CN107192826B - The colloid gold test paper and its preparation and detection method of a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate - Google Patents
The colloid gold test paper and its preparation and detection method of a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate Download PDFInfo
- Publication number
- CN107192826B CN107192826B CN201710542325.6A CN201710542325A CN107192826B CN 107192826 B CN107192826 B CN 107192826B CN 201710542325 A CN201710542325 A CN 201710542325A CN 107192826 B CN107192826 B CN 107192826B
- Authority
- CN
- China
- Prior art keywords
- dtpa
- diethylene triamine
- triamine pentacetic
- pentacetic acid
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The present invention relates to the colloid gold test paper and its preparation of a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate and detection methods；The colloid gold test paper includes reaction film, sample pad, conjugate release pad, water absorption pad and backing；There is coating diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object test section on the reaction film and be coated with the quality control region of sheep anti-mouse igg, the conjugate release pad is coated with diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object；When detection, sample is instilled in reagent card hole, when concentration is low in the sample for diethylene triamine pentacetic acid (DTPA) and its chelate, colloidal gold antibody can be fixed on the combination of the diethylene triamine pentacetic acid (DTPA) conjugate on reaction film in chromatography process, will appear each red stripes in the quality control region of test section；It is when concentration is high, then on the contrary；The present invention can accurately, quickly detect the animal derived foods such as chicken, pork, urine, honey, the residual content of diethylene triamine pentacetic acid (DTPA) and its chelate in detergent and water environment.
Description
Technical field
The present invention relates to a kind of colloid gold test paper, specifically a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate
Colloid gold test paper and its preparation and detection method.
Background technique
Diethylene triamine pentacetic acid (DTPA) (DTPA) is a kind of excellent complexones, sequestering strong, it and most of sun from
It is more stable than the corresponding chelate of ethylenediamine tetra-acetic acid that son was formed integrates object.As highly effective chelating agent, it is applied to acrylic fiber production process
Middle color inhibitors, paper industry, water softener, textile auxiliary, chelatometric titration agent, autochromy and food industry are medium.Simultaneously
It also has a wide range of applications in medical treatment, rare earths separation and agricultural production.
Diethylene triamine pentacetic acid (DTPA) also serves as metallic poison antidote, is often better than EDTA to the removal effect of metallic poison,
But its side effect is generally also larger, especially excludes to the acceleration of internal essential trace element, it is necessary to be observed.Flesh
Diethylene triamine pentacetic acid (DTPA) is infused, after one course for the treatment of of medication, serum Zn about drop by half, Fe decline 15%.When DTPA concentration is therapeutic dose
6-30 times when, can liver to mouse, kidney generate it is temporary have toxic effect, this may be that protein is caused to ooze out from cell membrane
Event.And evidence suggests DTPA can induce Vicia faba distortion, so DTPA may be dangerous to the mankind.High concentration
DTPA makes one distortion such as part, ring, the dicentromere that Lymphocytes Sister exchanges the increase of (SCE) amount and chromosome.
So the detection to diethylene triamine pentacetic acid (DTPA) need to be reinforced.
Currently, the detection method of diethylene triamine pentacetic acid (DTPA) and its chelate has gas chromatography, high performance liquid chromatography
(HPLC).But the detection of these methods is time-consuming and laborious and at high cost, and compared with other detection systems, one-time detection sample size is few, inspection
The survey time is long, is not suitable for field quick detection, only the confirmation method as a few experiments room, can not meet environment and agricultural production product examine
Survey fast and convenient needs.
And the immunologic detection method based on diethylene triamine pentacetic acid (DTPA) specific antibody provided for its detection it is a kind of new
Strategy.Immunization is increasingly taken seriously at present, and because its detection limit is low, single sample testing cost is low (such as ELISA method), especially
It is suitably applied the detection of gross sample, especially immunochromatographic method, detection speed is fast, high sensitivity, examines with HPLC, GC-MS
Survey has higher consistency, and is very suitable to the detection of living animal.And it is at low cost, it is not necessarily to large-scale instrument, and can be straight
It connects and the samples such as urine, feed, detergent, water sample is detected, be suitable for on-site test.But there is presently no detections two
The kit and test strips of the commercialization of ethylene pentaacetic acid and its chelate.Therefore, studying and establish has China autonomous
The diethylene triamine pentacetic acid (DTPA) and its chelate immunological detection method of intellectual property, to raising China's environment and agricultural product diethyl
Alkene pentaacetic acid and its chelate control and monitor level, guarantee environment and agricultural product quality and safety and the health tool of consumer
It is significant.
Summary of the invention
Object of the present invention is to deficiency to solve above-mentioned technical problem, provide a kind of detection diethylene triamine pentacetic acid (DTPA) and its
The colloid gold test paper of chelate and its preparation and detection method can accurately, quickly detect the animals such as chicken, pork, urine, honey
The residual content of diethylene triamine pentacetic acid (DTPA) and its chelate in derived food, detergent and water environment.
