CN108918888A - It is a kind of detect Endostatin latex enhancing immune than turbid kit and its application - Google Patents
It is a kind of detect Endostatin latex enhancing immune than turbid kit and its application Download PDFInfo
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- CN108918888A CN108918888A CN201810742366.4A CN201810742366A CN108918888A CN 108918888 A CN108918888 A CN 108918888A CN 201810742366 A CN201810742366 A CN 201810742366A CN 108918888 A CN108918888 A CN 108918888A
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- buffer
- endostatin
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- latex
- detection
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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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
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
-
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
Abstract
The invention discloses a kind of latex enhancing immunes for detecting Endostatin than turbid kit and its application, which includes reagent R1, reagent R2 and Endostatin standard solution, and wherein reagent R1 includes electrolyte, coagulant, preservative and buffer；Reagent R2 includes the coated latex particle of Endostatin, electrolyte, stabilizer, preservative and buffer；Standard items are the appropriate buffer containing various concentration Endostatin.This kit high sensitivity, high specificity, accuracy is good, and operation is quick and easy, can be used for various Biochemical Analyzers；The present invention is applied to the detection of Endostatin.
Description
Technical field
The present invention relates to a kind of latex enhancing immunes for detecting Endostatin than turbid kit and its application.
Background technique
The detection method of domestic Endostatin concentration is mainly enzyme-linked immunosorbent assay at present, but this method consumes
When and it is expensive.Immunoturbidimetry mainly includes turbidimetry, scattered light urbidmetry and immune latex turbidimetry method.Immune latex
Turbidimetry has high sensitivity, and the good feature of specificity is widely applied in large hospital clinical examination.But import tries
Agent is costly, increases the burden of patient.
Summary of the invention
In view of the deficiencies of the prior art mentioned above, problems solved by the invention is：A kind of detection Endostatin is provided
Latex enhancing immune is than turbid kit and application.
To solve the above problems, the technical solution adopted by the present invention is as follows：
It is a kind of detect Endostatin latex enhancing immune than turbid kit, including reagent R1, reagent R2, Endostatin mark
Quasi- product solution；The reagent R1 includes electrolyte, coagulant, preservative, buffer；The reagent R2 includes in recombined human
The coated latex particle of skin chalone antibody, electrolyte, stabilizer, preservative, buffer；The recombinant human endostatin antibody
The immunoglobulin that rabbit, sheep, chicken obtain through affinitive layer purification again is immunized in recombinant human endostatin for that will synthesize；The recombination
Human endostatin standard solution is the buffer containing various concentration recombinant human endostatin standard items.
Further, electrolyte described in the reagent R1 is selected from sodium chloride, magnesium chloride, potassium chloride, concentration 0.01%-
1.5%；Electrolyte described in reagent R2 is selected from sodium chloride, magnesium chloride, potassium chloride, concentration 0.01%-0.1%.
Further, coagulant described in the reagent R1 is selected from the mixed of the PEG6000 or difference PEG that concentration is 2.5%
It is fit.
Further, preservative described in the reagent R1 and R2 is selected from Sodium azide, concentration 0.05%-0.1%.
Further, buffer is selected from PBS buffer solution, MES buffer, Hepes buffer, sweet in the reagent R1 and R2
One of propylhomoserin buffer, Tris-Hcl buffer, DIPSO buffer or acetate buffer solution, buffer concentration 20- again
Between 200mM.
Further, stabilizer described in the reagent R2 is selected from EDTANa2 and bovine serum albumin(BSA), and concentration is
0.1%-1.0%.
Further, the polystyrene latex particles that latex particle is diameter range 0.05-0.20um in the reagent R2,
Latex particle concentration is 0.1%-0.8% in reagent R2.
Further, the latex particle is the polystyrene latex particles of diameter 0.13um, latex particle in reagent R2
Concentration is 0.3%.
Further, the coated latex particle of Endostatin antibody is being crosslinked by chemical cross-linking agent in the reagent R2
What buffer carried out；The chemical cross-linking agent is carbodiimides (EDAC) and n-hydroxysuccinimide (NHS)；Described
Cross-linking buffer be selected from PBS buffer solution, MES buffer, Hepes buffer, glycine buffer, Tris-Hcl buffer,
DIPSO buffer or acetate buffer solution, pH is between 6.0-8.0.
Further, the buffer of recombinant human endostatin standard items contains various concentration, and wherein Endostatin content is distinguished
For 0 μ g/mL, 1 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, buffer is PBS buffer solution, Hepes buffer
Or one or more of Tris-HCl buffer.
Further, reagent R1, R2 add take 225 μ L and 75 μ L respectively when detection, and sample pipetting volume amount is 4-12 μ L, or it is similar than
The whole increase and decrease of example.
