CN101738482A - Immunochromatographic test paper for testing abrin and preparation method thereof - Google Patents
Immunochromatographic test paper for testing abrin and preparation method thereof Download PDFInfo
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Abstract
The invention discloses immunochromatographic test paper for testing abrin and a preparation method thereof. The preparation method comprises the following steps of: preparing the gold colloid pad of the immunochromatographic test paper by marking colloidal gold by a mouse source monoclonal antibody which is specifically bound with the chain A of the abrin; coating a rabbit source anti-abrin polyclonal antibody on a nitrocellulose membrane serving as a testing line (T line); and assembling the T line and the gold colloid pad to form the immunochromatographic test paper. The test paper is suitable for an on-site test for a clinical test and emergency and an epidemiological survey, has the advantages of simple operation, high sensitivity, good stability, convenience, rapidness and the like, and has significance and an actual application value for the differential diagnosis of abrin toxication and timely cure.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of immune chromatography test paper that detects abrin and preparation method thereof.
Background technology
Abrin (abrin) is a kind of toxalbumin that is present in legume jequirity (the Abrus Precatorius L.) seed, it is to be the strongest a kind of phytotoxin of this toxicity of finding so far, toxicity is the hundred times of general chemistry weapon, the lethal dose that the adult takes in is 5.0～7.0 μ g/Kg, only need chew the seed of swallowing after mashed can die like a rat, and caused the serious device matter infringement of body when symptom occurring, bringing great difficulty for the treatment and the prevention of poisoning, is a kind of potential biotoxin weapon.Abrin is formed by connecting through a disulfide bond by two polypeptied chains of A, B, wherein abrin A chain has the N glycosidase activity, N-C glycosidic bond that can the 4324th adenylate of single-minded hydrolysis ribosomes 28S and make the ribosomes inactivation, and then influence the synthetic of protein.Abrin B chain is a kind of agglutinin that galactose is had special affinity, can contain the receptors bind of galactose residue with cell surface.As one of II type ribosome inactivating protein (RIP) member, the toxic action of abrin mechanism is similar to ricin (WA), but its toxicity is 75 times of ricin (WA), and the abrin antitumor activity also obviously is better than ricin (WA).Utilize abrin and abrin A chain to prepare immunotoxin and be used for the focus that tumor treatment is recent research, therefore the research of carrying out the abrin detection technique has crucial meaning.
Less about the biologic assay and the physical chemistry checking method report of abrin both at home and abroad, immunology detection mainly contains radioimmunoassay method (RIA) and enzyme linked immunosorbent assay (ELISA).The RIA technology mainly is by using radioelement
125The antibody of I mark takes the abrin in the sample such as detection by quantitative blood, the detection sensitivity height, and the I of abrin is surveyed concentration and is reached 50～100pg/mL in the blood plasma.Its shortcoming is to have the radiocontamination problem.The ELISA method detects that abrin has higher sensitivity and specificity but operating process is loaded down with trivial details, and the result judges length consuming time, is not suitable for on-the-spot the detection.Therefore, press for fast sensitivity, the method for the detection abrin that specificity is high.
Summary of the invention
The invention provides a kind of immune chromatography test paper that detects abrin, have high specificity, highly sensitive,, characteristics such as simple to operation.
The present invention also provides the preparation method of above-mentioned immune chromatography test paper, is suitable for suitability for industrialized production.
Detection abrin immune chromatography test paper of the present invention is characterized in that:
Be with the gold size pad of specificity in conjunction with immuno-chromatographic test paper strip among mouse resource monoclonal antibody mark Preparation of Colloidal Gold the present invention of abrin A chain, with the polyclonal antibody bag of the anti-abrin in rabbit source by on nitrocellulose filter as detection line (T line), with the polyclonal antibody bag of sheep anti-mouse igg by on nitrocellulose filter as nature controlling line (C line), be equipped to immuno-chromatographic test paper strip of the present invention together with the gold size pad.