Used technical solution is the present invention to solve above-mentioned technical problem:
A kind of colloid gold test paper detecting diethylene triamine pentacetic acid (DTPA) and its chelate, including reaction film, sample pad, combination
Object release pad, water absorption pad and backing；There is coating diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object survey on the reaction film
It tries area and is coated with the quality control region of sheep anti-mouse igg, the conjugate release pad is coated with diethylene triamine pentacetic acid (DTPA) monoclonal antibody-glue
Body gold marker；
The diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object passes through difunctional chelating by diethylene triamine pentacetic acid (DTPA)
Agent p-NH2- Bn-DTPA or P-SCN-Bn-DTPA [five second of 1- (the different sulphur cyanobenzyl of 4-) vinyl triamine-N, N, N', N, N'-
Acid] it is obtained with carrier protein couplet；
Diethylene triamine pentacetic acid (DTPA) monoclonal in the diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object
Antibody be using diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object as immunizing antigen immunity test animal after prepare.
The diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object is that diethylene triamine pentacetic acid (DTPA)-bovine serum albumin(BSA) is even
Join object, diethylene triamine pentacetic acid (DTPA)-human serum albumin conjugate, diethylene triamine pentacetic acid (DTPA)-keyhole limpet hemocyanin conjugate,
Five second of diethylene triamine pentacetic acid (DTPA)-ovalbumin conjugate, diethylene triamine pentacetic acid (DTPA)-ferritin conjugate or diethylenetriamine
Acid-poly-D-lysine conjugate.
The diethylene triamine pentacetic acid (DTPA)-keyhole limpet hemocyanin conjugate is the preparation method comprises the following steps: take 0.8-20 mg difunctional
Chelating agent is dissolved in 50 μ L dimethyl sulfoxides, prepares the P-SCN-Bn-DTPA of 16-400 mg/mL；Take 2.0-20.0 mg carrier egg
It is white to be dissolved in 1 mL HEPES buffer solution or carbonate buffer solution, prepare the keyhole limpet hemocyanin solution of 2-20 mg/mL；By this
Haptens solution is added dropwise in the keyhole limpet hemocyanin solution of 1 mL 2-20 mg/mL, in 4 DEG C or room temperature, pH7.5-
24 h of oscillating reactions under the conditions of 10.5；Unreacted lower-molecular substance, as divinyl three are removed with the method for ultrafiltration or dialysis
Triamine pentaacetic acid-keyhole limpet hemocyanin.
The diethylene triamine pentacetic acid (DTPA)-keyhole limpet hemocyanin conjugate is the preparation method comprises the following steps: take 0.8-20 mg difunctional
Chelating agent is dissolved in 50 μ L dimethyl sulfoxides, prepares the p-NH of 16-400 mg/mL2-Bn-DTPA；Take 2.0-20.0 mg key empty
Hemocyanin is dissolved in 1 mL HEPES buffer solution or carbonate buffer solution, prepares the carrier protein solution of 2-20 mg/mL；By 1
P-NH is added dropwise in 1% glutaraldehyde solution of mL2In-Bn-DTPA solution, left at room temperature over night is to activate amino.By the half of activation
Antigenic solution is added dropwise in the key sky hemocyanin solution of 1 mL 2-20 mg/mL, in 4 DEG C or room temperature, pH7.5-10.5
Under the conditions of 24 h of oscillating reactions；Unreacted lower-molecular substance, as diethylenetriamine five are removed with the method for ultrafiltration or dialysis
Acetic acid-keyhole limpet hemocyanin.
The diethylene triamine pentacetic acid (DTPA)-bovine serum albumin(BSA) conjugate is the preparation method comprises the following steps: take 0.8-20 mg difunctional
Chelating agent is dissolved in 50 μ L dimethyl sulfoxides, prepares the P-SCN-Bn-DTPA of 16-400 mg/mL；Take 2.0-20.0 mg cow's serum
Albumin is dissolved in 1 mL HEPES buffer solution or carbonate buffer solution, prepares the bovine serum albumin solution of 2-20 mg/mL；
This haptens solution is added dropwise in the bovine serum albumin solution of 1 mL 2-20 mg/mL, in 4 DEG C or room temperature,
24 h of oscillating reactions under the conditions of pH7.5-10.5；Unreacted lower-molecular substance, divinyl are removed with the method for ultrafiltration or dialysis
Pentaacetic acid-bovine serum albumin(BSA) conjugate.
The diethylene triamine pentacetic acid (DTPA)-bovine serum albumin(BSA) conjugate is the preparation method comprises the following steps: take 0.8-20 mg difunctional
Chelating agent is dissolved in 50 μ L dimethyl sulfoxides, prepares the p-NH of 16-400 mg/mL2-Bn-DTPA；Take 2.0-20.0 mg ox blood
Pure albumen is dissolved in 1 mL HEPES buffer solution or carbonate buffer solution, and the bovine serum albumin(BSA) for preparing 2-20 mg/mL is molten
Liquid；P-NH is added dropwise in 1 mL, 1% glutaraldehyde solution2In-Bn-DTPA solution, left at room temperature over night is to activate amino.It will
The haptens solution of activation is added dropwise in the bovine serum albumin solution of 1 mL 2-20 mg/mL, in 4 DEG C or room temperature,
24 h of oscillating reactions under the conditions of pH7.5-10.5；Unreacted lower-molecular substance, divinyl are removed with the method for ultrafiltration or dialysis
Pentaacetic acid-bovine serum albumin(BSA) conjugate.
