CN101819203A - Chemiluminescent kit for detecting abrin and preparation method thereof - Google Patents
Chemiluminescent kit for detecting abrin and preparation method thereof Download PDFInfo
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- CN101819203A CN101819203A CN201010132580A CN201010132580A CN101819203A CN 101819203 A CN101819203 A CN 101819203A CN 201010132580 A CN201010132580 A CN 201010132580A CN 201010132580 A CN201010132580 A CN 201010132580A CN 101819203 A CN101819203 A CN 101819203A
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Abstract
The invention discloses a chemiluminescent kit for detecting abrin and a preparation method thereof. Luminous plates are enveloped with mouse monoclonal antibodies of a specifically-bound abrin A chain, goat anti-rabbit IgG labeled by horse radish peroxidase is taken as an enzyme labeled antibody, luminal is taken as chemiluminescent substrate liquid, and then the chemiluminescent kit of the invention is formed by standard abrin protein products of which concentration is 0.5 ng/mL, 10 ng/mL, 20 ng/mL, 40 ng/mL and 80 ng/mL respectively. The chemiluminescent kit of the invention is suitable for quantitatively detecting a great number of samples clinically and in emergencies and surveying epidemic, has the characteristics of simple operation, high sensitivity, high stability, convenience, fastness and the like, and is of far-reaching importance and actual application value in the differential diagnosis and timely curing of abrin poisoning.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of chemical luminescence reagent kit that detects abrin, the present invention also provides the preparation method of this kit.
Background technology
Abrin (abrin) is a kind of toxalbumin that is present in the seed of legume jequirity (Abrus Precatorius L.), be all II type ribosome inactivating protein (RIP) family member with ricin (WA) (ricin), one of phytotoxin that the toxicity that is up to now to be found is the strongest, toxicity is the hundred times of general chemistry weapon.Because the different places of production or kind, the structure of the contained toxin of kindred plant and character may be slightly different, tell the four kinds of different toxin of abrin: abrin-a simultaneously in the jequirity that produce in Taiwan, abrin-b, abrin-c and abrin-d, relative molecular mass is between 63kDa～67kDa, wherein abrin-b and abrin-c have only more weak cytotoxic effect owing to its B chain agglutination activity is low, and abrin-a, abrin-d then has extremely strong cytotoxicity, 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, bring great difficulty for the treatment and the prevention of poisoning, 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 HPLC method is used for identification of proteins and detection specificity is better, but owing to need expensive equipment and more professional technique, is used for the laboratory confirmatory analysis usually more, is not suitable for the on-the-spot screening of batch samples.The ELISA method detect the abrin sensitivity and specificity higher, but quantitative test once was widely used in the on-the-spot screening of batch samples.Detection abrin chemical luminescence reagent kit of the present invention replaces chromogenic substrate among the traditional E LISA by detecting light signal that luminous substrate produces, can obviously improve detection sensitivity, have simultaneously special, fast, reliable characteristics.
Summary of the invention
The invention provides a kind of chemical luminescence reagent kit that detects abrin, have high specificity, characteristics such as highly sensitive, simple to operation.
The present invention also provides the preparation method of above-mentioned chemical luminescence reagent kit, is suitable for suitability for industrialized production.
Detection abrin chemical luminescence reagent kit of the present invention is characterized in that:
With specificity in conjunction with the mouse resource monoclonal antibody bag of abrin A chain by luminous plaque, be enzyme labelled antibody with the goat anti-rabbit igg of horseradish peroxidase-labeled, be chemical luminous substrate liquid with the luminol.With concentration be 0,5,10,20,40,80ng/mL abrin protein standard substance is equipped to chemical luminescence reagent kit of the present invention together.
