CN104655845A - Preparation of chemiluminescence immunoassay kit for detecting chloroquine - Google Patents
Preparation of chemiluminescence immunoassay kit for detecting chloroquine Download PDFInfo
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- CN104655845A CN104655845A CN201310584979.7A CN201310584979A CN104655845A CN 104655845 A CN104655845 A CN 104655845A CN 201310584979 A CN201310584979 A CN 201310584979A CN 104655845 A CN104655845 A CN 104655845A
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
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Abstract
The invention provides a preparation of a chemiluminescence immunoassay kit for detecting chloroquine and belongs to the field of immunological detection. The kit is composed of: a non-transparent white enzyme label plate coated by a chloroquine-carrier protein conjugate, a chloroquine standard substance, a chloroquine-peroxidase marked antibody operating fluid, a luminescent substrate liquid, a concentrated sample diluting liquid and a concentrated washing liquid. The chloroquine-carrier protein conjugate is prepared by coupling chloroquine to a carrier protein in a mixed anhydride method or a carbodiimide method. The concentrated washing liquid contains 0.05% of tween-20. The kit is higher in sensitivity, is short in detection time, is low in cost and can be used for detection of residual amount of the chloroquine in samples such as fruits, crops and vegetables and the like.
Description
Technical field
The present invention relates to a kind of chemiluminescence immune detection reagent kit detecting chloroquine, for detecting chloroquine content in fruit, vegetables, crops etc. or residual quantity.Belong to field of immunological detection.
Background technology
Chloroquine is comparatively slow human body metabolism, easily causes the reactions such as anorexia, n and V, diarrhoea when content exceedes a certain amount of; Also can occur that pruitus, purpura, depilation, hair bleach, eczema and exfoliative dermatitis, psoriasis; Nose heave, headache, giddy, tinnitus, dizzy, burnout, sleep-disorder, amentia, the visual field are reduced, cornea and retinosis etc.
Chloroquine retention analysis generally uses vapor-phase chromatography (GC), high performance liquid chromatography (HPLC) and gas chromatography combined with mass spectrometry technology (GC/MS), these methods are sensitive, accurate, can Simultaneously test multi-medicament, but need expensive instrument, sample pre-treatments is complicated, loaded down with trivial details time-consuming, testing cost is higher, and need professional to operate, be difficult to meet the needs carrying out scene to sample, detect in batches, fast.Therefore, develop a kind of analytical approach that is simple and quick, that be applicable to residues of pesticides on-site supervision to have important practical significance.
Chemiluminescence immunoassay detection technique is the product that chemoluminescence method and immunoassay combine, and therefore has the high sensitivity of chemiluminescence detection technology and the high specific of immuno analytical method simultaneously.The present invention has the chloroquine antibody of high-affinity, high specific by distinctive immune animal preparation, and adopt enzymic-labelled antibody, set up a kind of chemiluminescence immunoassay kit that can detect chloroquine, detection will be stayed to provide new method for chloroquine medicine in fruit, vegetables, crops etc., the features such as this method has easy and simple to handle, quick, and highly sensitive, specificity is good.
Summary of the invention
For problems of the prior art, the present invention mainly utilizes the ultimate principle of the specific immune response of antigen and antibody to realize.Chemiluminescence immune assay is the product that chemoluminescence method and immunoassay combine, and therefore has the high sensitivity of chemoluminescence method and the high specific of immunoassay simultaneously.In whole course of reaction, in sample, chloroquine content is higher, and in reaction system, luminous intensity is more weak; Otherwise in sample, chloroquine content is fewer, and luminous intensity is higher.