The preparation method of the colloid gold test paper of the detection diethylene triamine pentacetic acid (DTPA) and its chelate, including following step
It is rapid:
1) preparation coating diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has coating diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object test section and coating sheep anti-mouse igg
Quality control region reaction film；
And 2) 3) 1) conjugate release pad, reaction film and the sample pad, water absorption pad and the backing that prepare are assembled into examination
Paper.
The preparation method of the colloid gold test paper of the detection diethylene triamine pentacetic acid (DTPA) and its chelate, specific steps are such as
Under:
(1) by bifunctional chelating agent (P-SCN-Bn-DTPA or p-NH2- Bn-DTPA) and carrier protein couplet, it is formed
Diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object；
(2) mouse is immunized with diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object, by mouse boosting cell and mouse bone marrow cells
Oncocyte obtains the monoclonal antibody hybridoma cell strain of secretion diethylene triamine pentacetic acid (DTPA) by fusion, screening；
(3) it is reacted with trisodium citrate with gold chloride and prepares colloidal gold；
(4) the diethylene triamine pentacetic acid (DTPA) monoclonal antibody of preparation is added in the colloidal gold of step (3) preparation, is obtained
Diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object；
(5) diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object is coated in conjugate release pad, is used
The buffer that the disodium hydrogen phosphate and sodium dihydrogen phosphate of the ovalbumin containing 0.1-1.5% are prepared soaks 30 seconds, and it is spare that 37 oC dry 2h；
(6) diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object is coated on reaction film and constitutes test section, and by sheep
Anti- mouse IgG, which is coated on reaction film, constitutes quality control region, is closed with the confining liquid containing 0.1% cow's serum；
(7) by sample pad: rabbit serum proteins, 1-1.5% Tween-20 disodium hydrogen phosphate and the biphosphate of 0.1-1.5%
The buffer that sodium is prepared impregnates 2h, and 37 DEG C of baking 2h are spare；
(8) assembling of diethylene triamine pentacetic acid (DTPA) and its chelating analyte detection colloidal gold test paper card: on backing in order
Successively adhesive reaction film, water absorption pad, conjugate release pad and sample pad are then cut into the small item of 3mm wide, add plastic casing, vacuum
Packaging.Sample pad is covered conjugate release pad by test card of the invention can extend the observing time of testing result, sample pad
Liquid can also be will test and fully absorb to completely attach to gold labeling antibody and sufficiently react, reduce error.
The method of the colloid gold test paper detection diethylene triamine pentacetic acid (DTPA) and its chelate are as follows: sample is instilled into reagent card
In hole, when concentration is low in the sample for diethylene triamine pentacetic acid (DTPA) and its chelate, colloidal gold antibody can quilt in chromatography process
The diethylene triamine pentacetic acid (DTPA) conjugate being fixed on reaction film combines, and will appear each one in test section (T) quality control region (C)
Red stripes.If diethylene triamine pentacetic acid (DTPA) and its chelate are when concentration is high in the sample, colloidal gold antibody and divinyl three
Triamine pentaacetic acid and its chelate all combine, thus because competitive reaction will not be with diethylene triamine pentacetic acid (DTPA) idol in the area (T)
Join object to combine without there are red stripes；Negative sample is in the detection process due to lacking antigen-antibody competitive reaction, it will
(T) there are red stripes in area and the area (C)；
It is positive: when the area (C) shows red stripes, and the area (T) does not develop the color, to be judged to the positive；
Negative: when the area (C) shows red stripes, the area (T) also shows that red stripes simultaneously, and (T) area's color is close
(C) line or when being shallower than (C) line, is judged to the positive；
Invalid: whether the area (C) does not show red stripes, then no matter the area (T) shows red stripes, which sentences
It is invalid.
The p-NH2- Bn-DTPA, full name: S-2- (4-Aminobenzyl)-diethylenetriamine
Pentaacetic acid, molecular formula: C21H30N4O104HCl, molecular weight: 644.3, structural formula is。
The p-SCN-Bn-DTPA, full name: S-2- (4-Isothiocyanatobenzyl)-
Diethylenetriamine pentaacetic acid, molecular formula: C22H28N4O10S3HCl, molecular weight:
649.9 structural formula is。
Beneficial effect is:
1, the quick detection test paper of diethylene triamine pentacetic acid (DTPA) of the invention and its chelate is using the anti-of high degree of specificity
Body-antigen-reactive and immunochromatographiassays assays technology are coated with diethylene triamine pentacetic acid (DTPA)-carrier protein couplet in reaction film test section
The diethylene triamine pentacetic acid (DTPA) monoclonal antibody of antigen, conjugate release pad coating colloid gold label, is detected using competition law
Whether contain diethylene triamine pentacetic acid (DTPA) in sample to be tested.It is reacted by diethylene triamine pentacetic acid (DTPA) in sample to be tested with being coated on
Diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object on film competes diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid jointly
Golden marker, according to test section red stripes whether there is or not or shade judge whether to contain diethylenetriamine in analyte sample fluid
How much are pentaacetic acid and its chelate or content.Comlete antigen well known to those skilled in the art, being obtained using different raw materials, i.e.,
Diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object, structure is necessarily not exactly the same, due to the incomplete phase of the antigenic structure of acquisition
Together, the technical effect being used in conjunction with antibody and sheep anti-mouse igg is not also identical, and antigen prepared by the application and antibody are mutually tied
It closes, has been finally reached the technical purpose of accurate and sensitive detection diethylene triamine pentacetic acid (DTPA).
