CN1279399A - Reagent kit for discriminating diabetes and its preparing process - Google Patents
Reagent kit for discriminating diabetes and its preparing process Download PDFInfo
- Publication number
- CN1279399A CN1279399A CN 99113082 CN99113082A CN1279399A CN 1279399 A CN1279399 A CN 1279399A CN 99113082 CN99113082 CN 99113082 CN 99113082 A CN99113082 A CN 99113082A CN 1279399 A CN1279399 A CN 1279399A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- antibody
- dilution
- bcg
- linked reaction
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims description 12
- 206010012601 diabetes mellitus Diseases 0.000 title abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 4
- 102000004190 Enzymes Human genes 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 14
- 102000002812 Heat-Shock Proteins Human genes 0.000 claims abstract description 12
- 108010004889 Heat-Shock Proteins Proteins 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 210000002966 serum Anatomy 0.000 claims abstract description 9
- 239000000758 substrate Substances 0.000 claims abstract description 9
- 239000000872 buffer Substances 0.000 claims abstract description 5
- 238000010790 dilution Methods 0.000 claims description 10
- 239000012895 dilution Substances 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 7
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 238000008157 ELISA kit Methods 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 2
- 241000283707 Capra Species 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 2
- 235000019800 disodium phosphate Nutrition 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 235000015097 nutrients Nutrition 0.000 claims description 2
- 244000309466 calf Species 0.000 claims 1
- 239000007788 liquid Substances 0.000 abstract description 4
- 238000005406 washing Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 abstract 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 13
- 238000002360 preparation method Methods 0.000 description 13
- 208000035408 type 1 diabetes mellitus 1 Diseases 0.000 description 10
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- 230000009885 systemic effect Effects 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 2
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 208000030172 endocrine system disease Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
A reagent kit for diagnosing diabetes is composed of BCG vaccine 65KD heat shock protein coated enzyme-linked reaction board, HRP-IgG, washing liquid, diluted enzyme-linked antibody liquid, diluted substrate liquid, substrate OPD, stop buffer and positive serum. Its diagnostic specificity for diabetes A can reach 94% or more and the sensitivity can reach 83% or more. Its advantages include easily preparing it, low cost and higher added value.
Description
Reagent kit for discriminating diabetes and preparation method thereof, relate to and utilize Bacille Calmette-Guerin (BCG) 65KD heat shock protein as antigen, utilization ELISA method detects 64KD antibody in diabetic's serum, a kind of kit of the type of antidiastole diabetes clinically and preparation method thereof.
At present differentiate that clinically the index of diabetes mainly contains insular cellular antibody (ICA), insulin autoantibody (IAA), and 64KD antibody.It is low that ICA and IAA deposit among the insulin-dependent diabetes mellitus (IDDM) patients serum positive rate, and morbidity back recall rate descends, and ICA and IAA also are present in other endocrine system diseases; So fail clinically to apply.Detecting 64KD antibody has utilization glutamate decarboxylase (GAD) in the prior art as antigen, high because of price, is difficult to be generalized to clinical use.ELISA kit of detection 64KD antibody of the present invention and preparation method thereof is a new technical method of producing a kind of New Product.Detection 64KD antibody (65KDHSP antibody) kit with the present invention's preparation has detected 30 routine insulin-dependent diabetes mellitus (IDDM), 30 routine type II diabetess, 5 routine systemic worker's yabbi sores, 8 routine hyperthyroidisms, 44 routine healthy people, 64KD antibody positive recall rate is respectively 83.3%, 13.3%, 0.00%, 0.00%, 2.27%; The type II diabetes of 24 routine 64KD antibody positives has 22 (91.7%) examples to need insulinize, so can identify the tardy systemic autoimmune diabetes of adult from type II diabetes.Specificity to the insulin-dependent diabetes mellitus (IDDM) diagnosis is 94.6%, and the susceptibility of diagnosis is 83.3%, and the diagnosis index is 177.9%.
The object of the present invention is to provide a kind of with Bacille Calmette-Guerin (BCG) 65KD heat shock protein as antigen, and detect the kit of 64KD antibody in diabetic's serum with the ELISA method, be used for the type of antidiastole diabetes clinically; This kit can not only be diagnosed insulin-dependent diabetes mellitus (IDDM), and can discern adult's tardy systemic autoimmune diabetes (LADA) from type II diabetes.The preparation of antigen Bacille Calmette-Guerin (BCG) 65KD heat shock protein is simple, and cost is lower.
