.
Also include preparing quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody and amount first in step A
The mixture of the vitamin D antibody of son point mark, specific method include following operating procedure：
a：Modified cyclodextrin is added in latex particle suspension, is mixed 24 hours, purifying, obtains modified cyclodextrin-latex
Compound suspension；
b：Quantum dot is added in modified cyclodextrin-latex compound suspension prepared by step a, is mixed 24 hours, it is pure
Change, obtain quantum dot-modified cyclodextrin-latex compound suspension；
c：Activator, activation 15 are added in quantum dot-modified cyclodextrin-latex compound suspension prepared by step b
Minute, purifying, calcitonin antibody being added afterwards, after mixing 2~4 hours, centrifuging to obtain precipitate particles, precipitate particles are sealed
Liquid resuspension is closed, room temperature mixes 1 hour, purified again afterwards, produce quantum dot-labeled calcitonin antibody；
d：Quantum dot-labeled folic acid antibody and quantum dot-labeled dimension are prepared according to the same methods of above-mentioned steps a~c
Raw plain D antibody, then by quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody and 25 quantum dot-labeled hydroxyls
Base vitamine D3 antibody mixes, standby；
The modified cyclodextrin is carboxymethyl-modification cyclodextrin or amination modified cyclodextrin.
The activator is 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides.
The mass ratio of quantum dot-modified cyclodextrin-latex compound and activator described in step c is 1: 10.
Confining liquid used is bovine serum albumin solution in step B.
Confining liquid used is bovine serum albumin solution in step c.
The mass ratio of modified cyclodextrin and latex particle described in step a is 20: 1；Quantum dot described in step b is pasted with ring
The mass ratio of essence-latex compounds is 10: 1；Calcitonin antibody described in step c and quantum dot-modified cyclodextrin-latex are compound
The mass ratio of thing is 1: 20.
Purification process is carried out before latex particle uses in step a, the specific method of the purification process is：Take latex particle
Add in the first buffer solution, centrifuge, supernatant discarding, then precipitate particles are resuspended with the first buffer solution, then centrifuge again point
From, supernatant discarding, then precipitate particles are resuspended with the first buffer solution, 4 DEG C save backup.
Above-mentioned latex particle after purification takes latex particle when in use, is diluted to latex particle using the second buffer solution
Concentration is 10mg/ml.
Also include, first by the modified cyclodextrin prepared in step a-latex compound suspension, using in above-mentioned steps b
Second buffer solution is diluted to the suspension that concentration is 10mg/ml, then adds quantum dot.
Also include in above-mentioned steps c first that the quantum dot-modified cyclodextrin-latex compound prepared in step b is suspended
Liquid, use the second buffer solution to be diluted to suspension of the concentration for 10mg/ml, then add activator activation.
The specific method that is purified is after being closed described in step c：15min is centrifuged under the conditions of 8000rpm, is discarded
Clearly, precipitation is redissolved in dispersion liquid.
By quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody, 25 quantum dot-labeled hydroxyls in step A
Base vitamine D3 antibody mixes the specific method being coated on glass fibre element film：Quantum dot-labeled calcitonin will be resisted
Body, quantum dot-labeled folic acid antibody and the quantum dot-labeled mixtures of antibodies even application of 25(OH)VD 3 are in glass fibers
Tie up on plain film.
The specific method purified described in step a and step b is：Centrifuged under the conditions of 8000rpm, supernatant discarding, precipitation is used
First buffer solution is resuspended, and is centrifuged afterwards under the conditions of 8000rpm, supernatant discarding, and precipitation is redissolved in the first buffer solution.
The specific method that purifies is after being activated in step c：Supernatant discarding is centrifuged under the conditions of 8000rpm, precipitation is slow with first
Fliud flushing is resuspended.