The preparation method that the present invention detects the abrin chemical luminescence reagent kit is as follows:
Adopt gel chromatography to prepare abrin from jequirity, prepare toxoid with the formaldehyde attenuation treatment, immunizing rabbit and Balb/C mouse prepare polyclonal antibody and monoclonal antibody respectively.With specificity in conjunction with the mouse resource monoclonal antibody bag of abrin A chain by luminous plaque, be enzyme labelled antibody with the goat anti-rabbit igg of horseradish peroxidase-labeled, be chemical luminous substrate liquid with the luminol.With concentration be 0,5,10,20,40,80ng/mL abrin protein standard substance is equipped to chemical luminescence reagent kit of the present invention together.
Mainly may further comprise the steps:
(1) adopts 5% cold acetate solution extracting, and prepare slightly poison of abrin with 35%～95% ammonium sulfate precipitation; Through Sepharose 6B affinity chromatography, DEAE-Sepharose FF ion-exchange chromatography and Superdex 75 gel filtrations three steps chromatography prepare abrin again;
(2) abrin that will prepare from step (1) prepares toxoid with 1% formaldehyde attenuation treatment, is used for immune Balb/C mouse and prepares monoclonal antibody;
(3) filter out the monoclonal antibody of specificity in the monoclonal antibody that adopts the Westerblot method from step (2), to prepare in conjunction with abrin A chain;
(4) the monoclonal antibody bag that obtains with step (3) is by luminous plaque, 10% sheep blood serum sealing preparation luminous plaque;
(5) the abrin toxoid immunizing rabbit for preparing in the step (2) is prepared the anti-abrin polyclonal antibody in rabbit source;
(6) preparation chemical luminous substrate liquid, enzyme labelled antibody, dilution, 10 times of concentrated cleaning solutions;
(7) with specificity in conjunction with the mouse resource monoclonal antibody bag of abrin A chain by luminous plaque;
(8) goat anti-rabbit igg with horseradish peroxidase-labeled is an enzyme labelled antibody;
(9) utilize the middle bag of step (8) by the enzyme labelled antibody for preparing in good chemiluminescence plate and the step (9), with the luminol is chemical luminous substrate liquid, with concentration be 0,5,10,20,40,80ng/mL abrin protein standard substance is equipped to chemical luminescence reagent kit of the present invention together.
The above-mentioned abrin chemical luminescence reagent kit that the present invention relates to, the wherein preparation method of abrin:
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 with 35%～95% ammonium sulfate precipitation, resolution of precipitate is in 5mmol/L PB (pH8.0) solution, with Sepharose 4B affinitive layer purification, purified product is crossed DEAE-Sepharose FF ion exchange column, collects first eluting peak and is abrin, adding final concentration after desalting processing is 0.01% thimerosal sodium salt ,-20 ℃ of preservations.
The above-mentioned abrin chemical luminescence reagent kit that the present invention relates to, the wherein preparation method of polyclonal antibody of the anti-abrin in rabbit source:
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/, the back cardiac puncture blood sampling of one week, separation of serum, serum is handled through the 50% ammonium sulfate precipitation precipitation method, the centrifugal 20min of 5000g/min, abandon supernatant, precipitation is resuspended with PBS (0.02mol/L pH7.0), is the polyclonal antibody of slightly carrying, and crude extract prepares highly purified antibody with Hitrap rProteinA FF or Hitrap rProtein G FF prepacked column.
The above-mentioned abrin chemical luminescence reagent kit 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 with specificity in conjunction with the mouse resource monoclonal antibody bag of abrin A chain by the chemiluminescence plate, with the anti-abrin polyclonal antibody in rabbit source as enzyme labelled antibody, with the luminol is chemical luminous substrate liquid, adopt double antibody sandwich method principle assembling chemical luminescence reagent box, be used 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 chemical luminescence reagent kit of abrin is: detection specificity height, highly sensitive, good stability, convenient, fast, just can detect the jequirity poison by fast quantification in conjunction with the chemiluminescence measuring instrument.Have simple to operate, detect fast, the result is easy to judge and convenient the preservation, is suitable for clinical and detection accident.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.
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.