The present invention is a kind of chemiluminescence immune detection reagent kit detecting chloroquine, it is characterized in that containing following composition:
1, the opaque white color ELISA Plate of chloroquine-carrier protein couplet thing is coated with; Described chloroquine-carrier protein couplet thing is obtained by mixed anhydride method or carbodlimide method coupling chloroquine and carrier protein, and described carrier protein is human serum albumins, bovine serum albumin(BSA), egg albumin, mouse haemocyanin or rabbit serum proteins;
2, chloroquine standard items;
3, chloroquine-peroxidase labeled antibodies: this composition is the chloroquine antibody with peroxidase labelling, and described chloroquine antibody is monoclonal antibody or polyclonal antibody;
4, luminous substrate liquid: the Chemoluminescent substrate that this luminous substrate liquid is is luminous agent with the different luminol of luminol goods, is divided into A liquid and B liquid to preserve, presses 1:1 before use used in combination; Wherein A liquid level luminescence enhancer adds luminol goods luminescence enhancer and adds different luminol, and B liquid is superoxol or urea hydrogen peroxide solution;
5,2 times of concentration and dilution liquid;
6,20 times of concentrated cleaning solutions.
In the present invention, described opaque white color ELISA Plate is the detachable of 96 holes or non-removable opaque white color ELISA Plate.
In the present invention, described chloroquine standard items, are made up of the chloroquine standard items of a series of variable concentrations, and concentration is the concentration ranges of 0.01 ~ 25 ng/mL.
In the present invention, described chloroquine-peroxidase labeled antibodies is the chloroquine antibody with peroxidase labelling, as the chloroquine antibody that horseradish peroxidase (HRP) marks.
In the present invention, described luminous substrate liquid is any one Chemoluminescent substrate being luminous agent with the different luminol of luminol goods commercial.
In the present invention, described 2 times of concentration and dilution liquid, its composition is the phosphate buffer of 0.01mol/L, pH7.4, Glycine-HCl buffer or Tris-HCl damping fluid, please dilutes (1 part of concentrating sample dilution+1 part of deionized water) by 1:1 before using.
In the present invention, described 20 times of concentrated cleaning solutions, it comprises 0.05% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0-7.5, please dilutes (1 part of concentration and dilution liquid+19 parts of deionized waters) by 1:19 before using.
When the chemiluminescence immune detection reagent kit of chloroquine of the present invention is applied to the detection of chloroquine, detecting step is:
(1) pre-service testing sample is fluid sample by sample preparation to be tested, or uses Solvent Extract methods testing sample, and nitrogen dries up and redissolved in sample diluting liquid working fluid;
(2) required reagent is taken out from cold storage environment, be placed in room temperature (20 ~ 25 DEG C) and balance 30 more than min, notice that often kind of liquid reagent must shake up before using;
(3) get bag by the ELISA Plate of chloroquine antigen, add standard items/testing sample 50 μ L/ hole in the micropore of correspondence, standard items and each concentration of sample do two parallel laboratory tests;
(4) add chloroquine antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 45 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;
(5) carefully open cover plate film, liquid in hole is dried, with wash operating solution 250 μ L/ hole, fully washs 4 ~ 5 times, every minor tick 10 s, pat dry (bubble be not eliminated after patting dry can be poked with original rifle head) with thieving paper;
(6) add luminous substrate liquid mixed liquor (A liquid mixes by 1:1 before use with B liquid) 100 μ L/ hole, mixing of vibrating gently, detects luminous intensity (RLU) in chemiluminescence detector after mixing;
(7) calculating of testing result: calculate with the ratio of the standard solution obtained and sample solution luminous value and blank solution.See following formula:
Relative luminous intensity=RLU/RULmax
In formula:
The luminous intensity values of RLU=standard (or sample) solution;
The luminous intensity values of RLUmax=blank (concentration is the standard solution of 0).
The natural logarithm of corresponding for the relative luminous intensity value calculated chloroquine (μ g/L) is made semilog coordinate system curve figure.The chloroquine concentration of each testing sample is found on typical curve according to its RLU value, or is calculated by the corresponding equation of typical curve.As having dilution in sample preparation, the sample concentration that should draw according to typical curve will be multiplied by its extension rate again.Be the actual concentrations of chloroquine in sample.