Calcitonin secondary antibody, folic acid secondary antibody, vitamin D antigen, sheep anti mouse lgG are coated on cellulose nitrate respectively in step B
Specific method on the detection line 1 of plain film, detection line 2, detection line 3 and control line is：The drop that concentration is 1mg/ml is taken respectively
Calcium element secondary antibody, folic acid secondary antibody, the antigen of 25(OH)VD 3 and sheep anti-mouse igg, with 1ul/cm, the speed of 10cm/ seconds is sprayed respectively
It is coated onto on detection line 1, detection line 2, detection line 3 and the control line of cellulose nitrate.
Drying described in step 1) and step 2) is dried 3~5 hours for 37 DEG C.
Above-mentioned first buffer solution is 0.02M, pH7.4 PB buffer solutions.Above-mentioned second buffer solution is 0.02M, pH6.1's
MES buffer solutions.
Testing sample when in use, is added kit by above-mentioned calcitonin, folic acid, VITAMIN D COMBINATION detection with kit
Sample pad on, calcitonin antibody, quantum dot-labeled folic acid antibody, the quantum dot of quantization on glass fibre element film mark
The mixtures of antibodies of the 25(OH)VD 3 specific first calcitonin captured in testing sample, folic acid and 25 hydroxyls of mark
Base vitamine D3 antibody, then when reaching detection line 3, quantum dot-labeled vitamin D antibody and 25 hydroxyls in detection line 3
Vitamine D3 antigentic specificity combines；In detection line 2, on quantum dot-labeled folic acid antibody-folic acid conjugate and detection line 2
Folic acid secondary antibody specific binding；In detection line 1, quantum dot-labeled calcitonin antibody-calcitonin conjugate and detection line 1
On calcitonin secondary antibody specific binding；Afterwards by detecting the quantum dot combined in detection line 1, detection line 2 and detection line 3
Fluorescent effect, qualitatively and quantitatively judge calcitonin in testing sample, folic acid and vitamin D.
Calcitonin of the present invention, folic acid, VITAMIN D COMBINATION detection kit, with the calcitonin antibody of quantization mark, amount
Folic acid antibody, the quantum dot-labeled mixtures of antibodies of 25(OH)VD 3 of son point mark are coated on glass fibre element film,
As detection probe, realized using the fluorescent characteristic of double antibody/antigen sandwich principle incorporating quantum point using fluorescence immunoassay layer
The method of analysis carries out quick, special, easy, sensitive joint-detection to calcitonin, folic acid and vitamin D.Calcitonin of the present invention,
Folic acid, VITAMIN D COMBINATION detection are low with kit detection line, and detection sensitivity is high.
Calcitonin of the present invention, folic acid, the preparation method of VITAMIN D COMBINATION detection kit, it is easy to operate, it is easy to control
System, suitable for industrial application.
It is further preferred that in preparation method of the present invention, quantum dot-labeled technology is innovated, using carboxy methylation or amination
Modified cyclodextrin modified quantum dot, then calcitonin antibody, folic acid antibody, the antibody of 25(OH)VD 3 are bonded respectively to
On the carboxymethyl group or amino group of modified cyclodextrin, realization prepares quantum dot-labeled calcitonin antibody, quantum dot
The folic acid antibody of mark, the quantum dot-labeled antibody of 25(OH)VD 3.
Brief description of the drawings
Fig. 1 is the luminous value of kit detection calcitonin and the mark curve of actual concentrations prepared using embodiment 1；
Fig. 2 is the luminous value of kit detection folic acid and the mark curve of actual concentrations prepared using embodiment 1；
Fig. 3 is the luminous value of kit detection vitamin D and the mark curve of actual concentrations prepared using embodiment 1.
Embodiment
Technical scheme is described in detail below by specific embodiment.