2, the pH value 7.5-10.5 in reaction process of the present invention and bifunctional chelating agent, buffer, bovine serum albumin(BSA),
The dosage or concentration of the raw materials such as key sky hemocyanin, glutaraldehyde, it is ensured that each raw material is made full use of in holoantigen preparation process, and
Ensure yield with higher.
3, test paper provided by the invention can be used for detecting the animal derived foods such as chicken, pork, urine, honey, detergent
And the residual content of diethylene triamine pentacetic acid (DTPA) and its chelate in water environment, and market value of the present invention has easy to operate, cost
Low, high sensitivity, detection speed are fast, are suitble to that large-scale promotion, storage be simple, shelf-life tea row, and can on-site test, be suitble to
Great amount of samples screening is not required to specialized instrument and equipment and professional, easy promotion and implementation.
Detailed description of the invention
Fig. 1 is the assembling schematic diagram of Test paper card of the present invention;
Fig. 2 is Analysis of test results schematic diagram of the invention.
Specific embodiment
Embodiment 1
The preparation of diethylene triamine pentacetic acid (DTPA) and its chelate Test paper card:
1. diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object synthesis and identification
(1) diethylene triamine pentacetic acid (DTPA)-keyhole limpet hemocyanin conjugates synthesis
It can prepare according to the following steps: 0.8-20 mgP-SCN-Bn-DTPA being taken to be dissolved in 50 μ L dimethyl sulfoxides, prepare 16-
The P-SCN-Bn-DTPA of 400 mg/mL；2.0-20.0 mg carrier protein is taken to be dissolved in 1 mL HEPES buffer solution or carbonate
Buffer prepares the keyhole limpet hemocyanin solution of 2-20 mg/mL；This haptens solution is added dropwise to 1 mL 2-20 mg/
In the keyhole limpet hemocyanin solution of mL, 24 h of oscillating reactions under the conditions of 4 DEG C or room temperature, pH7.5-10.5；With ultrafiltration or dialysis
Method remove unreacted lower-molecular substance, obtain diethylene triamine pentacetic acid (DTPA)-keyhole limpet hemocyanin conjugates, i.e. DTPA's
Comlete antigen.
2. the preparation of diethylene triamine pentacetic acid (DTPA) monoclonal antibody
(1) animal immune
Immunizing antigen (comlete antigen) is injected into Balb/c Mice Body, immunizing dose is 50 μ g/, keeps its generation more
Clonal antibody.
(2) cell fusion and clone
Immune Balb/c mouse boosting cell is taken, is merged in 5:1 ratio with SP2/0 myeloma cell, using indirect competition
ELISA method measures cell supernatant, screens positive hole.Screening diethylene triamine pentacetic acid (DTPA) solution concentration is still positive lower than 3nm
The cell strain of reaction.Cloning is carried out to positive hole using limiting dilution assay, until obtaining the miscellaneous of stably excreting monoclonal antibody
Hand over tumor cell strain.
(3) specific detection of hybridoma cell strain
With the diethylene triamine pentacetic acid (DTPA) solution and diethyl of indirect competitive ELISA method measurement cell supernatant and various concentration
Alkene pentaacetic acid-metal ion (such as Cu2+、Zn2+、Ni2+、Co2+、Fe3+、Cr3+、Mg2+、Hg2+、Ca2+、Cd2+、Pb2+、In3+、K+、Na+) chelate solution reactivity.Selection cross reactivity reaches 90% or more cell strain.
(4) cell cryopreservation and recovery
The monoclonal hybridoma strain of anti-diethylene triamine pentacetic acid (DTPA) is made 1 × 10 with frozen stock solution8The cell of a/ml
Suspension saves for a long time in liquid nitrogen.Cryopreservation tube is taken out when recovery, is immediately placed in 37 DEG C of water-bath middling speeds and is melted, centrifugation removal frozen stock solution
Afterwards, culture culture in glassware is moved into.
(5) preparation and purification of monoclonal antibody
Balb/c mouse peritoneal is injected into sterilizing paraffin oil 0.5ml/ only, anti-five second of diethylenetriamine is injected intraperitoneally after 7 days
The monoclonal hybridoma 10 of acid5A/only, ascites is acquired after 7 days.Purified with octanoic acid-saturation sulfuric acid by method, after purification
Ascites be put into -20 DEG C of environment and save.
3. the preparation of diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object
(1) preparation of colloidal gold
1% gold chloride is diluted to 0.01 % with double distilled deionized water, sets to stir on magnetic force heating rod blender and boil, often
1% trisodium citrate of 2ml is added in 0.01 % gold chloride of 100ml, continues to boil, until stopping heating when liquid takes on a red color, is cooled to
Dehydration is supplied after room temperature.The colloidal gold appearance prepared answer it is pure, bright, without precipitating and floating material, validity period be one month.
(2) diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object preparation
Under magnetic stirring, it with the pH value of 0.1M potassium carbonate tune colloidal gold to 8.2, is added by 1 ~ 2 μ g/ml antibody colloidal gold
Anti- diethylene triamine pentacetic acid (DTPA) monoclonal antibody continues to stir and evenly mix 30min, 10% BSA to final concentration of 1% is added, stands
30min.12000rpm, 4oC are centrifuged 30min, abandon supernatant, precipitating is washed twice with redissolution buffer, with initial colloid gold body
Precipitating is resuspended the redissolution buffer of product 1/20, sets 4 DEG C of spare, preservations 60 days.