Kit of the present invention can be used for the residues detection of chloroquine in the samples such as fruit, vegetables, crops.Detect the comparison of chloroquine residual quantity with existing other, kit of the present invention has following advantage:
(1) kit of the present invention of Chemiluminescence immunoassay is adopted, more more fast and convenient than chromatographic process (efficient liquid phase, LC-MS, gas chromatography mass spectrometry), capillary electrophoresis method, required instrument is more simple, and testing cost is more cheap, has high-throughout feature simultaneously;
(2) adopt the kit of the present invention of Chemiluminescence immunoassay, more sensitiveer than ELISA method, can detect that the chloroquine of lower concentration and content remains, the range of linearity is wider simultaneously;
(3) adopt the kit of the present invention of Chemiluminescence immunoassay, decrease two anti-use links, thus shorten detection time; Opaque white color ELISA Plate adds chemiluminescence detector sensitivity.In addition, with chloroquine-carrier protein couplet thing but not chloroquine antibody wrap by opaque white color ELISA Plate, decrease the instability of chloroquine antibody, ensure that the long-term effectiveness of kit.
Embodiment
Below by way of specific embodiment, the invention will be further described.These embodiments only for illustration of the present invention, and are not used for limiting the scope of the invention.
Embodiment 1
1, the preparation of each component of kit
(1) the haptenic preparation of chloroquine: by chloroquine acidifying, with sodium nitrite effect in 4 DEG C of unglazed low temperature environments, generates the intermediate containing diazo positive ion.Diazotizing chloroquine, as haptens, is used and is synthesized immunizing antigen and envelope antigen later;
(2) chloroquine-bovine serum albumin(BSA) (BSA) immunogenic preparation: adopt diazotising method to carry out coupling chloroquine and bovine serum albumin(BSA) (BSA) and obtain immunizing antigen.
The preparation of chloroquine-ovoserum albumin (OVA) envelope antigen: adopt diazotising method to carry out coupling chloroquine and ovoserum albumin (OVA) and obtain envelope antigen.
The preparation of chloroquine-peroxidase labeled antibodies: (g), heavy dose of immunization protocol is body weight 18 ~ 20, and first immunisation 160 μ g chloroquine-BSA mix with equivalent Freund's complete adjuvant, hypodermic injection to the female BAl BIc/c mouse in 6 ~ 8 week age.After 3 weeks, then mix with equivalent Freund's complete adjuvant with 80 μ g chloroquine-BSA, hypodermic injection.After this mixed with equivalent Freund's complete adjuvant with 80 μ g chloroquine-BSA every 3 weeks, lumbar injection.Last immunity in the spleen 80 μ g chloroquine-BSA is as booster immunization.Put to death mouse after three days, get its spleen, with myeloma cell fusion.Positive hybridoma cell is screened with indirect ELISA method.Prepare mouse ascites in a large number by mouse peritoneal injection hybridoma, ascites, after filtration, centrifugal preliminary purification, adopts sad method and affinity chromatography purifying ascites, then obtains the chloroquine monoclonal antibody of purifying through dialysis.Chloroquine monoclonal antibody and horseradish peroxidase, thus obtain chloroquine-peroxidase labeled antibodies.
Be coated with the opaque white color ELISA Plate preparation of chloroquine-OVA conjugate: be used for wrapping the detect aperture by opaque white color ELISA Plate after being diluted by chloroquine-OVA conjugate with damping fluid, 4 DEG C spend the night after use PBST buffer solution, then 180 μ L confining liquids (5% skim milk powder solution) are added, 37 DEG C of incubation 1.5 h, incline liquid in hole, and thieving paper pats dry rear sealing and preserves.
2, the establishment of the chemiluminescence immune detection reagent kit of chloroquine is detected
The chemiluminescence immune detection reagent kit of the detection chloroquine set up, contains following ingredient:
(1) 96 hole opaque white color ELISA Plate (8 hole × 12) is coated with chloroquine-OVA conjugate, uses aluminium foil bag vacuum sealed package;
(2) chloroquine standard solution 6 bottles, concentration is respectively:
0 ng/mL、0.01 ng/mL、0.05ng/mL、0.5ng/mL、5ng/mL、25ng/mL
(3) chloroquine-Horseradish Peroxidase Conjugates solution;
(4) luminous substrate A liquid (luminol and reinforcing agent), luminous substrate B liquid (urea hydrogen peroxide);
(5) 2 times of concentrating sample dilutions.Please dilute (1 part of concentration and dilution liquid+1 part of deionized water) by 1:1 before using and become working prototype dilution, the working prototype dilution after its dilution is the PBST damping fluid of 0.05 mol/L, pH 7.4;
(6) 20 times of concentrated cleaning solutions.Please dilute (1 part of concentration and dilution liquid+19 parts of deionized waters) by 1:19 before using and become work cleansing solution, the work cleansing solution after its dilution is between pH value range 7.0-7.5, containing 0.05% Tween-20, and the PBST damping fluid of 0.01mol/L.
3, chemiluminescence immune detection reagent kit on probation of chloroquine
1) pre-treatment of sample:
(1) prepared and diluted liquid: water-acetonitrile-methanesulfonic acid solution (7mL methane-sulforic acid joins in 4mL glacial acetic acid, is diluted with water to 100mL)-diethylamine solution (2mL is diluted to 20mL, shakes up) (86:10:2:2);
(2) representative sample or by tissue homogenate is ground;
(3) sample that weighing 160mg handles well adds 80mL methanol solution;
(4) mix in the container of sealing, concussion vortex 30min;
(5) room temperature centrifugal more than 4000r/min, 10min; Get 3ml supernatant;
(6) with the dilution proportion supernatant of 1:9, mix to be measured.
2) chemiluminescence immune detection reagent kit
The hole bar of standard and sample requirement is inserted in microwell plate framework, the position of record standard and sample.50 μ L/ hole chloroquine standard solution and testing samples are added respectively in suitable micropore.Add 50 μ L/ hole chloroquine-Horseradish Peroxidase Conjugates in each micropore, fully after mixing, under room temperature, lucifuge leaves standstill incubation 45 min.Liquid in hole is dried, fully washs 4 ~ 5 times with wash operating solution.Remove the liquid in hole completely, pat dry with thieving paper, add luminous substrate liquid mixed liquor (A liquid mixes by 1:1 before use with B liquid) 100 μ L/ hole.In chemiluminescence detector, luminous intensity (RLU) is detected immediately after mixing;
3) computational analysis of testing result
Calculate with the ratio of obtained standard solution and sample solution luminous value and blank solution.See following formula:
Relative luminous intensity=RLU/RULmax
In formula:
The luminous intensity values of RLU=standard (or sample) solution;
The luminous intensity values of RLUmax=blank (concentration is the standard solution of 0).
The natural logarithm of corresponding for the relative luminous intensity value calculated chloroquine (μ g/L) is made semilog coordinate system curve figure.The chloroquine concentration of each testing sample is found on typical curve according to its RLU value, or is calculated by the corresponding equation of typical curve.As sample have passed through beforehand dilution, the sample concentration that should draw according to typical curve will be multiplied by its extension rate again.
Embodiment 2
Detect the chemiluminescence immune detection reagent kit of chloroquine, contain following ingredient:
(1) 96 hole opaque white color ELISA Plate (8 hole × 12) is coated with chloroquine-OVA conjugate, uses aluminium foil bag vacuum sealed package;
(2) chloroquine standard solution 6 bottles, concentration is respectively:
0 ng/mL、0.01 ng/mL、0.05ng/mL、0.5ng/mL、5ng/mL、25ng/mL
(3) chloroquine-Horseradish Peroxidase Conjugates solution;
(4) luminous substrate A liquid (luminol and reinforcing agent), luminous substrate B liquid (urea hydrogen peroxide);
(5) 2 times of concentrating sample dilutions.Please dilute (1 part of concentration and dilution liquid+1 part of deionized water) by 1:1 before using and become working prototype dilution, the working prototype dilution after its dilution is the Tris-HCl damping fluid of 0.05 mol/L, pH 7.4;
(6) 20 times of concentrated cleaning solutions.Please dilute (1 part of concentration and dilution liquid+19 parts of deionized waters) by 1:19 before using and become work cleansing solution, the work cleansing solution after its dilution is between pH value range 7.0-7.5, containing 0.05% Tween-20, and the PBST damping fluid of 0.01mol/L.