The preparation method that the present invention detects the abrin immune chromatography test paper is as follows:
With the mouse resource monoclonal antibody mark Preparation of Colloidal Gold gold size pad of specificity in conjunction with abrin A chain; With the polyclonal antibody bag of the anti-abrin in rabbit source by on nitrocellulose filter as detection line (T line); With the polyclonal antibody bag of sheep anti-mouse igg by on nitrocellulose filter as nature controlling line (C line), promptly get and detect test paper.
Mainly may further comprise the steps:
(1) abrin that will purifying prepares from jequirity with 1% formaldehyde attenuation treatment after immune Balb/C mouse prepare monoclonal antibody;
(2) filter out the monoclonal antibody of specificity in the monoclonal antibody that adopts the Westerblot method from step (1), to prepare in conjunction with abrin A chain;
(3) the labeling of monoclonal antibody collaurum that obtains with (2) is prepared into colloidal gold probe, it is sprayed on makes the collaurum pad on the glass fibre membrane;
(4) abrin that will purifying prepares from jequirity with 1% formaldehyde attenuation treatment after immunizing rabbit prepare the anti-abrin polyclonal antibody in rabbit source;
(5) with the polyclonal antibody bag of rabbit source anti-abrin by on nitrocellulose filter as detection line (T line);
(6) with the polyclonal antibody bag of sheep anti-mouse igg by on nitrocellulose filter as nature controlling line (C line);
(7) with sample pad, pad, nitrocellulose filter, absorbent filter by being fixed in successively on the PVC lamina membranacea from top to bottom, be prepared into immune chromatography test paper.
The above-mentioned abrin immune chromatography test paper that the present invention relates to, the preparation method of abrin wherein: the jequirity processing of shelling, remove kind of a benevolence 200g, be dipped in the 1L 5% cold acetic acid, 4 ℃ are spent the night, high-speed homogenization is handled, the centrifugal 20min of 10000g/min, supernatant is used Sepharose 4B affinitive layer purification with 35%～95% ammonium sulfate precipitation, resolution of precipitate in 5mmol/L PB (pH8.0) solution, purified product is crossed DEAE-Sepharose FF ion exchange column, collect first eluting peak and be abrin, adding final concentration after desalting processing is 0.01% thimerosal sodium salt ,-20 ℃ of preservations.
The above-mentioned abrin immune chromatography test paper that the present invention relates to, the preparation method of polyclonal antibody of the anti-abrin in rabbit source wherein: abrin is through the formaldehyde attenuation treatment, divide three immunizing rabbits, first immunisation dosage is 500 μ g/, booster immunization carries out after 2 weeks in first immunisation, dosage is 300 μ g/, booster immunization 2 weeks of every interval carry out once, 8 Zhou Houyong do not contain the toxoid solution of adjuvant and do superpower immunity immunity through the ear vein injection, dosage is 800 μ g/, week back cardiac puncture blood sampling, separation of serum, serum is handled through the 50% ammonium sulfate precipitation precipitation method, the centrifugal 20min of 5000g/min abandons supernatant, and precipitation is resuspended with PBS (0.02mol/L pH7.0), be the polyclonal antibody of slightly carrying, crude extract prepares highly purified antibody with Hitrap rProtein A FF or Hitrap rProtein G FF prepacked column.
The above-mentioned abrin immune chromatography test paper that the present invention relates to, wherein specificity is in conjunction with the mouse resource monoclonal antibody preparation method of abrin A chain: abrin is with 1% formaldehyde attenuation treatment, Balb/C mouse immune to 6～8 ages in week, pass through Fusion of Cells, the ELISA screening, the clone, and adopt the Westerblot method to identify its specificity, the Westerblot that learns from else's experience is accredited as and can secretes the cell line of specificity in conjunction with the mouse resource monoclonal antibody of abrin A chain, adopt external evoked method, prepare ascites with injecting for 6～8 ages in week after the cell strain of monoclonal antibody enlarged culture in the Balb/C mouse peritoneal, the ascites of preparation is after ammonium sulfate salting-out process is slightly carried, be further purified with Hitrap rProtein A FF gel affinity column, promptly obtain highly purified monoclonal antibody.