Embodiment 1
The present embodiment calcitonin, folic acid, VITAMIN D COMBINATION detection kit, including plastic plate, be arranged on plastic plate
Glass fibre element film, the nitrocellulose filter that is arranged on glass fibre element film, one end is provided with the nitrocellulose filter
Sample pad, the corresponding other end are provided with adsorptive pads, and the quantum dot mark that mass ratio is 1: 1: 1 is coated with the glass fibre element film
The mixture of the calcitonin antibody of note, quantum dot-labeled folic acid antibody and the quantum dot-labeled antibody of 25(OH)VD 3；
Detection line 1, detection line 2, detection line 3 and control line are provided with the nitrocellulose filter, drop is coated with the detection line 1
Calcium element secondary antibody, folic acid secondary antibody is coated with the detection line 2, the antigen of 25(OH)VD 3, institute are coated with the detection line 3
State and sheep anti mouse lgG is coated with control line；The quantum dot is CdSe quantum dots.
The present embodiment calcitonin, folic acid, the preparation method of VITAMIN D COMBINATION detection kit, concrete operation step are：
1) glass fibre element film is prepared：
a：The unmarked latex particles of 1ml are taken to add 10ml, 0.02M, in pH7.4PB buffer solutions；Centrifuge 10000rpm from
Heart 20min；Remove supernatant, then precipitation particulate matter is resuspended with 10ml, 0.02M, pH7.4PB buffer solutions；Centrifuge again afterwards
10000rpm centrifuges 20min, removes supernatant and precipitation particulate matter, 4 DEG C of preservations are resuspended with 5ml 0.02M, pH7.4, PB buffer solutions
It is stand-by；
b：Take the latex particle 0.02M, pH6.1 of step a preparations MES buffer solutions that latex particle is diluted into 10mg/
Ml, the latex particle suspension after 0.05ml dilutions is taken to add 10mg carboxymethyl-modification cyclodextrin, after mixing 24 hours, centrifugation
Machine 8000rpm is centrifuged, supernatant discarding, and with 0.02M, pH7.4 PB buffer solutions, which are resuspended, precipitates particulate matter, afterwards centrifuge again
8000rpm is centrifuged, supernatant discarding, and precipitation particulate matter is redissolved in 0.02M, pH7.4 PB buffer solutions, obtains carboxymethyl-modification ring
Dextrin-latex compound suspension；
c：Carboxymethyl-modification cyclodextrin-latex compound the suspension for taking step b to prepare, with 0.02M, pH6.1 MES
Buffer solution is diluted to 10mg/ml, is taken carboxymethyl-modification cyclodextrin-compound suspension of latex after 20ml dilutions, is added
20mg CdSe quantum dots, mix 24 hours, centrifuge 8000rpm centrifugations, supernatant discarding, and delayed with 0.02M, pH7.4 PB
Precipitation particulate matter is resuspended in fliud flushing, and centrifuge 8000rpm is centrifuged again afterwards, supernatant discarding, and precipitation particulate matter is redissolved in 0.02M,
In pH7.4 PB buffer solutions, quantum dot-carboxymethyl-modification cyclodextrin-latex compound suspension is obtained；
d：Quantum dot-carboxymethyl-modification cyclodextrin-latex compound the suspension for taking step c to prepare, with 0.02M,
PH6.1 MES buffer solutions are diluted to 10mg/ml, then according to quantum dot-modified cyclodextrin-latex compound and activation
The mass ratio of agent is 1: 10, adds 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, after activating 15 minutes,
8000rpm is centrifuged, supernatant discarding, and precipitate particles are resuspended, are resisted afterwards according to calcitonin with 0.02M, pH7.4 PB buffer solutions
Body：Quantum dot-carboxymethyl-modification cyclodextrin-latex compound=1: 20 ratio adds calcitonin antibody, is mixed 3 hours
Afterwards, 8000rpm is centrifuged 15 minutes, and precipitate particles are resuspended with confining liquid, and room temperature mixes 1 hour, 8000rpm centrifugations afterwards
15min, precipitation particulate matter are redissolved in dispersion liquid, produce quantum dot-labeled calcitonin antibody；