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 Sepharose6B or Sepharose 4B agarose affinity chromatography post, DEAE-Sepharose FF anion-exchange column and Hiload26/60Superdex75 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 if tire
-4Promptly 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 GFF affinity column purifying, purity is identified in the SDS-PAGE gel electrophoresis, and the BCA method is measured its concentration; It is 1: 3.2 * 10 that indirect elisa method detection ascites is tired
5, 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/LpH7.0), 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.
The preparation of chemical luminescence reagent kit
1, utilize the test of the anti-abrin polyclonal antibody of monoclonal antibody and rabbit concentration gradient, determine that finally monoclonal antibody best effort concentration is 4 μ g/mL, the anti-abrin antibody of rabbit best effort concentration is 2.15 μ g/mL.
2, luminous plaque preparation is diluted to 4 μ g/mL bag by luminous plaque with the monoclonal anti body and function coating buffer liquid of purifying, and seals with 10% sheep blood serum.
3, the preparation of standard items is diluted to abrin 0,5,10,20,40 respectively, 80ng/mL.
4, the preparation of the anti-abrin antibody of rabbit is got the anti-abrin antibody of purified rabbit (20mg/mL) and is diluted to 2.15 μ g/mL with dilution (0.5%BSA-PBST).
5, enzyme labelled antibody be the goat anti-rabbit igg of horseradish peroxidase-labeled available from Sigma company, face with preceding with deionized water dilution in 1: 3000.
6, the preparation of chemical luminous substrate liquid, enzyme labelled antibody, dilution, 10 times of concentrated cleaning solutions
(1) dilution: pH7.4PBS
KH
2PO
4 0.2g
Na
2HPO
4.12H
2O 2.9g
NaCl 8.0g
KCl 0.2g
BSA (bovine serum albumin(BSA)) 0.5g
(2) 10 times of concentrated cleaning solutions
KH
2PO
4 0.2g
Na
2HPO
4.12H
2O 2.9g
NaCl 8.0g
KCl 0.2g
Deionized water dissolving and constant volume value 100mL, regulating pH is 7.4, adds 1mL Tween-20
(3) chemical luminous substrate A liquid
Luminol 8.858g
To iodophenol 0.66g
Tris 12.114g
Deionized water is settled to 1L, transfers pH to 8.5 with concentrated hydrochloric acid
Chemical luminous substrate B liquid: 30% H
2O
2Available from Sigma company, A liquid mixed with B liquid in 100: 1 by volume during use.
Detect the test of abrin chemical luminescence reagent kit
1, sample preparation
Tracer liquid breast sample goods: look sample concentration and viscosity is suitably diluted with dilution, gained solution promptly can be used as detection liquid.
Detect blood sample: 1 volume blood sample adds the dilution 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 the dilution 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, sample detection
Chemical luminescence reagent kit of the present invention detects the method for abrin: the luminous plaque of getting monoclonal antibody bag quilt, the every hole 50 μ L of standard items 0,5,10,20,40,80ng/mL and testing sample that add each concentration in the reacting hole respectively, 37 ℃ of insulation 45min, wash 3 times, 4min/ time, on thieving paper, pat dry.The anti-abrin antibody of the rabbit 100 μ L/ holes that add purifying in the reacting hole, 37 ℃ of insulation 45min wash 3 times, 4min/ time, pat dry on thieving paper.Enzyme labelled antibody with 5000 times of diluted, is added in the reacting hole, 100 μ L/ holes, 37 ℃ of insulation 45min wash 3 times, 4min/ time, pat dry on thieving paper.Every hole adds luminous substrate liquid 100 μ L, 37 ℃ of lucifuge reaction 5-10min.On the chemiluminescence measuring instrument, measure the luminous intensity (RLU) in each hole successively, detection time 1 second/hole.Concentration and corresponding RLU value by standard items are set up typical curve, determine the concentration of abrin in the testing sample by curvilinear equation.