The technical scheme of kit of the present invention and preparation method thereof is as follows:
1) preparation Bacille Calmette-Guerin (BCG) 65KD heat shock protein
BCG is inoculated in and cultivates 10～14 days in the nutrient culture media, and equipment uses the constant-temperature shaking culture case, 9 of (Shanghai make a leapleap forward medicine equipment two factories) capacity 500ml Erlenmeyer flasks;
Constant water bath box (model: ZB11241-89), 40～45 ℃ of water-baths 1～4 hour;
Collect supernatant with hydro-extractor (Beijing Medical Centrifugal Machine Factory produces the LD4-2 type);
With ammonium sulphate precipitation method condensing protein, concentration is 1～500mg/ml;
With Protein Recovery groove (Liuyi Instruments Plant, Beijing produces DYY-III 43 types) purifying, perhaps use Waters chromatograph (chromatography) purifying Bacille Calmette-Guerin (BCG) 65KD heat shock protein;
Identify its molecular weight with the SDS-PAGE method, identify its immunity (antigen) activity with immune animal (rabbit or small white mouse);
Promptly produce Bacille Calmette-Guerin (BCG) the 65KD heat shock protein of usefulness required for the present invention after qualified; Quantity is about 0.5 gram of preparation in everyone every month; Three people cooperate preparation in about every month 3 grams.
2) preparation enzyme-linked reaction plate
Use the carbonate buffer solution of PH (8-10) quantitatively to 1 μ g～1000 μ g/ml Bacille Calmette-Guerin (BCG) 65KD heat shock protein;
Bag is by enzyme-linked reaction plate 0.1～50 μ g/ hole;
With the phosphate buffer sealing that contains 1～5%BSA (bSA), sealing condition be 37 ℃ following 1 hour, or 4 ℃ following 12 hours;
Washing; Cleansing solution is the PH7.4 that contains 0.1% Tween-20,0.01mol/L phosphate buffer washing 3 times, each 5 minutes;
Through air dry,, preserve standby down at 4 ℃ with polyvinyl chloride plastic pocket sterilization enclosed package;
1 gram Bacille Calmette-Guerin (BCG) 65KD heat shock protein can wrap by 100,000 person-portions.
3) preparation kit
Two kinds of templates have been wrapped by the enzyme-linked reaction plate of Bacille Calmette-Guerin (BCG) 65KD heat shock protein: removable 96 holes or removable 48 holes;
HRP-goat anti-human igg (goat anti-human igg of horseradish peroxidase mark), dilution in 1: 800, pipe is adorned two kinds of specifications: 15 μ l or 7.5 μ l;
Cleansing solution, bottled 30ml or 15ml, 20 times concentrate the PH7.4 contain 0.05% Tween-20,0.01mol/L phosphate buffer; (dilution is 20 times during use)
The enzyme labelled antibody dilution; (cleansing solution adds 1%～2% bSA 200 μ l)
The substrate dilution; (the sodium hydrogen phosphate 10.28ml of 0.1mol/L adds 0.05mol/L citric acid 9.72ml)
Substrate OPD; (o-phenylenediamine, sheet, 3, imported product, Sigma company produces)
Stop buffer 2mol/L sulfuric acid;
Positive serum, 50 μ l.
The present invention is used for clinical detection 64KD antibody process:
1) enzyme-linked reaction plate adds serum to be checked (1: 10)-(1: 100) 100 μ l, hatches 1 hour, and washes plate for 37 ℃;
2) add the anti-human IgG (Military Medical Science Institute's product) of HRP mark, hatched 1 hour, and washed plate for 37 ℃;
3) add substrate OPD liquid 100 μ l, 37 ℃ of black outs developed the color 10～20 minutes;
4) add stop buffer 2mol/LH
2SO
450 μ l;
5) 492nm place colorimetric (using Nanjing East China Electronics Co., Ltd instrument plant to produce enzyme-linked immunosorbent assay instrument).
Benefit of the present invention has: (1) is particularly suitable for clinical expansion and uses owing to take the measure of pre-envelope antigen plate technique, and is easy to operate simple: (2) preparation technology is simply ripe, raw material is easy to get, cost is lower, specific new technology innovative content is arranged, the added value of product height; (3) the insulin-dependent diabetes mellitus (IDDM) specificity is reached more than 94%, diagnostic sensitivity reaches more than 83%, can identify the tardy systemic autoimmune diabetes of adult to type II diabetes.
The kit and the method for making of the present invention's narration describe in detail in technical scheme, can produce the kit of the present invention's narration with the method for making of the present invention's narration by its explanation.
Claims (5)
1) the ELISA kit of detection 64KD antibody, comprise (a) enzyme-linked reaction plate, (b) HRP-goat anti-human igg, (c) cleansing solution, (d) enzyme labelled antibody dilution, (e) substrate dilution, (f) substrate 0PD, (g) stop buffer, (h) positive serum is characterized in that: with Bacille Calmette-Guerin (BCG) 65KD heat shock protein bag quilt (a) enzyme-linked reaction plate; (b) HRP-goat anti-human igg (goat anti-human igg of horseradish peroxidase mark), dilution in 1: 800, pipe is adorned two kinds of specifications: 15 μ l or 7.5 μ l; (c) cleansing solution, bottled 30ml or 15ml, 20 times concentrate the PH74 contain 0.05% Tween-20,0.01 mol/L phosphate buffer (dilution is 20 times during use); (d) enzyme labelled antibody dilution (cleansing solution adds 1%～2% bSA 200 μ l); (e) substrate dilution (the sodium hydrogen phosphate 10.28ml of 0.1mol/L adds 0.05mol/L citric acid 9.72ml); (f) substrate OPD (o-phenylenediamine, sheet, 3, imported product, Sigma company product); (g) stop buffer 2mol/l sulfuric acid; (h) positive serum, 50 μ l.