e：Quantum dot-labeled folic acid antibody and quantum dot-labeled 25 are prepared according to the same methods of above-mentioned steps a~c
Hydroxycholecalciferol antibody, then by quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody and quantum dot mark
The antibody of 25(OH)VD 3 mixing of note, is sprayed on glass fibre element mould, 37 DEG C of dryings 4 hours, produces quantum dot-labeled
Calcitonin antibody, quantum dot-labeled folic acid antibody and the quantum dot-labeled mixtures of antibodies of 25(OH)VD 3 mark
Glass fibre element film；
2) nitrocellulose filter is prepared：Nitrocellulose filter is taken, interval 6mm divides detection line 3, detection line 2, detection successively
Line 1 and control line, calcitonin secondary antibody, folic acid secondary antibody, the antigen of 25(OH)VD 3, goat-anti that concentration is 1mg/ml are taken afterwards
Mouse IgG, with 1ul/cm, the speed of 10cm/ seconds sprays to the detection line 1 of nitrocellulose membrane, detection line 2, detection line 3 and control respectively
On line processed, nitrocellulose filter is put into bovine serum albumin solution closed afterwards, 37 DEG C of dryings 3 hours, produced described
Nitrocellulose filter；
3) assemble：Glass fibre element mould prepared by step 1) is placed on plastic plate, in glass fibre prepared by step 1)
Nitrocellulose filter prepared by step 2) is placed on plain film, sample pad is then placed in one end of nitrocellulose, it is corresponding another
Adsorptive pads are placed in one end, 37 DEG C of dryings 5 hours, that is, complete.
Embodiment 2
The present embodiment calcitonin, folic acid, VITAMIN D COMBINATION detection kit, the place different from embodiment is, adopts
Quantum dot is cadmiumsulfide quantum dot, and the modified cyclodextrin used prepares calcitonin antibody, amount for amination dryness cyclodextrin
During the folic acid antibody and the quantum dot-labeled antibody of 25(OH)VD 3 of son point mark, in the quantum dot-change of activation
Property cyclodextrin-latex suspension in add the mixing time of antibody or antigen be 2 hours, other are the same as embodiment 1.
Embodiment 3
The present embodiment calcitonin, folic acid, VITAMIN D COMBINATION detection kit, the place different from embodiment are, make
During standby calcitonin antibody, quantum dot-labeled folic acid antibody and the quantum dot-labeled antibody of 25(OH)VD 3,
The mixing time that antibody or antigen are added in quantum dot-modified cyclodextrin-latex suspension of activation is 4 hours, and other are the same as real
Apply example 1.
Test example：
Calcitonin is diluted to 7.8125ug/L, 15.625ug/L, 31.25ug/L, 62.5ug/L, 125ug/L, 250ug/
L；Folic acid be diluted to 3.90625ug/L, 7.8125ug/L, 15.625ug/L, 31.25ug/L, 62.5ug/L, 125ug/L,
250ug/L；25(OH)VD 3 be diluted to 1.5625ug/L, 3.125ug/L, 6.25ug/L, 12.5ug/L, 25ug/L,
50ug/L, 100ug/L, 200ug/L prepare blank solution simultaneously as standard solution.
The standard items 100ul of each concentration is taken to be added separately in the sample pad of kit prepared by embodiment 1 respectively, 5min
The kit for the completion that develops the color is put into fluorescence immune chromatography readout instrument (the sincere bio tech ltd of Nanjing Mei Ningkang) weight afterwards, read
The luminous value of corresponding detection line is taken, and records the corresponding pass between the luminous value and actual concentrations of each concentration standard sample detection
System, standard curve is drawn, as shown in FIG. 1 to 3, the standard curve regression equation of calcitonin is：Y=-0.00009x2+
0.04228x-0.01248, R2=0.99956；The standard curve regression equation of folic acid is：Y=-0.00008x2+0.03915x+
0.09601, R2=0.99928；The standard curve regression equation of vitamin D is：Y=-0.00007x2+0.02681x+
0.06104, R2=0.99885；Understand that the kit of the invention prepared is directed to the minimum detection limit of calcitonin by the standard curve
For 1ng/L, folic acid lowest detection be limited to 2 μ g/L, the lowest detection of vitamin D is limited to 2 μ g/L.
In addition, taking blank solution to be added in the kit of the preparation of embodiment 1, fluorescent effect is not detected by.
100 random clinical samples are taken, the kit prepared using the embodiment of the present invention 1 is to calcitonin, folic acid and dimension
Raw plain D concentration carries out joint-detection.Detection method is：100ul testing samples (50ul sample+50ul sample diluting liquids) instill
In kit sample pad, the test strips of the develop the color standard items completed and testing sample are put into fluorescence immune chromatography reading after 5min
In instrument (the sincere bio tech ltd of Nanjing Mei Ningkang), the luminous value of T/C lines is read, and is recorded, by testing sample T/C value bands
Enter in standard curve, obtain the concentration of calcitonin, folic acid and vitamin D, it is as a result as shown in table 1 below：
Table 1
As can be seen here, this method can detect calcitonin, folic acid, vitamin D with direct quantitative, be not required to professional training, operate
Convenient, quickly, 10min can obtain result, be adapted to promote and use in basic hospital and medical center.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
The present invention is described in detail with reference to the foregoing embodiments, it will be understood by those within the art that：It still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic；
And these modification or replace, do not make appropriate technical solution essence depart from various embodiments of the present invention technical scheme spirit and
Scope.
Claims (9)
1. a kind of calcitonin, folic acid, the preparation method of VITAMIN D COMBINATION detection kit, it is characterised in that the kit
Including plastic plate, the glass fibre element film being arranged on plastic plate, the nitrocellulose filter being arranged on glass fibre element film, institute
State one end on nitrocellulose filter and be provided with sample pad, the corresponding other end is provided with adsorptive pads, it is characterised in that the glass fibre
Quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody and 25 quantum dot-labeled hydroxyls dimension are coated with plain film
The mixture of raw plain D3 antibody；Detection line 1, detection line 2, detection line 3 and control line, institute are provided with the nitrocellulose filter
State and calcitonin secondary antibody is coated with detection line 1, folic acid secondary antibody is coated with the detection line 2,25 are coated with the detection line 3
Hydroxycholecalciferol antigen, sheep anti mouse lgG is coated with the control line；
Its preparation method includes following operating procedure：
1) preparation of raw material：
A：Prepare glass fibre element film：By quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody, quantum dot mark
The antibody of 25(OH)VD 3 mixing of note is coated on glass fibre element film, is dried, standby；
B：Prepare nitrocellulose filter：Nitrocellulose filter is taken, interval marks off detection line 1, detection line 2, detection line 3 and control
Line, calcitonin secondary antibody, folic acid secondary antibody, the antigen of 25(OH)VD 3, sheep anti mouse lgG are then coated on nitrocellulose respectively
On the detection line 1 of film, detection line 2, detection line 3 and control line, the nitrocellulose filter after coating is put into confining liquid afterwards
Closing, dry, it is standby；
2) assemble：Glass fibre element mould prepared by step 1) is placed on plastic plate, the glass fibre then prepared in step 1)
Nitrocellulose filter prepared by step 1) is placed on plain film, sample pad is then placed in one end of nitrocellulose, it is corresponding another
Adsorptive pads are placed in one end, dry, that is, complete；
Wherein, also include preparing quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody and amount first in step A
The mixture of the antibody of 25(OH)VD 3 of son point mark, specific method include following operating procedure：
a：Modified cyclodextrin is added in latex particle suspension, is mixed 24 hours, purifying, it is compound to obtain modified cyclodextrin-latex
Thing suspension；
b：Quantum dot is added in modified cyclodextrin-latex compound suspension prepared by step a, is mixed 24 hours, purifying, is obtained
Quantum dot-modified cyclodextrin-latex compound suspension；
c：Activator is added in quantum dot-modified cyclodextrin-latex compound suspension prepared by step b, is activated 15 minutes,
Purifying, calcitonin antibody is added afterwards, after mixing 2~4 hours, precipitate particles are centrifuged to obtain, by precipitate particles confining liquid
It is resuspended, room temperature mixes 1 hour, is purified again afterwards, produces quantum dot-labeled calcitonin antibody；
d：Quantum dot-labeled folic acid antibody and quantum dot-labeled vitamin D are prepared according to the same methods of above-mentioned steps a~c
Antibody, then quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody and 25 quantum dot-labeled hydroxyls are tieed up
Raw plain D3 antibody mixing, it is standby.