The mensuration and the practice of abrin immune chromatography test paper performance
1, detects the specific mensuration of abrin chemical luminescence reagent kit
When the detection abrin chemical luminescence reagent kit 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 chemical luminescence reagent kit susceptibility
Detect abrin with the present invention, minimum detectable activity is 0.1ng/mL, abrin concentration luminous intensity (RLU) logarithm value of concentration logarithm value and measurement in 0.1-80ng/mL scope correlativity that is in line.
3, detect abrin chemical luminescence reagent kit stability
(1) same batch chemical luminescence reagent kit all carries out the detection of chemical luminescence reagent kit every day during 37 ℃ of failure tests, and the intensity of test strips color decreases in the time of the 6th day.Illustrate that test strips can preserve 6 days at 37 ℃.
(2) chemical luminescence reagent kit in 4 ℃ preserve 10 months during, the luminous intensity value stabilization of the result standard product of Ce Dinging weekly; Illustrate that kit can preserve 10 months at 4 ℃, sensitivity descends to some extent after the 11st month.
(3) during 13 months, respectively every two weeks one detects same batch chemical luminescence reagent kit in-20 ℃ of preservations, and color intensity decreases in the time of 11st month; Illustrate that chemical luminescence reagent kit can preserve 11 months at-20 ℃, sensitivity descends to some extent after the 11st month.
5, detect abrin chemical luminescence reagent kit manual simulation pattern detection
(1) manual simulation's sausage toxin sample detection result is shown that recall rate is 100% when toxin sample concentration during greater than 1ng/mL.
(2) manual simulation's milk toxin sample detection is carried out discrimination test with the chemical luminescence reagent kit of ELISA test and preparation, the result shows that the coincidence rate of two kinds of detection methods is 100% when toxin sample concentration during greater than 1ng/mL.
Claims (6)
1. chemical luminescence reagent kit that detects abrin is characterized in that:
With specificity in conjunction with the mouse resource monoclonal antibody bag of abrin A chain by luminous plaque, be enzyme labelled antibody with the goat anti-rabbit igg of horseradish peroxidase-labeled, be chemical luminous substrate liquid with the luminol; With concentration be 0,5,10,20,40,80ng/mL abrin protein standard substance is equipped to kit together.
2. the preparation method of the described detection abrin of claim 1 chemical luminescence reagent kit may further comprise the steps:
Adopt gel chromatography to prepare abrin from jequirity, prepare toxoid with the formaldehyde attenuation treatment, immunizing rabbit and Balb/C mouse prepare polyclonal antibody and monoclonal antibody respectively; With specificity in conjunction with the mouse resource monoclonal antibody bag of abrin A chain by luminous plaque, be enzyme labelled antibody with the goat anti-rabbit igg of horseradish peroxidase-labeled, be chemical luminous substrate liquid with the luminol; With concentration be 0,5,10,20,40,80ng/mL abrin protein standard substance is equipped to kit together.
3. the described preparation method of claim 2 mainly may further comprise the steps:
(1) adopts 5% cold acetate solution extracting, and prepare slightly poison of abrin with 35%～95% ammonium sulfate precipitation; Through Sepharose 6B affinity chromatography, DEAE-Sepharose FF ion-exchange chromatography and Superdex 75 gel filtrations three steps chromatography prepare abrin again;
(2) abrin that will prepare from step (1) prepares toxoid with 1% formaldehyde attenuation treatment, is used for immune Balb/C mouse and prepares monoclonal antibody;
(3) filter out the monoclonal antibody of specificity in the monoclonal antibody that adopts the Westerblot method from step (2), to prepare in conjunction with abrin A chain;
(4) the monoclonal antibody bag that obtains with step (3) is by luminous plaque, 10% sheep blood serum sealing preparation luminous plaque;
(5) the abrin toxoid immunizing rabbit for preparing in the step (2) is prepared the anti-abrin polyclonal antibody in rabbit source;
(6) preparation chemical luminous substrate liquid, enzyme labelled antibody, dilution, 10 times of concentrated cleaning solutions;
(7) with specificity in conjunction with the mouse resource monoclonal antibody bag of abrin A chain by luminous plaque;
(8) goat anti-rabbit igg with horseradish peroxidase-labeled is an enzyme labelled antibody;
(9) utilize the middle bag of step (8) by the enzyme labelled antibody for preparing in good chemiluminescence plate and the step (9), with the luminol is chemical luminous substrate liquid, with concentration be 0,5,10,20,40,80ng/mL abrin protein standard substance is equipped to kit together.