Further, the latex enhancing immune of detection detection Endostatin is as follows than turbid kit forms：
R1 reagent：0.05mol/L PBS, 2.5%PEG6000,0.05%NaN3, 0.015%NaCl；
R2 reagent：Sensitizing latex, 30mmol/L DIPSO buffer, 0.1%NaN3, 0.01%EDTANa2With 0.09%
BSA；
Recombinant human endostatin standard solution：Endostatin is as antigen standard, 50mM PBS buffer solution.
Further, in the present invention, recombinant human endostatin and the anti-recombinant human endostatin of the coated chicken of latex particle are more
Antigen-antibody reaction occurs for clonal antibody, forms antigen-antibody-latex particle compound, turbidity is caused to rise, it is preferred that
Absorbance of this turbidity at 450-600nm (most preferred, 510nm) wavelength is measured, reference standard curve can calculate sample
Middle Endostatin content.
It is a kind of detect Endostatin latex enhancing immune than turbid kit application, which is characterized in that be applied to endothelium
The detection of chalone.
Beneficial effects of the present invention
1. current immunoturbidimetry mainly includes turbidimetry, scattered light urbidmetry and immune latex turbidimetry method.Immune glue
Newborn turbidimetry has high sensitivity, and the good feature of specificity is widely applied in large hospital clinical examination.But import
Reagent is costly, increases the burden of patient.At present Endostatin generally use the method for enzyme-linked immunosorbent assay into
Row detection, elapsed time is long in the detection process for this method, and price is higher, and the latex enhancing immune ratio developed in this experiment
Turbid method detection kit is easy to operate, and the time is short, easy to detect, and reagent is easy to save, "dead", and can use full-automatic life
Change the survey that analyzer carries out mass, there is certain researching value.
2. the latex microsphere of the bright selection appropriate particle size of research and development, is crosslinked recombinant human endostatin chicken antibody (IgY), will be original
Rabbit antibody (IgG) in research changes IgY into, is since IgY has the advantage that, it can effectively avoid mending in human body fluid first
The interference of body, rheumatoid factor or Fc receptor is to guarantee that reagent has higher specificity.Secondly genetically with human evolution
The antibody that the biggish birds of relationship difference generate has compatibility more higher than mammal polyclonal antibody.In addition such antibody
The high dosage relative reduction of potency, reagent cost aspect also have some superiority.
3. the present invention carries out discussion research to latex-enhanced turbidimetry measurement recombinant human endostatin, so that it is determined that best anti-
Condition is answered, the research and development of the domestic recombinant human endostatin detection kit of the split excellent low price of hair quality have directive significance.Yin Benfa
Bright reagent raw material are mostly domestic and self-control, and cost is relatively low, can achieve the purpose for reducing medical treatment cost.
Detailed description of the invention
Fig. 1 is the latex enhancing immune of Endostatin than turbid kit detectable concentration standard curve.
R2 prepared by the latex that Fig. 2 is 0.13um compares in different latex concentration testing results.
Fig. 3 is configured to R2 for the latex of 0.3% concentration of 0.13um and compares in different wave length testing result.
Specific embodiment
The content of present invention is described in further detail with reference to the accompanying drawing.
Preparation of the latex enhancing immune of 1 Endostatin of embodiment than turbid kit
Reagent R1：Reagent R1：50mM PBS buffer solution (pH7.4), 2.5%PEG6000,0.015%NaCl, 0.5%
NaN3, the reagent are colourless transparent solution.
Reagent R2：Recombinant human endostatin antibody is coated with latex particle (0.13 μm of partial size) 0.3%, 30mM, pH7.4's
Hepes or DIPSO buffer, 0.1%NaN3, 0.01%EDTANa2And 0.09%BSA.
The antibody of recombinant human endostatin utilizes chemical cross-linking agent carbodiimides (EDAC) and n-hydroxysuccinimide
(NHS) preparation method of the directional at-tachment on styrene latex particle：
(1) it takes 1mL 0.05mol/L MES buffer (pH6.0) into 10mL centrifuge tube, the latex of 60 μ L10% is added,
It mixes up and down, 15000rpm/min is centrifuged 30min, removes supernatant；
(2) it weighs 3.0mg EDAC to be added in 1mL MES buffer, the 100 μ L solution is then taken to be added to 900 μ L MES
Solution is made in buffer；
(3) solution matched in 1mL step (2) is added in step (1) centrifuge tube, is blown and beaten and is mixed with pipette tips, 22 DEG C
16rpm/min is protected from light revolving reaction 10min；
(4) 100 μ L 1.35mg/mL NHS are added in above-mentioned centrifuge tube, and (MES for weighing 13.5mgNHS addition 10mL is slow
In fliud flushing), 22 DEG C of 16rpm/min are protected from light revolving reaction 30min；14000rpm is centrifuged 30min, removes supernatant；
(5) 1mL 0.05M PBS (pH7.4) is added in above-mentioned centrifuge tube, is mixed up and down with pipette tips, 15000rpm/min
It is centrifuged 30min, removes supernatant；
(6) 1mL 0.05M PBS solution (PH7.4) is added in above-mentioned centrifuge tube, ultrasound mixes, and antibody 20 is then added
μ g, after mixing, 37 DEG C of 16rpm are protected from light revolving reaction 2h；
(7) 10 μ L terminate liquids are added in above-mentioned centrifuge tube, 16rpm/min is protected from light revolving reaction 1h under the conditions of 37 DEG C；
15000rpm is centrifuged 30min, removes supernatant；
(8) in above-mentioned centrifuge tube be added 1mL 30mmoL/L DIPSO buffer, ultrasound mix, 15000rpm/min from
Heart 30min；
(9) 1ml 30mmoL/LDIPSO buffer is added, ultrasound mixes, 4 DEG C of preservations.