2. calcitonin as claimed in claim 1, folic acid, the preparation method of VITAMIN D COMBINATION detection kit, its feature exist
In the modified cyclodextrin is carboxymethyl-modification cyclodextrin or amination modified cyclodextrin.
3. calcitonin as claimed in claim 1, folic acid, the preparation method of VITAMIN D COMBINATION detection kit, its feature exist
In the mass ratio of modified cyclodextrin and latex particle described in step a is 20：1；Quantum dot described in step b and cyclodextrin-breast
The mass ratio of glue compound is 10：1；Calcitonin antibody described in step c and quantum dot-modified cyclodextrin-latex compound
Mass ratio is 1：20.
4. calcitonin as claimed in claim 1, folic acid, the preparation method of VITAMIN D COMBINATION detection kit, its feature exist
In quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody, quantum dot-labeled vitamin D are resisted in step A
Body mixes the specific method being coated on glass fibre element film：By quantum dot-labeled calcitonin antibody, quantum dot-labeled
Folic acid antibody and quantum dot-labeled vitamin D mixtures of antibodies even application are on glass fibre element film.
5. calcitonin as claimed in claim 1, folic acid, the preparation method of VITAMIN D COMBINATION detection kit, its feature exist
In, in step B by calcitonin secondary antibody, folic acid secondary antibody, the antigen of 25(OH)VD 3, sheep anti mouse lgG be coated on respectively nitric acid fibre
The specific method tieed up on detection line 1, detection line 2, detection line 3 and the control line of plain film is：It is 1mg/ml's to take concentration respectively
Calcitonin secondary antibody, folic acid secondary antibody, the antigen of 25(OH)VD 3 and sheep anti-mouse igg, with 1ul/cm, the speed of 10cm/ seconds is distinguished
Spray on detection line 1, detection line 2, detection line 3 and the control line of cellulose nitrate.
6. a kind of calcitonin, folic acid, VITAMIN D COMBINATION detection kit, it is characterised in that by any one of Claims 1 to 5
Described preparation method is prepared.
7. calcitonin as claimed in claim 6, folic acid, VITAMIN D COMBINATION detection kit, it is characterised in that the glass
Coated quantum dot-labeled calcitonin antibody, quantum dot-labeled folic acid antibody and 25 quantum dot-labeled hydroxyls on glass fiber
Quantum dot-labeled calcitonin antibody in the mixture of vitamine D3 antibody, quantum dot-labeled folic acid antibody and quantum dot-labeled
The antibody of 25(OH)VD 3 mass ratio be 1：1：1.
8. calcitonin as claimed in claim 6, folic acid, VITAMIN D COMBINATION detection kit, it is characterised in that the amount
Son point is cadmium selenide or cadmiumsulfide quantum dot.
9. calcitonin as claimed in claim 6, folic acid, VITAMIN D COMBINATION detection kit, it is characterised in that the nitre
Between the detection line 1 and detection line 2 that are set on acid cellulose film, between detection line 2 and detection line 3, detection line 1 and control line it
Between interval be not less than 5mm.
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