4. claim 2 or 3 described preparation methods is characterized in that the preparation process of abrin is as follows:
Kind of a benevolence 200g is removed in the jequirity processing of shelling, and is dipped in the 115% cold acetic acid, 4 ℃ are spent the night, high-speed homogenization is handled, the centrifugal 20min of 10000g/min, and supernatant is with 35%～95% ammonium sulfate precipitation, resolution of precipitate is in 5mmol/LPB (pH8.0) solution, with Sepharose 4B affinitive layer purification, purified product is crossed DEAE-Sepharose FF ion exchange column, collects first eluting peak and is abrin, adding final concentration after desalting processing is 0.01% thimerosal sodium salt ,-20 ℃ of preservations.
5. claim 2 or 3 described preparation methods is characterized in that the preparation method of polyclonal antibody of the anti-abrin in rabbit source:
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/, the back cardiac puncture blood sampling of one week, separation of serum, serum is handled through the 50% ammonium sulfate precipitation precipitation method, the centrifugal 20min of 5000g/min, abandon supernatant, precipitation is resuspended with PBS (0.02mol/L pH7.0), is the polyclonal antibody of slightly carrying, and crude extract prepares highly purified antibody with HitraprProtein A FF or Hitrap rProtein G FF prepacked column.
6. claim 2 or 3 described preparation methods is characterized in that specificity is as follows 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 further purified with Hitrap rProtein A FF gel affinity column after ammonium sulfate salting-out process is slightly carried, and promptly gets monoclonal antibody.
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| CN201010132580A CN101819203A (en) | 2010-03-26 | 2010-03-26 | Chemiluminescent kit for detecting abrin and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106771258A (en) * | 2017-02-16 | 2017-05-31 | 广州赛太特生物医学科技有限公司 | The detection kit and its methods and applications of a kind of M2BP |
| CN107643398A (en) * | 2017-09-11 | 2018-01-30 | 武汉市中西医结合医院（武汉市第医院） | A kind of MFG E8 detection method |
| CN111505304A (en) * | 2019-01-31 | 2020-08-07 | 艾维可生物科技有限公司 | Kit for detecting galectin-3 by chemiluminescence method and use method thereof |
| CN114437209A (en) * | 2022-01-25 | 2022-05-06 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to abrin and application thereof |
-
2010
- 2010-03-26 CN CN201010132580A patent/CN101819203A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 李小兵 等: "相思子毒素-a单克隆抗体的制备与鉴定", 《中国兽医学报》 * |
| 罗胜军 等: "相思子毒素BSA-ELISA检测试剂盒的研制", 《中国畜牧兽医学会家畜内科学分会第6届会员代表大会暨学术研讨会会议论文》 * |
| 高荣 等: "化学发光免疫分析技术应用研究进展", 《中国生物制品学杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106771258A (en) * | 2017-02-16 | 2017-05-31 | 广州赛太特生物医学科技有限公司 | The detection kit and its methods and applications of a kind of M2BP |
| CN107643398A (en) * | 2017-09-11 | 2018-01-30 | 武汉市中西医结合医院（武汉市第医院） | A kind of MFG E8 detection method |
| CN111505304A (en) * | 2019-01-31 | 2020-08-07 | 艾维可生物科技有限公司 | Kit for detecting galectin-3 by chemiluminescence method and use method thereof |
| CN114437209A (en) * | 2022-01-25 | 2022-05-06 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to abrin and application thereof |
| CN114437209B (en) * | 2022-01-25 | 2022-09-20 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to abrin and application thereof |
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