The present invention prepares colloidal gold probe with specificity in conjunction with the mouse resource monoclonal antibody of abrin A chain, with the anti-abrin polyclonal antibody in rabbit source as detection line, adopt the double antibody sandwich method principle to detect abrin, both strengthen its detection sensitivity, also kept the characteristics of its high specificity.
The good effect that the present invention detects the immune chromatography test paper of abrin is: detection specificity height, highly sensitive, good stability, convenient, fast.Have simple to operate, detect fast, the result is easy to judge and convenient the preservation, and need not advantage such as any instrument and equipment, the scene that is particularly suitable for clinical and accident is detected, and is fit to the epidemiology survey large-scale application.
Description of drawings
Fig. 1 is abrin monoclonal antibody Westerblot qualification result figure.
Wherein the 1st swimming lane is low-molecular-weight Marker; 2nd, 3 swimming lanes are that complete abrin Westerblot detects the colour developing result; 4th, 5 swimming lanes are abrin after beta-mercaptoethanol is handled, and abrin A chain Westerblot detects the colour developing result.
Fig. 2 is the testing result figure of colloidal gold strip.
1, negative findings; 2,5ng/mL toxin sample detection result; 3,50ng/mL toxin sample detection result.
Embodiment
The following example is intended to further illustrate, rather than restriction the present invention.It will be appreciated by those skilled in the art that, under the prerequisite that does not deviate from the spirit and principles in the present invention, all will fall in the claim scope that awaits the reply of the present invention any parallel change of the present invention and change.
Embodiment 1
Abrin Antibody Preparation and evaluation
1, the preparation of abrin
Adopt slightly poison of 5% acetic acid extracting, through high speed centrifugation (10,000g, abandon sediment 20min), supernatant is with 35%～95% saturated ammonium sulfate fractional precipitation, with sediment with the PBS 72h that fully dialyses, the gained extract of crude toxin adopts Sepharose 6B or Sepharose 4B agarose affinity chromatography post, DEAE-Sepharose FF anion-exchange column and Hiload 26/60Superdex75 gel permeation chromatography prepacked column further separate, the result adopts SDS-PAGE electrophoresis analyzing molecules amount, gel thin-layer sweep measuring purity, the N terminal amino acid sequence is measured and mass spectrum peptide fingerprint map analyzing is identified, the result shows and prepares high-purity abrin.
2, specificity is in conjunction with the preparation of the mouse resource monoclonal antibody of abrin A chain
Abrin is behind 1% formaldehyde phosphate buffer attenuation treatment 24h, and immune Balb/C mouse prepares monoclonal antibody.The immunity immune programme for children is: first immunisation: get 100 μ L antigens (50 μ g) and the complete Fei Shi adjuvant of 100 μ L mixing, subcutaneous multi-point injection for every.Booster immunization: exempt from the back in head and carried out in 2 weeks, every get 100 μ L antigens (30 μ g) and the incomplete Fei Shi adjuvant of equivalent mixing after, lumbar injection.After this per 2 all immunity are once exempted from four times altogether, and last immunity docking blood sampling in back 7 days separation of serum with the former poison of 2 μ g/mL (without the hydroformylation attenuation treatment) coated elisa plate, detects anti-abrin serum antibody titer with indirect elisa method.Be higher than 1 * 10-4 if tire and promptly can be used for Fusion of Cells.Superpower immunity: the no adjuvant antigen 50 μ g of each injection of 3～4d tail vein before Fusion of Cells, 200 μ L/ only.Revulsion in the body is adopted in the monoclonal antibody hybridoma cell strain of adopting the mouse hybridoma cell technology to prepare anti-abrin, respectively hybridoma is inoculated in the Balb/c mouse and has prepared ascites; Detect the subclass of mAb with mouse mAb subgroup identification kit, ascites is after ammonium sulfate salting-out process is slightly carried, adopt Hitrap rProtein A FF affinity column or Hitrap rProtein G FF affinity column purifying, purity is identified in the SDS-PAGE gel electrophoresis, and the BCA method is measured its concentration; Indirect elisa method detects ascites and tires and be 1:3.2 * 105, and with abrus agglutinin, ricin (WA) and three kinds of equal no cross reactions of analog of ricinus agglutinin; Adopt the Westerblot method to identify its specificity, the result shows that but preparation-obtained monoclonal antibody specificity is in conjunction with abrin A chain, with equal no cross reaction such as abrus agglutinin, ricin (WA) and ricinus agglutinin, show this monoclonal antibody specificity height.See Fig. 1: among the figure, the 1st swimming lane, low-molecular-weight Marker; 2nd, 3 swimming lanes, complete abrin Westerblot detect the colour developing result; 4th, 5 swimming lanes, abrin are after beta-mercaptoethanol is handled, and abrin A chain Westerblot detects the colour developing result.