Endostatin standard solution：Endostatin is added in 50mM PBS buffer solution as standard items, keeps it dense eventually
Degree is respectively 0 μ g/mL, 1 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL.And as calibration, carry out sample
Relative quantification detection.
Detecting step and Specification Curve of Increasing of the 2 Endostatin latex enhancing immune of embodiment than turbid kit
Detection wavelength：510nm.
Sample size：8μL；R1：250μL；R2：75μL；R1 and R2 composition is the same as embodiment 1.
By taking 7080 Biochemical Analyzer of Hitachi as an example, R1,37 DEG C of incubation 30s are firstly added, standard items are added later (to test sample
This), it is incubated for 5min, adds R2, it is poor to calculate absorbance for the absorbance value (respectively A1, A2) of measurement reaction 10s and 5min
Value OD510=A2-A1；
Specification Curve of Increasing：Using standard concentration as abscissa, absorbance difference OD510 is that ordinate draws detectable concentration
Standard curve simultaneously establishes regression equation and obtains calibration curve, sees Fig. 1；
Calculation method：The sample to be tested absorbance difference OD510 measured substitution regression equation can be calculated to test sample
Endostatin concentration in product.
Screening of the 3 Endostatin latex enhancing immune of embodiment than turbid reagent constituents
The screening that the R1 prepared and R2 is carried out to reagent constituents by taking 7080 Biochemical Analyzer of Hitachi as an example, is firstly added
Standard items (sample to be tested) is added later in R1,37 DEG C of incubation 30s, is incubated for 5min, adds R2, measurement reaction 10s and 5min
Absorbance value (respectively A1, A2), calculate absorbance difference OD510=A2-A1.
The absorbance difference for calculating the latex detection of different-grain diameter, prepares from preferably 0.13 μm of linear relationship of latex
R2。
In other condition phases likewise it is preferred that R2 prepared by 0.13 μm of latex, carries out same in different latex concentrations
As a result Fig. 2 is shown in upper detection, it is best that the latex of preferably 0.3% concentration is configured to R2 linear relationship in detection range.
It determines in R2 after the concentration of latex, carries out most suitable Detection wavelength, as a result as shown in Figure 3, when other conditions are identical,
Between 450-600 select 3 common wavelengths detected, comparatively 510 in detection range at linear relationship it is best,
Therefore select the detection of wavelength progress kit.
4 Endostatin latex enhancing immune of embodiment is than turbid kit performance detection
The present embodiment investigates kit performance based on the testing conditions that embodiment 4 optimizes
Statistical analysis：Data are expressed as average ± standard misuse 19.0 software of (X ± SE) SPSS and carry out single factor test independence
T is tested and analyzed, and P < 0.05 is determined as significant difference, and P >=0.05 is determined as that difference is not significant.
1. kit detection limit
With 20 zero standard product of kit measurement of the present invention, it is as shown in the table, and 20 blank sample detection limit average values are
0.185 μ g/mL, standard deviation is 0.036 μ g/mL, therefore the detection of kit is limited to 0.185+3 × 0.036=0.293 μ g/mL,
There are error when detecting operation and application in view of reagent, kit detection limit is determined as 0.5 μ g/mL, and kit detection is linear
Range is 0.5-20.00 μ g/mL.
The detection limit detection of the kit of the present invention of table 1
2. kit accuracy
The kit prepared with the present invention is 2.5 μ g/mL to known Endostatin concentration, 5 μ g/mL, 10 μ g/mL are carried out
It detects (n=5), accuracy is determined with the rate of recovery and the coefficient of variation (CV).The result shows that rate of recovery range is that CV is respectively less than
10%, illustrate that kit accuracy is preferable.
The kit accuracy of the present invention of table 2 detection
3. kit accuracy
Taking the detection kit of the present invention of 3 different batches is 2.5 μ g/mL, 5 μ g/mL, 10 to known Endostatin concentration
μ g/mL is surveyed (n=5), with the rate of recovery and the coefficient of variation (CV) determine accuracy.Detected value batch in and batch between variation lines
Number is respectively less than 10%, illustrates that the precision of kit is higher.
The detection of the kit accuracy of the present invention of table 3
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. it is a kind of detect Endostatin latex enhancing immune than turbid kit, which is characterized in that including reagent R1, reagent R2,
Endostatin standard solution；The reagent R1 includes electrolyte, coagulant, preservative, buffer；The reagent R2 packet
The coated latex particle of antibody containing recombinant human endostatin, electrolyte, stabilizer, preservative, buffer；In the recombined human
Skin chalone antibody is that the immune globulin that rabbit, sheep, chicken obtain through affinitive layer purification again is immunized in the recombinant human endostatin that will be synthesized
It is white；The recombinant human endostatin standard solution is the buffer containing various concentration recombinant human endostatin standard items.
2. the latex enhancing immune of detection Endostatin according to claim 1 is than turbid kit, which is characterized in that described
Reagent R1 described in electrolyte be selected from sodium chloride, magnesium chloride, potassium chloride, concentration 0.01%-1.5%；Described in reagent R2
Electrolyte be selected from sodium chloride, magnesium chloride, potassium chloride, concentration 0.01%-0.1%.
3. the latex enhancing immune of detection Endostatin according to claim 1 is than turbid kit, which is characterized in that described
Reagent R1 described in coagulant be selected from concentration be 2.5% PEG6000 or difference PEG mixture.
4. the latex enhancing immune of detection Endostatin according to claim 1 is than turbid kit, which is characterized in that described
Reagent R1 and R2 described in preservative be selected from Sodium azide, concentration 0.05%-0.1%.
5. the latex enhancing immune of detection Endostatin according to claim 1 is than turbid kit, which is characterized in that described
Reagent R1 and R2 in buffer be selected from PBS buffer solution, MES buffer, Hepes buffer, glycine buffer, Tris-Hcl
One of buffer, DIPSO buffer or acetate buffer solution, buffer concentration is again between 20-200mM.
6. the latex enhancing immune of detection Endostatin according to claim 1 is than turbid kit, which is characterized in that described
Reagent R2 described in stabilizer be selected from EDTANa2 and bovine serum albumin(BSA), concentration 0.1%-1.0%.
7. the latex enhancing immune of detection Endostatin according to claim 1 is than turbid kit, which is characterized in that described
Reagent R2 in latex particle be diameter range 0.05-0.20um polystyrene latex particles, latex particle is dense in reagent R2
Degree is 0.1%-0.8%.
8. the latex enhancing immune of detection Endostatin according to claim 7 is than turbid kit, which is characterized in that described
Latex particle be diameter 0.13um polystyrene latex particles, latex particle concentration is 0.3% in reagent R2.
9. the latex enhancing immune of detection Endostatin according to claim 1 is than turbid kit, which is characterized in that described
Reagent R2 in the coated latex particle of Endostatin antibody be to be carried out by chemical cross-linking agent in cross-linking buffer；Described
Chemical cross-linking agent is carbodiimides (EDAC) and n-hydroxysuccinimide (NHS)；The cross-linking buffer is slow selected from PBS
Fliud flushing, MES buffer, Hepes buffer, glycine buffer, Tris-Hcl buffer, DIPSO buffer or acetate buffer
Liquid, pH is between 6.0-8.0.
10. a kind of latex enhancing immune of detection Endostatin according to claim 1 is than the application of turbid kit, special
Sign is, the detection applied to Endostatin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810742366.4A CN108918888A (en) | 2018-07-09 | 2018-07-09 | It is a kind of detect Endostatin latex enhancing immune than turbid kit and its application |
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|---|---|---|---|
| CN201810742366.4A CN108918888A (en) | 2018-07-09 | 2018-07-09 | It is a kind of detect Endostatin latex enhancing immune than turbid kit and its application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112730845A (en) * | 2020-12-16 | 2021-04-30 | 广东海大畜牧兽医研究院有限公司 | Detection kit for Aeromonas schubertii latex agglutination antibody, and preparation method and application thereof |
-
2018
- 2018-07-09 CN CN201810742366.4A patent/CN108918888A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 于晓珠: "内皮抑素胶乳增强免疫比浊法检测试剂盒和糖化白蛋白酶法检测试剂盒的研究", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112730845A (en) * | 2020-12-16 | 2021-04-30 | 广东海大畜牧兽医研究院有限公司 | Detection kit for Aeromonas schubertii latex agglutination antibody, and preparation method and application thereof |
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Application publication date: 20181130 |