5, the coating of conjugate release pad
Conjugate release pad is soaked in the disodium hydrogen phosphate of the ovalbumin containing 0.1-1.5% and sodium dihydrogen phosphate is matched
It is soaked in the buffer of system 30 seconds, 37 DEG C of baking 2h;With point film instrument by the diethylene triamine pentacetic acid (DTPA) prepared monoclonal antibody-glue
Body gold marker is uniformly coated in conjugate release pad, vacuum drying, Vacuum Package, set 4 DEG C it is spare.
6, the processing of nitrocellulose filter
Processing: coating diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object constitutes test section, and coating sheep anti-mouse igg is constituted
Quality control region, then closed with the confining liquid containing 0.1% cow's serum.
Coating process: use phosphate buffer (methanol containing 3%) by diethylene triamine pentacetic acid (DTPA)-nitrocellulose filter
Hemocyanin (KLH) conjugate is diluted to 10mg/mL, and point film instrument is used to be coated in nitrocellulose filter as test section, packet
It is measured as 0.8 μ g/cm2, test section is close to bonding pad end, away from bonding pad end about 8mm;With 7.4 PBS buffer solution of 0.01M pH
Sheep anti-mouse igg antibody is diluted to 150 μ g/ml by (methanol containing 3%), and point film instrument is used to be coated in cellulose membrane as Quality Control
Area, package amount are 0.8 μ g/cm2, quality control region is close to water absorption pad, away from absorption pad about 8mm, two linear distance 5-8mm.37 DEG C of drying, envelope
Dress.
7, the assembling of colloid gold immune test paper card
Nitrocellulose filter, water absorption pad, conjugate release pad and sample pad are adhered in order on PVC backing, as schemed institute
Show, gold conjugation pad is conjugate release pad in figure;NC film is reaction film；PVC offset plate is backing.Patch sample pad is covered
Conjugate release pad is then cut into the small item of 2-3mm wide, adds plastic casing, vacuum packaging.Original packing should be in 18-25 DEG C of environment
Middle storage, is valid for one year.Sample pad is covered conjugate release pad by test card of the invention can extend testing result observation
Time, sample pad can also will test liquid and fully absorb and can completely attach to gold labeling antibody, sufficiently react, to reduce mistake
Difference.
The method of the colloid gold test paper detection diethylene triamine pentacetic acid (DTPA) and its chelate are as follows: sample is instilled into reagent card
In hole, when concentration is low in the sample for diethylene triamine pentacetic acid (DTPA) and its chelate, colloidal gold antibody can quilt in chromatography process
The diethylene triamine pentacetic acid (DTPA) conjugate being fixed on reaction film combines, and will appear each one in test section (T) quality control region (C)
Red stripes.If diethylene triamine pentacetic acid (DTPA) and its chelate are when concentration is high in the sample, colloidal gold antibody and divinyl three
Triamine pentaacetic acid and its chelate all combine, thus because competitive reaction will not be with diethylene triamine pentacetic acid (DTPA) idol in the area (T)
Join object to combine without there are red stripes；Negative sample is in the detection process due to lacking antigen-antibody competitive reaction, it will
(T) there are red stripes in area and the area (C)；
It is positive: when the area (C) shows red stripes, and the area (T) does not develop the color, to be judged to the positive；
Negative: when the area (C) shows red stripes, the area (T) also shows that red stripes simultaneously, and (T) area's color is close
(C) line or when being shallower than (C) line, is judged to the positive；
Invalid: whether the area (C) does not show red stripes, then no matter the area (T) shows red stripes, which sentences
It is invalid.
P-SCN-Bn-DTPA described in the present embodiment is bought in SIGMA company, the U.S., purity: >=94%
Embodiment 2
Diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object, that is, diethylene triamine pentacetic acid (DTPA)-key sky blood is blue in the present embodiment
The synthetic method of protein conjugate are as follows: take 0.8-20 mg bifunctional chelating agent to be dissolved in 50 μ L dimethyl sulfoxides, prepare 16-400
The p-NH of mg/mL2-Bn-DTPA；2.0-20.0 mg key sky hemocyanin is taken to be dissolved in 1 mL HEPES buffer solution or carbonate
Buffer prepares the carrier protein solution of 2-20 mg/mL；P-NH is added dropwise in 1 mL, 1% glutaraldehyde solution2-Bn-DTPA
In solution, left at room temperature over night is to activate amino.The haptens solution of activation is added dropwise to the key of 1 mL 2-20 mg/mL
In empty hemocyanin solution, 24 h of oscillating reactions under the conditions of 4 DEG C or room temperature, pH7.5-10.5；With the method for ultrafiltration or dialysis
Unreacted lower-molecular substance is removed, diethylene triamine pentacetic acid (DTPA)-key sky limpet hemocyanin conjugate is obtained, is i.e. DTPA's is completely anti-
It is former.