2) the ELISA kit of detection 64KD antibody according to claim 1, it is characterized in that: Bacille Calmette-Guerin (BCG) the 65KD hot body gram albumen of bag quilt uses the carbonate buffer solution of PH (8-10) quantitatively to 1 μ g～1000 μ g/ml on the enzyme-linked reaction plate, bag is sealed from the phosphate buffer of albumen with the calf serum that contains 1～5% by enzyme-linked reaction plate 0.1～50 μ g/ hole.
3) according to the ELISA kit of the described detection of claim 1 or claim 2 64KD antibody, it is characterized in that: with the condition of phosphate buffer sealing be 37 ℃ following 1 hour.
4) according to the ELISA kit of the described detection of claim 1 or claim 2 64KD antibody, it is characterized in that: with the condition of phosphate buffer sealing be 4 ℃ following 12 hours.
5) a kind of method for making of making the described kit of claim 1, it is characterized in that: Bacille Calmette-Guerin (BCG) the 65KD heat shock protein of bag quilt makes with following method on the enzyme-linked reaction plate: BCG was inoculated in the nutrient culture media 10～14 days, and water-bath is 1～4 hour under 40 ℃～45 ℃ conditions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99113082 CN1279399A (en) | 1999-07-05 | 1999-07-05 | Reagent kit for discriminating diabetes and its preparing process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99113082 CN1279399A (en) | 1999-07-05 | 1999-07-05 | Reagent kit for discriminating diabetes and its preparing process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1279399A true CN1279399A (en) | 2001-01-10 |
Family
ID=5276317
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99113082 Pending CN1279399A (en) | 1999-07-05 | 1999-07-05 | Reagent kit for discriminating diabetes and its preparing process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1279399A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100415899C (en) * | 2001-09-05 | 2008-09-03 | 普赖德普罗特奥米克斯公司 | Type II diabetes protein |
| CN104849466A (en) * | 2014-02-14 | 2015-08-19 | 张曼 | Application of urine vitronectin in diagnosis and treatment of type 2 diabetes mellitus combined early renal injury |
-
1999
- 1999-07-05 CN CN 99113082 patent/CN1279399A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100415899C (en) * | 2001-09-05 | 2008-09-03 | 普赖德普罗特奥米克斯公司 | Type II diabetes protein |
| CN104849466A (en) * | 2014-02-14 | 2015-08-19 | 张曼 | Application of urine vitronectin in diagnosis and treatment of type 2 diabetes mellitus combined early renal injury |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Freed et al. | Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods | |
| CN107340386A (en) | SST2 detection kits based on microparticle chemiluminescence immunoassay | |
| CN102338801B (en) | High-sensitivity immunochip detection system and application method thereof | |
| EP1107773B1 (en) | Method for detection of legionella bacteria employing purified antigen-specific antibodies | |
| CN102087294B (en) | Immunochromatographic test strip for rapidly detecting malaria and preparation method thereof | |
| CN101571539A (en) | Elisa kit for detecting cephalo-type medicine and application thereof | |
| CN101477127A (en) | Human brain natriuretic non-competitive double-antibody sandwich method ELISA detection reagent kit | |
| CN104792996B (en) | A kind of rabies virus antibodies（IgG）Enzyme-linked immunologic detecting kit and its detection method | |
| CN102331495A (en) | Food allergen assay kit and preparation method thereof | |
| CN102735835B (en) | Disposable whole-blood uric acid detection test strip and manufacturing method thereof | |
| CN102236021A (en) | Human immunodeficiency virus (HIV) antibody time resolved fluoroimmunoassay method and kit | |
| Ibrahim | A review of immunoassays and their application to salmonellae detection in foods | |
| CN102735849A (en) | Enzymatic chemiluminescence immunoassay method for human heart-type fatty acid binding protein and reagent kit | |
| CN109298181A (en) | A kind of ELISA kit and its preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody | |
| CN102183647A (en) | Kit and method for detecting hepatitis B virus surface antigen (HBsAg) | |
| CN1122845C (en) | Test strip for detecting more antigens of typhoid bacillus | |
| CN1279399A (en) | Reagent kit for discriminating diabetes and its preparing process | |
| CN1580772A (en) | SARS virus antibody double antigen sandwich ELISA detecting method | |
| CN101377494B (en) | Chemiluminescence immune analytic reagent kit for detecting tuberculosis antibody | |
| CN105092563A (en) | Enzyme linked immunosorbent assay kit, preparation method thereof and method for detecting pepsase through same | |
| CN102043049A (en) | In vitro detection method and application of antibody | |
| CN1866021A (en) | Test paper for quick detection of cholera vibrio 01 group, 0139 group and cholera toxin | |
| CN103645310A (en) | Macrodantin chemiluminescence detection kit | |
| JPS62156560A (en) | Method and kit for executing test of ligand/non-ligand | |
| CN105699643B (en) | Pretreatment solution and pretreatment method before analysis of pathogenic bacteria samples |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |