CN106990234B - A kind of detection reagent and method of lipoprotein (a) - Google Patents
A kind of detection reagent and method of lipoprotein (a) Download PDFInfo
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- CN106990234B CN106990234B CN201710399760.8A CN201710399760A CN106990234B CN 106990234 B CN106990234 B CN 106990234B CN 201710399760 A CN201710399760 A CN 201710399760A CN 106990234 B CN106990234 B CN 106990234B
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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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention relates to technical field of in vitro diagnostic reagents, in particular to the detection reagent and method of a kind of lipoprotein (a).The detection reagent is made of reagent 1 and reagent 2；Wherein, reagent 1 includes MOPS buffer, Tween80, preservative and water；Reagent 2 is coated with latex microsphere, glycine buffer, Tween80, the preservative, glycerol and water of lipoprotein (a) monoclonal antibody.The reagent and method testing result accuracy that the present invention detects lipoprotein (a) are high, sensitivity is good, precision is good, the range of linearity is wide, and stability is good, and strong antijamming capability, specificity are high.
Description
Technical field
The present invention relates to technical field of in vitro diagnostic reagents, in particular to the detection reagent and method of a kind of lipoprotein (a).
Background technique
Lipoprotein (a) is a kind of special plasma lipoprotein.Think that Lpa is that artery is athero- in researchs such as Dalhen in 1975
The risk factor of hardening.1978, Mclean etc., which studies the Apo (a) in lipoprotein (a), had height with plasminogen (PLG)
Homology, it is thus regarded that lipoprotein (a) is not only the risk factor of atherosclerosis, and may be related with fibrinolytic system.
Lipoprotein (a) is predicting the generation of atherosclerotic cardiovascular disease, coronary atherosclerotic heart disease (coronary disease
Disease) send out again, the generation of phlebothrombosis event and kidney transplant prognosis etc. have important clinical value.
Electrophoresis sensitivity used in early detection lipoprotein (a) is low, is chiefly used in qualitative detection.It then has developed a variety of
The immunochemistry detection method that lipoprotein (a) can directly be measured, such as radial immunodiffusion (RID), electroimmunodiffusion
(EID), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immune turbidimetry [including immune scattering turbidimetry
Method (INA) and Immunity transmission turbidity (ITA)], dissociation enhancing ligand fluorescence immunoassay (DELFIA) etc..RID method and EID
Method is not required to special installation because of easy operation, still has some grass-roots unit laboratories to use, but the disadvantage is that sensitivity is low.RIA method
The disadvantage is that manipulation is complicated, there is radionuclide contamination.DELFIA method is because needing specific apparatus, domestic also less application.Wherein it is immunized
Turbidimetry, it is fast and convenient, precision is high, be easy to automate, be suitable for high-volume sample while detection, convenient for promote, be suitble to face
Bed detection.But the antibody dosage needed is big, high (should have high specific, high titre and high-affinity) to antibody preparation, particle
Lp (a) of different sizes can generate inconsistent light scattering and light absorption, and more apparent, appearance is influenced by the matrix in sample
Vulnerable to the interference of rheumatoid factor, so researching and developing a kind of at low cost, detection method good, that deviation is small of specificity is current city
Field demand.
A kind of detection kit of lipoprotein (a) is disclosed in Publication No. CN103604930A, composition includes: Tris-
HCl buffer, lipoprotein (a) 100mg/dL, sodium chloride 150mM, sucrose 5.0%, BSA1.0%, EDTA10mM, sodium azide
0.1%.But there is also certain shortcomings in terms of anti-interference ability and precision for the kit.
Summary of the invention
In view of this, the present invention provides a kind of detection reagent of lipoprotein (a) and methods.The detection reagent and method are anti-
Interference performance is strong, specificity is high, accuracy is high, stability is good.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of detection reagent of lipoprotein (a), which is made of reagent 1 and reagent 2；
Wherein, reagent 1 includes MOPS buffer, Tween80, preservative and water；
Reagent 2 is coated with the latex microsphere of lipoprotein (a) monoclonal antibody, glycine buffer, Tween80, prevents
Rotten agent, glycerol and water.
Preferably, in reagent 1 each component dosage are as follows:
MOPS buffer: 50~200mmol/L；
Tween80:0.5~5vt%；
Preservative: 0.05~0.2wt%；
Preferably, in reagent 1 each component dosage are as follows:
MOPS buffer: 50~100mmol/L；
Tween80:1~3vt%；
Preservative: 0.05wt%.
Preferably, the concentration of Tween80 is 1~2vt% in reagent 1.