In the present embodiment, using the method for colloid gold test paper detection diethylene triamine pentacetic acid (DTPA) and its chelate are as follows: by sample
Product instill in reagent card hole, and when concentration is low in the sample for diethylene triamine pentacetic acid (DTPA) and its chelate, colloidal gold antibody is in layer
The diethylene triamine pentacetic acid (DTPA) conjugate that can be fixed on reaction film during analysis combines, in test section (T) quality control region (C)
It will appear each red stripes.If diethylene triamine pentacetic acid (DTPA) and its chelate, when concentration is high in the sample, colloidal gold is anti-
Body is all combined with diethylene triamine pentacetic acid (DTPA) and its chelate, thus because competitive reaction will not be with divinyl in the area (T)
Pentaacetic acid conjugate is combined without there are red stripes；Negative sample is in the detection process due to lacking antigen-antibody competition
Reaction, it will red stripes occur in the area (T) and the area (C)；
It is positive: when the area (C) shows red stripes, and the area (T) does not develop the color, to be judged to the positive；
Negative: when the area (C) shows red stripes, the area (T) also shows that red stripes simultaneously, and (T) area's color is close
(C) line or when being shallower than (C) line, is judged to the positive；
Invalid: whether the area (C) does not show red stripes, then no matter the area (T) shows red stripes, which sentences
It is invalid.
Testing result is as shown in Figure 2.
P-NH described in the present embodiment2- Bn-DTPA is bought in SIGMA company, the U.S., purity: >=94%
Embodiment 3
Diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object in the present embodiment, i.e. diethylene triamine pentacetic acid (DTPA)-ox blood are pure
The synthetic method of protein conjugate are as follows: can prepare according to the following steps: 0.8-20 mg bifunctional chelating agent is taken to be dissolved in 50 μ L diformazans
Base sulfoxide prepares the P-SCN-Bn-DTPA of 16-400 mg/mL；2.0-20.0 mg bovine serum albumin(BSA) is taken to be dissolved in 1 mL
HEPES buffer solution or carbonate buffer solution prepare the bovine serum albumin solution of 2-20 mg/mL；By this haptens solution by
It is added dropwise into the bovine serum albumin solution of 1 mL 2-20 mg/mL, is vibrated under the conditions of 4 DEG C or room temperature, pH7.5-10.5
React 24 h；Unreacted lower-molecular substance is removed with the method for ultrafiltration or dialysis, obtains diethylene triamine pentacetic acid (DTPA)-carrier egg
White conjugate, the i.e. comlete antigen of DTPA.
Embodiment 4
Diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object is diethylene triamine pentacetic acid (DTPA)-cow's serum in the present embodiment
The synthetic method of albumin conjugate are as follows: take 0.8-20 mg bifunctional chelating agent to be dissolved in 50 μ L dimethyl sulfoxides, prepare 16-
The p-NH of 400 mg/mL2-Bn-DTPA；2.0-20.0 mg bovine serum albumin(BSA) is taken to be dissolved in 1 mL HEPES buffer solution or carbon
Phthalate buffer prepares the bovine serum albumin solution of 2-20 mg/mL；P- is added dropwise in 1 mL, 1% glutaraldehyde solution
NH2In-Bn-DTPA solution, left at room temperature over night is to activate amino.The haptens solution of activation is added dropwise to 1 mL 2-
In the bovine serum albumin solution of 20 mg/mL, 24 h of oscillating reactions under the conditions of 4 DEG C or room temperature, pH7.5-10.5；Use ultrafiltration
Or the method for dialysis removes unreacted lower-molecular substance, obtains diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object, i.e. DTPA
Comlete antigen.
Coherent detection test:
The detection of diethylene triamine pentacetic acid (DTPA) in test one, sample
1.0g (being accurate to ± 0.01g) sample is weighed in 50mL boiling flask, after suitable quantity of water dissolution or homogenate, filtering
Or 3000r/min is centrifuged 5-10min, takes supernatant, using the glue of detection diethylene triamine pentacetic acid (DTPA) and its chelate of the invention
The detection of body gold test paper point sample, the color of the interior observation detection line of 30s.When the concentration of diethylene triamine pentacetic acid (DTPA) and its chelate reaches
When 1200ng/ml, detection line color still naked eyes are visible.Within this method time-consuming 15min, the rate of recovery is up to 95% or more.
The detection of diethylene triamine pentacetic acid (DTPA) in test two, sample
1.0g (being accurate to ± 0.01g) sample is weighed in 50mL boiling flask, after suitable quantity of water dissolution or homogenate, filtering
Or 3000r/min is centrifuged 5-10min, takes supernatant, with formic acid tune pH to 0.5-2.0, filtering or 3000r/min are centrifuged 5-10min,
Precipitating is collected with the dissolution of HEPES buffer solution pH 4.5；Detect the colloid gold test paper point of diethylene triamine pentacetic acid (DTPA) and its chelate
Sample detection, the color of the interior observation detection line of 30s.When the concentration of diethylene triamine pentacetic acid (DTPA) and its chelate reaches 1200ng/ml
When, detection line color still naked eyes are visible.Within this method time-consuming 15min, the rate of recovery reaches 85%-95%.