3, the anti-abrin Polyclonal Antibody Preparation in rabbit source
Abrin is prepared into the abrin toxoid through the formaldehyde attenuation treatment.With this toxoid through subcutaneous and intramuscular injection immunizing rabbit, first immunisation dosage is 500 μ g/, get the equal-volume complete Freund's adjuvant and mix also emulsification fully with toxoid solution after the hypodermic injection immunity, reinforced immunological carries out after injecting for 2 weeks first, be two weeks immune interval time, dosage is 300 μ g/, get that the equal-volume incomplete Freund's adjuvant mixes with toxoid solution and fully emulsified after subcutaneous or intramuscular injection is immune, after 8 weeks of immunity, do superpower immunity with the toxoid solution that does not contain adjuvant through the ear vein injection, last immune 1 week is after heart blood sampling preparation antiserum.Serum is handled through 50% ammonium sulfate precipitation method, the centrifugal 20min of 5000g/min, abandon supernatant, precipitation is resuspended with PBS (0.02mol/L pH7.0), be the polyclonal antibody of slightly carrying, crude extract prepares highly purified antibody with Hitrap rProtein A FF or Hitrap rProtein G FF gel affinity chromatography.The anti-abrin polyclonal antibody in rabbit source adopts agar immunodiffusion and ELISA method to identify, the result shows and abrus agglutinin, ricin (WA) and the equal no cross reaction of ricinus agglutinin.
Embodiment 2
The preparation of colloidal gold probe
1, the preparation of colloid gold particle
Get one of the 250mL triangular flask of silication, add 100mL deionized water and 1mL 1% gold chloride, ebuillition of heated; Stir the accurate down 1.5mL1% of adding trisodium citrate, continue to boil 15min, the cooling back returns to the colloid gold particle that original volume can obtain particle diameter 25nm with deionized water.By regulating the colloid gold particle that the amount that adds 1～2mL1% trisodium citrate can be prepared particle diameter 15～40nm.
2, the colloid gold label specificity is in conjunction with the mouse resource monoclonal antibody of abrin A chain
Get one of the 250mL triangular flask of silication, add 100mL particle diameter 25nm collaurum; Add an amount of 0.2mol/LK2CO3 pH is adjusted into 8.7, slowly dropwise adding specificity is 1mg/mL in conjunction with mouse resource monoclonal antibody to the antibody final concentration of abrin A chain, places 15～25min on the room temperature shaking table; Add 10%BSA to final concentration be 5%, prevent antibody protein and collaurum polymerization and precipitation.
3, the preparation of gold mark pad
The plain film of glass fibre is cut into 0.8 * 30cm/ bar in Biohazard Safety Equipment, stand-by after 4 ℃ of vacuum are drained.The colloidal gold probe that purifying is good is uniformly coated on the plain film of glass fibre by the amount of 4mL/ bar, and aeration-drying is spent the night in Biohazard Safety Equipment, and drying condition is preserved standby down.
Detect the immune chromatography test paper assembling and the test of abrin
1, the preparation of detection line and nature controlling line on the nitrocellulose filter
It is 1mg/mL, 1.5mg/mL that anti-abrin polyclonal antibody in rabbit source that purifying is good and sheep anti-mouse igg polyclonal antibody are diluted to final concentration respectively with PBS (0.01mol/mL.pH7.5) solution.The rabbit source anti-abrin polyclonal antibody solution that dilution is the good BIODOT that packs into draws film machine shower nozzle 2, be fixed on the position of nitrocellulose filter lower limb 1.1cm, the sheep anti-mouse igg polyclonal antibody that dilution the is good BIODOT that packs into draws film machine shower nozzle 1, is fixed on the position of nitrocellulose filter lower limb 1.6cm.Parameter is 1.0 μ L/cm and is sprayed on the nitrocellulose membrane.Will spray good nitrocellulose membrane vacuum drying 2 hours (24 ℃ of relative humidity are 40% below), outward appearance, length and drying regime etc. on inspection, the semi-manufacture lot number is indicated in the aluminium foil bag sealing of qualified back, places the preservation of material working area.
2, the assembling of immune chromatography test paper
The assemble method of immune chromatography test paper: every operation all carries out under free from dust, aseptic, no oarse-grained situation, get size and be the PVC base plate of 30cm * 80mm, nitrocellulose filter (30cm * 25mm) be assembled on the viscosity PVC base plate with coated antibody, require its lower limb must align density bullet line on the mould and careful floating face.With adsorptive pads (30cm * 35mm) be assembled on the adhesive base, and carefully floating near the mould coboundary.To be coated with the plain film of golden labeling antibody glass fibre (30cm * 8mm) be assembled on the adhesive base, and carefully floating near the scale lower limb.With sample pad (30cm * 25mm) be assembled on the adhesive base, and carefully floating near the mould lower limb.Be cut into the wide test paper of 4.5mm with cutting cutter, in the assembly section test paper that cuts merged 0.5g drying agent one bag and put into packaging bag ,-20 ℃ of preservations.
3, test result
(1) sample preparation
Tracer liquid breast sample: look sample concentration and viscosity and do suitable dilution with 0.01mol/L PBS (pH7.5) solution, gained solution promptly can be used as detection liquid.
Detect blood sample: 1 volume blood sample adds 0.01mol/L PBS (pH7.5) solution of 1 volume, and static 10 minutes of mixing is got supernatant and promptly be can be used as detection liquid after the centrifugal treating.
Detect foodstuff samples: food is cooked 0.01mol/L PBS (pH7.5) the solution mixing that adds appropriate amount after the pulverization process, and static 30min under the room temperature gets supernatant and promptly can be used as detection liquid after the centrifugal treating.
(2) pattern detection
Get a toxin sample drop to be checked and be added in the sample application zone of the test strips for preparing, sample begins to spread on nitrocellulose filter, treat that gold mark pad discharges fully after, T line and C line clearly appear on the nitrocellulose filter.When not containing abrin in the sample, detection line is colourless and nature controlling line has color when detecting with this test strips.See Fig. 2.
Among Fig. 2, test paper 1: negative control; Test paper 2:5ng/mL; Test paper 3:50ng/mL.Wherein a is a detection line; B is a nature controlling line.
Embodiment 4
The mensuration and the practice of abrin immune chromatography test paper performance
1, detects the specific mensuration of abrin immune chromatography test paper
When the detection abrin immune chromatography test paper of application preparation detects the abrus agglutinin nearer with the character of abrin molecular structure, ricin (WA), ricinus agglutinin, the result is all negative, show and they between do not have cross reaction, the test strips specificity is good.
2, detect abrin immune chromatography test paper susceptibility
Use above-mentioned two kinds of antibody, when adopting the double-antibody sandwich elisa method to detect the abrin sample, lowest detection is limited to 1ng/mL.Detect the abrin immune chromatography test paper with the present invention, it is unintelligible that test strips detected colour developing when abrin concentration was lower than 5ng/mL in the sample; The detection line colour developing is clear but more of light color than nature controlling line when the toxin sample concentration is between 5～40ng/mL; When the color of toxin sample concentration detection line when 40ng/mL is above darker than nature controlling line color.The sensitivity of immune chromatography test paper is a little less than the ELISA method.
3, detect abrin immune chromatography test paper repeatability
Get same sample and carry out revision test five times, testing result is consistent, illustrates that this method has repeatability.
4, detect abrin immune chromatography test paper stability
(1) same batch immune chromatography test paper all carries out the detection of test strips every day during 37 ℃ of failure tests, and the intensity of test strips color decreases in the time of the 12nd day.Illustrate that test strips can preserve 11 days at 37 ℃.
(2) same batch immune chromatography test paper in 4 ℃ preserve 6 months during, the develop the color color of band of the result of Ce Dinging is all very clear weekly, color intensity decreases in the time of six month; Illustrate that test strips can preserve 5 months at 4 ℃, sensitivity descends to some extent after the 6th month.
(3) during 11 months, respectively every two weeks one detects same batch immune chromatography test paper in-20 ℃ of preservations, and color intensity decreases in the time of 10th month; Illustrate that test strips can preserve 10 months at-20 ℃, sensitivity descends to some extent after the 11st month.
5, detect abrin immune chromatography test paper manual simulation pattern detection
Manual simulation's sausage toxin sample detection is carried out discrimination test with the immune chromatography test paper of ELISA test and preparation, and the result shows that the coincidence rate of two kinds of detection methods is 100% when toxin sample concentration during greater than 5ng/mL.
Manual simulation's milk toxin sample detection is carried out discrimination test with the immune chromatography test paper of ELISA test and preparation, and the result shows that the coincidence rate of two kinds of detection methods is 100% when toxin sample concentration during greater than 5ng/mL.
Claims (2)
1. immune chromatography test paper that detects abrin, it is characterized in that: with the mouse resource monoclonal antibody mark Preparation of Colloidal Gold gold size pad of specificity in conjunction with abrin A chain, with the polyclonal antibody bag of the anti-abrin in rabbit source by on nitrocellulose filter as detection line (T line), with the polyclonal antibody bag of sheep anti-mouse igg by on nitrocellulose filter as nature controlling line (C line).
2. the preparation method of the described immune chromatography test paper of claim 1 may further comprise the steps:
(1) abrin that will purifying prepares from jequirity with 1% formaldehyde attenuation treatment after immune Balb/C mouse prepare monoclonal antibody;
(2) filter out the monoclonal antibody of specificity in the monoclonal antibody that adopts the Westerblot method from step (1), to prepare in conjunction with abrin A chain;
(3) the labeling of monoclonal antibody collaurum that obtains with (2) is prepared into colloidal gold probe, it is sprayed on makes the collaurum pad on the glass fibre membrane;
(4) abrin that will purifying prepares from jequirity with 1% formaldehyde attenuation treatment after immunizing rabbit prepare the anti-abrin polyclonal antibody in rabbit source;
(5) with the polyclonal antibody bag of rabbit source anti-abrin by on nitrocellulose filter as detection line (T line);
(6) with the polyclonal antibody bag of sheep anti-mouse igg by on nitrocellulose filter as nature controlling line (C line);
(7) with sample pad, pad, nitrocellulose filter, absorbent filter by being fixed in successively on the PVC lamina membranacea from top to bottom, be prepared into immune chromatography test paper.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101887061A (en) * | 2010-07-26 | 2010-11-17 | 东北农业大学 | Colloidal gold immuno-chromatography test paper strip used for detecting escherichia coil F5 pilus and preparation method thereof |
| CN102020714A (en) * | 2010-09-26 | 2011-04-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Colloidal gold test paper for jointly detecting abrin and ricin and special monoclonal antibody |
| CN104914224A (en) * | 2015-07-08 | 2015-09-16 | 河南省农业科学院 | Colloidal gold immunochromatographic test paper capable of quickly detecting soybean Bowman-Brik trypsin inhibiting factor and preparation method |
-
2010
- 2010-01-08 CN CN201010030819A patent/CN101738482A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101887061A (en) * | 2010-07-26 | 2010-11-17 | 东北农业大学 | Colloidal gold immuno-chromatography test paper strip used for detecting escherichia coil F5 pilus and preparation method thereof |
| CN102020714A (en) * | 2010-09-26 | 2011-04-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Colloidal gold test paper for jointly detecting abrin and ricin and special monoclonal antibody |
| CN104914224A (en) * | 2015-07-08 | 2015-09-16 | 河南省农业科学院 | Colloidal gold immunochromatographic test paper capable of quickly detecting soybean Bowman-Brik trypsin inhibiting factor and preparation method |
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Application publication date: 20100616 |