In embodiment provided by the invention, the dosage of each component in reagent 1 are as follows:
MOPS buffer: 100mmol/L；
Tween80:1.5vt%；
Preservative: 0.05wt%.
Preferably, the pH value of reagent 1 is 6~8.
Preferably, the pH value of reagent 1 is 6.5~7.5.
In embodiment provided by the invention, the pH value of reagent 1 is 7.0.
Preferably, in reagent 2 each component dosage are as follows:
It is coated with the latex microsphere of lipoprotein (a) monoclonal antibody: 0.001~0.02g/L；
Glycine buffer: 10~100mmol/L；
Tween80:0.5~5vt%；
Preservative: 0.05~0.2wt%；
Glycerol: 2~10vt%；
Preferably, in reagent 2 each component dosage are as follows:
It is coated with the latex microsphere of lipoprotein (a) monoclonal antibody: 0.001~0.01g/L；
Glycine buffer: 10~50mmol/L；
Tween80:1~5vt%；
Preservative: 0.1wt%；
Glycerol: 2~10vt%.
Preferably, the concentration of Tween80 is 2~5vt% in reagent 2.
In embodiment provided by the invention, the dosage of each component in reagent 2 are as follows:
It is coated with the latex microsphere of lipoprotein (a) monoclonal antibody: 0.001g/L；
Glycine buffer: 50mmol/L；
Tween80:2vt%；
Preservative: 0.1wt%；
Glycerol: 3vt%.
Preferably, the pH value of reagent 2 is 7~9.
Preferably, the pH value of reagent 2 is 7~8.
In embodiment provided by the invention, the pH value of reagent 2 is 7.5.
In the present invention, the selection of appropriate pH improves the storage stability of reagent.
Preferably, preservative is Sodium azide.
In embodiment provided by the invention, the latex microsphere for being coated with lipoprotein (a) monoclonal antibody is by lipoprotein
(a) monoclonal antibody and latex microsphere are obtained using chemical crosslink technique.
In embodiment provided by the invention, the system of the latex microsphere of lipoprotein (a) monoclonal antibody is coated in reagent 2
Preparation Method are as follows: mix lipoprotein (a) monoclonal antibody with the latex microsphere by MES activation, 30 DEG C of crosslinkings obtain.
Preferably, lipoprotein (a) monoclonal antibody is anti-human apolipoprotein (a) monoclonal antibody of mouse.
Preferably, latex microsphere is hydrophobic microballoon.The selection of latex microsphere improves the efficiency of chemical crosslinking, reduces
The loss of antibody, reduces costs, and improves sensitivity and precision.
Preferably, hydrophobic microballoon is one or more kinds of with sulfonic group, carboxylic group or aldehyde groups
Latex microsphere.
Preferably, hydrophobic microballoon is the latex microsphere with sulfonic group and/or carboxylic group.
Preferably, the average grain diameter of latex microsphere is 100~300nm.
Preferably, the average grain diameter of latex microsphere is 200~300nm.
Preferably, the mass ratio of lipoprotein (a) monoclonal antibody and latex microsphere is (0.5~1): 100.
Preferably, the mass ratio of lipoprotein (a) monoclonal antibody and latex microsphere is 0.8:100.
The present invention also provides a kind of detection methods of lipoprotein (a) content, comprising: sample to be tested is added in reagent 1, incubates
The formation agglutination of reagent 2 is added after educating 5~10min, measures absorbance under 660nm wavelength, the standard for passing through lipoprotein (a) is bent
Line obtains the content of lipoprotein (a) in sample to be tested；
Wherein, reagent 1 includes MOPS buffer, Tween80, preservative and water；
Reagent 2 is coated with the latex microsphere of lipoprotein (a) monoclonal antibody, glycine buffer, Tween80, prevents
Rotten agent, glycerol and water.
Preferably, the temperature being incubated for is 37 DEG C.
Preferably, the volume ratio of reagent 1 and reagent 2 is 3:1 in the detection process.
The present invention provides a kind of detection reagent of lipoprotein (a) and methods.The detection reagent is by reagent 1 and 2 groups of reagent
At；Wherein, reagent 1 includes MOPS buffer, Tween80, preservative and water；Reagent 2 is coated with lipoprotein (a) monoclonal
The latex microsphere of antibody, glycine buffer, Tween80, preservative, glycerol and water.The present invention at least have following advantage it
One:
(1) reagent of present invention detection lipoprotein (a) and method testing result accuracy are high, sensitivity is good, precision is good,
The range of linearity is wide, and stability is good, long shelf-life, at low cost convenient for promoting the use of, and can be widely applied to situation of all-level hospitals, health
The content of lipoprotein (a) in prevention department and medical biotechnology R&D institution measurement human serum.