Claims (4)
1. the colloid gold test paper of a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate, including reaction film, sample pad, conjugate
Release pad, water absorption pad and backing, it is characterised in that: there is coating diethylene triamine pentacetic acid (DTPA)-carrier protein on the reaction film
The test section of conjugate and the quality control region of coating sheep anti-mouse igg, the conjugate release pad are coated with diethylene triamine pentacetic acid (DTPA) list
Clonal antibody-colloid gold label object；
The diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object is diethylene triamine pentacetic acid (DTPA)-bovine serum albumin(BSA) coupling
Object or diethylene triamine pentacetic acid (DTPA)-keyhole limpet hemocyanin conjugate；
Preparation method are as follows: take 0.8-20mg p-NH2- Bn-DTPA is dissolved in 50 μ L dimethyl sulfoxides, prepares 16-400mg/mL's
p-NH2-Bn-DTPA；Take 2.0-20.0 mg keyhole limpet hemocyanin or bovine serum albumin(BSA) be dissolved in 1mL HEPES buffer solution or
Carbonate buffer solution is configured to the carrier protein solution of 2-20mg/mL；P-NH is added dropwise in 1% glutaraldehyde solution of 1mL2-
In Bn-DTPA solution, left at room temperature over night is to activate amino；The haptens solution of activation is added dropwise to 1mL 2-20 mg/
In the carrier protein solution of mL, oscillating reactions is for 24 hours under the conditions of 4 DEG C or room temperature, pH7.5-10.5；With the method for ultrafiltration or dialysis
Unreacted lower-molecular substance is removed, diethylene triamine pentacetic acid (DTPA)-keyhole limpet hemocyanin conjugate or diethylenetriamine five are obtained
Acetic acid-bovine serum albumin(BSA) conjugate；
Diethylene triamine pentacetic acid (DTPA) monoclonal antibody in the diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object
Be using diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object as immunizing antigen immunity test animal after prepare.
2. the preparation method of the colloid gold test paper of detection diethylene triamine pentacetic acid (DTPA) and its chelate as described in claim 1,
It is characterized in that, comprising the following steps:
1) preparation coating diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has coating diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object test section and is coated with the matter of sheep anti-mouse igg
Control the reaction film in area；
And 2) 3) 1) conjugate release pad, reaction film and the sample pad, water absorption pad and the backing that prepare are assembled into test paper.
3. the preparation method of the colloid gold test paper of detection diethylene triamine pentacetic acid (DTPA) and its chelate as claimed in claim 2,
It is characterized in that: the following steps are included:
(1) by difunctional diethylene triamine pentacetic acid (DTPA) and carrier protein couplet, diethylene triamine pentacetic acid (DTPA)-carrier protein is formed
Conjugate；
(2) mouse is immunized with diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object, mouse boosting cell and mouse myeloma is thin
Born of the same parents obtain the monoclonal antibody hybridoma cell strain of secretion diethylene triamine pentacetic acid (DTPA) by fusion, screening；Culture secretion second two
The monoclonal antibody hybridoma cell strain of ethylene pentaacetic acid, obtains the monoclonal antibody of diethylene triamine pentacetic acid (DTPA)；
(3) it is reacted with trisodium citrate with gold chloride and prepares colloidal gold；
(4) the diethylene triamine pentacetic acid (DTPA) monoclonal antibody of preparation is added in the colloidal gold of step (3) preparation, obtains diethyl
Alkene pentaacetic acid monoclonal antibody-colloid gold label object；
(5) diethylene triamine pentacetic acid (DTPA) monoclonal antibody-colloid gold label object is coated in conjugate release pad, with containing
The buffer that the disodium hydrogen phosphate and sodium dihydrogen phosphate of 0.1-1.5% ovalbumin are prepared soaks 30 seconds, and it is spare that 37 oC dry 2h；
(6) diethylene triamine pentacetic acid (DTPA)-carrier protein couplet object is coated on reaction film and constitutes test section, and by sheep anti mouse
IgG, which is coated on reaction film, constitutes quality control region, is closed with the confining liquid containing 0.1% cow's serum；
(7) by sample pad: rabbit serum proteins, 1-1.5% Tween-20 disodium hydrogen phosphate and the sodium dihydrogen phosphate of 0.1-1.5% is matched
The buffer of system impregnates 2h, 37 DEG C of baking 2h, spare；
(8) it the assembling of diethylene triamine pentacetic acid (DTPA) and its chelating analyte detection colloid gold test paper: is successively glued in order on backing
Reaction enclosure film, water absorption pad, conjugate release pad and sample pad are then cut into the small item of 3mm wide, add plastic casing, vacuum packaging.