Claims (7)
1. detect a chemiluminescence immune detection reagent kit for chloroquine, it is characterized in that the following composition in kit:
(1) the opaque white color ELISA Plate of chloroquine-carrier protein couplet thing is coated with; Described chloroquine-carrier protein couplet thing is obtained by mixed anhydride method or carbodlimide method coupling chloroquine and carrier protein, and described carrier protein is human serum albumins, bovine serum albumin(BSA), egg albumin, mouse haemocyanin or rabbit serum proteins;
(2) chloroquine standard items;
(3) chloroquine-peroxidase labeled antibodies: this composition is the chloroquine antibody with peroxidase labelling, and described chloroquine antibody is monoclonal antibody;
(4) luminous substrate liquid: the Chemoluminescent substrate that this luminous substrate liquid is is luminous agent with the different luminol of luminol goods, is divided into A liquid and B liquid to preserve, presses 1:1 before use used in combination; Wherein A liquid is that luminescence enhancer adds luminol goods luminescence enhancer and adds different luminol, and B liquid is superoxol or urea hydrogen peroxide solution;
(5) 2 times of concentration and dilution liquid;
(6) 20 times of concentrated cleaning solutions.
2. the chemiluminescence immune detection reagent kit of chloroquine according to claim 1, wherein, described opaque white color ELISA Plate is the detachable of 96 holes or non-removable opaque white color ELISA Plate.
3. the chemiluminescence immune detection reagent kit of chloroquine according to claim 1, wherein, the concentration of described chloroquine standard items is the concentration ranges of 0.01 ~ 25 ng/mL.
4. the chemiluminescence immune detection reagent kit of chloroquine according to claim 1, wherein, described chloroquine-peroxidase labeled antibodies working fluid is diluted to 1:20000 ratio with antibody diluent.
5. the chemiluminescence immune detection reagent kit of chloroquine according to claim 1, wherein, described luminous substrate liquid is any one Chemoluminescent substrate being luminous agent with the different luminol of luminol goods commercial.
6. the chemiluminescence immune detection reagent kit of chloroquine according to claim 1, wherein, described 2 times of concentration and dilution liquid, its composition is 0.01mol/L, the phosphate buffer of pH7.4, Glycine-HCl buffer or Tris-HCl damping fluid, please dilute (1 part of concentrating sample dilution+1 part of deionized water) by 1:1 before using.
7. the chemiluminescence immune detection reagent kit of chloroquine according to claim 1, wherein, described 20 times of concentrated cleaning solutions, it comprises 0.05% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0-7.5, please dilutes (1 part of concentration and dilution liquid+19 parts of deionized waters) by 1:19 before using.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017200489A1 (en) * | 2016-05-18 | 2017-11-23 | Singapore Health Services Pte. Ltd. | A pharmaceutical composition and the use thereof in the treatment of autoimmune diseases |
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| CN1569835A (en) * | 2004-05-10 | 2005-01-26 | 浙江大学 | Production method and use for dichloro quinolinic acid artificial hapten, artificial antigen and specific antibody |
| CN101413952A (en) * | 2008-11-27 | 2009-04-22 | 上海交通大学 | Chemiluminescence immune detection reagent kit for detecting ractopamine |
| CN103018454A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit |
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- 2013-11-20 CN CN201310584979.7A patent/CN104655845A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1569835A (en) * | 2004-05-10 | 2005-01-26 | 浙江大学 | Production method and use for dichloro quinolinic acid artificial hapten, artificial antigen and specific antibody |
| CN101413952A (en) * | 2008-11-27 | 2009-04-22 | 上海交通大学 | Chemiluminescence immune detection reagent kit for detecting ractopamine |
| CN103018454A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017200489A1 (en) * | 2016-05-18 | 2017-11-23 | Singapore Health Services Pte. Ltd. | A pharmaceutical composition and the use thereof in the treatment of autoimmune diseases |
| US11421000B2 (en) | 2016-05-18 | 2022-08-23 | Singapore Health Services Pte. Ltd. | Pharmaceutical composition and the use thereof in the treatment of autoimmune diseases |
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Application publication date: 20150527 |