4. using the method for the detection diethylene triamine pentacetic acid (DTPA) of colloid gold test paper described in claim 1 and its chelate, feature
It is: sample is instilled in reagent card hole, when concentration is low in the sample for diethylene triamine pentacetic acid (DTPA) and its chelate, colloidal gold
Antibody can be fixed on the combination of the diethylene triamine pentacetic acid (DTPA) conjugate on reaction film in chromatography process, in test section and Quality Control
It will appear each red stripes in area；If diethylene triamine pentacetic acid (DTPA) and its chelate are when concentration is high in the sample, colloid
Golden antibody is all combined with diethylene triamine pentacetic acid (DTPA) and its chelate, thus because competitive reaction will not be with two in test section
Ethylene pentaacetic acid conjugate is combined without there are red stripes；Negative sample is in the detection process due to lacking antigen-antibody
Competitive reaction, it will occur red stripes in Qu Yuqu；
When quality control region shows red stripes, and test section is not developed the color, it is judged to the positive；
When quality control region shows red stripes, test section also shows that red stripes simultaneously, and test section color is close to nature controlling line
Or when being shallower than nature controlling line, it is judged to the positive；
Whether quality control region does not show red stripes, then no matter test section shows red stripes, which is judged in vain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710542325.6A CN107192826B (en) | 2017-07-05 | 2017-07-05 | The colloid gold test paper and its preparation and detection method of a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710542325.6A CN107192826B (en) | 2017-07-05 | 2017-07-05 | The colloid gold test paper and its preparation and detection method of a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107192826A CN107192826A (en) | 2017-09-22 |
| CN107192826B true CN107192826B (en) | 2019-04-16 |
Family
ID=59881409
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710542325.6A Expired - Fee Related CN107192826B (en) | 2017-07-05 | 2017-07-05 | The colloid gold test paper and its preparation and detection method of a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107192826B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108383903B (en) * | 2018-02-09 | 2021-01-15 | 浙江工商大学 | Preparation method of heavy metal mercury artificial antigen and application of DOTA in preparation of heavy metal mercury artificial antigen reagent |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948807A (en) * | 2010-08-24 | 2011-01-19 | 北京市农林科学院 | Heavy metal mercury ion antibody and application thereof |
| CN102260347A (en) * | 2011-06-13 | 2011-11-30 | 上海交通大学 | Synthesis and application method of antigen for multiple heavy metals |
| CN102692503A (en) * | 2012-01-15 | 2012-09-26 | 河南科技大学 | Colloidal gold test paper card for detecting dimethyl phthalate and preparation method of colloidal gold test paper card |
| CN104749364A (en) * | 2013-12-25 | 2015-07-01 | 深圳先进技术研究院 | Immune analysis method and immune analysis kit for detecting zinc |
-
2017
- 2017-07-05 CN CN201710542325.6A patent/CN107192826B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948807A (en) * | 2010-08-24 | 2011-01-19 | 北京市农林科学院 | Heavy metal mercury ion antibody and application thereof |
| CN102260347A (en) * | 2011-06-13 | 2011-11-30 | 上海交通大学 | Synthesis and application method of antigen for multiple heavy metals |
| CN102692503A (en) * | 2012-01-15 | 2012-09-26 | 河南科技大学 | Colloidal gold test paper card for detecting dimethyl phthalate and preparation method of colloidal gold test paper card |
| CN104749364A (en) * | 2013-12-25 | 2015-07-01 | 深圳先进技术研究院 | Immune analysis method and immune analysis kit for detecting zinc |
Non-Patent Citations (1)
| Title |
|---|
| 衍生化－气相色谱－质谱法测定染整助剂中的乙二胺四乙酸盐和二乙烯三胺五乙酸盐;汤娟等;《分析试验室》;20160930;1019-1023 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107192826A (en) | 2017-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101477124B (en) | Method for detecting melamine and its special test paper | |
| CN101661043A (en) | Method for detecting fumonisin by colloidal gold immunochromatographic test | |
| CN101477123B (en) | ELISA reagent kit for detecting two oxyethyl group thiophosphate organophosphorus pesticide | |
| CN103575889A (en) | Test strip and method for detecting vancomycin | |
| CN102879574A (en) | Bar/pat gene transfer plant and rapid test strip for derivative product of bar/pat transgenic plant | |
| CN102876634B (en) | PMSG (pregnant mare serum gonadotropin) double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) kit | |
| CN106918705B (en) | Test paper for detecting fenpropathrin and application thereof | |
| CN103575887A (en) | Test paper card for detecting aflatoxin B1 and application of test paper card | |
| CN112142833B (en) | Furosemide artificial antigen, antibody and application thereof in detection of furosemide | |
| CN107192826B (en) | The colloid gold test paper and its preparation and detection method of a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate | |
| CN103995107B (en) | A kind of method detecting lincomycin and special test paper thereof | |
| CN103728449A (en) | Test paper and method for detecting florfenicol and thiamphenicol | |
| CN102520179A (en) | Quantitative detection method of fumonisins B1 | |
| CN103105490B (en) | A kind of kit and method detecting tetracycline medication | |
| CN103376316A (en) | Test strip for detecting streptomycin and method | |
| CN103364546A (en) | Kit for detecting furazolidone metabolin and method thereof | |
| CN102520177A (en) | Method for quantitative detection of zearalenone | |
| CN202486140U (en) | Kit for detecting tetracyclines antibiotic | |
| CN201181296Y (en) | Colloidal gold test paper card for detecting streptomycin medicine residue | |
| CN105440137B (en) | The antibody of anti-Ractopamine and its application | |
| CN103364555A (en) | Kit and method for detecting furacilin metabolin | |
| CN107247142B (en) | The colloid gold test paper and its preparation and detection method of a kind of detection ethylenediamine tetra-acetic acid and its chelate | |
| CN105092849B (en) | A kind of test strips detecting phenylethanolamine A and method | |
| CN111454912A (en) | Cyperazine monoclonal antibody hybridoma cell strain and application thereof | |
| CN104330569A (en) | Test strip for quickly detecting antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190416 Termination date: 20200705 |