(2) detection reagent strong antijamming capability of the present invention, specificity are high.The selection of surfactant, it is possible to reduce sample
Influence of the turbidity to experimental result；The selection specificity of antibody is more preferable, it is possible to reduce nonspecific interference and rheumatoid factor
Interference.
(3) time for simplifying processing latex microsphere and labelled antibody in reagent preparation process, process flow is simplified, is made
It operates easier.
Specific embodiment
The invention discloses a kind of detection reagent of lipoprotein (a) and method, those skilled in the art can be used for reference herein
Content is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
It is it will be apparent that they are considered as being included in the present invention for member.Method and application of the invention has passed through preferably real
It applies example to be described, related personnel can obviously not depart from the content of present invention, in spirit and scope to methods herein and answer
With being modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Agents useful for same or instrument are available on the market in the detection reagent and method of lipoprotein (a) provided by the invention.
The formula of commercially available domestic lipoprotein (a) kit (latex enhancing immune turbidimetry) is as follows in following embodiment:
Reagent 1:40mmol/L contains 1.0%NaN3Glycine buffer；
Reagent 2: the sensitization latex particle suspension of anti-human lipoprotein (a)-IgG.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1: the present invention detects the reagent and method of lipoprotein (a)
It is coated with the preparation of the latex particle of anti-human lipoprotein (a) monoclonal antibody of mouse:
0.01% carboxylic group latex microsphere is activated with the MES solution of equivalent, then the anti-human lipoprotein (a) of 5mg/mL mouse
Monoclonal antibody is mixed with the latex microsphere through overactivation, and 30 DEG C of crosslinkings obtain.
The present embodiment reagent includes:
Reagent 1:100mmol/L MOPS buffer, Tween80 1.2%, preservative Sodium azide 0.05%, pH 7.0；
Reagent 2: being coated with the latex particle 0.001g/L of anti-human lipoprotein (a) monoclonal antibody of mouse, and partial size is 200~
300nm, 50mol/L glycine buffer, 0.1% Sodium azide, 2%Tween80,3% glycerol, pH value 7.5.
Detection method: sample to be tested serum is added in reagent 1, is incubated in 5min and adds the formation agglutination of reagent 2, reagent 1
Volume ratio with reagent 2 is 3:1, measures absorbance under 660nm wavelength, calculates sample by the standard curve of lipoprotein (a)
The content of middle lipoprotein (a).
Embodiment 2: the present invention detects the reagent and method of lipoprotein (a)
It is coated with the preparation of the latex particle of anti-human lipoprotein (a) monoclonal antibody of mouse:
0.01% sulfonic group latex microsphere is activated with the MES solution of equivalent, then the anti-human lipoprotein of 5mg/mL mouse
(a) monoclonal antibody is mixed with the latex microsphere through overactivation, and 30 DEG C of crosslinkings obtain.
The present embodiment reagent includes:
Reagent 1:100mmol/L MOPS buffer, Tween80 1.2%, preservative Sodium azide 0.05%, with reference to pH
7.0；
Reagent 2: being coated with the latex particle 0.001g/L of anti-human lipoprotein (a) monoclonal antibody of mouse, and partial size is 200~
300nm, 50mol/L glycine buffer, 0.1% Sodium azide, 2%Tween80,3% glycerol, pH value 7.5.
Detection method: sample to be tested serum is added in reagent 1, is incubated in 5min and adds the formation agglutination of reagent 2, reagent 1
Volume ratio with reagent 2 is 3:1, measures absorbance under 660nm wavelength, calculates sample by the standard curve of lipoprotein (a)
The content of middle lipoprotein (a).
Embodiment 3: the present invention detects the reagent and method of lipoprotein (a)
It is coated with the preparation of the latex particle of anti-human lipoprotein (a) monoclonal antibody of mouse:
0.01% carboxyl and sulfonic group latex microsphere are activated with the MES solution of equivalent, it is then that 5mg/mL mouse is anti-human
Lipoprotein (a) monoclonal antibody is mixed with the latex microsphere through overactivation, and 30 DEG C of crosslinkings obtain.
The present embodiment reagent includes:
Reagent 1:100mmol/L MOPS buffer, Tween80 1.2%, preservative Sodium azide 0.05%, with reference to pH
7.0；
Reagent 2: being coated with the latex particle 0.001g/L of anti-human lipoprotein (a) monoclonal antibody of mouse, and partial size is 200~
300nm, 50mol/L glycine buffer, 0.1% Sodium azide, 2%Tween80,3% glycerol, pH value 7.5.
Detection method: sample to be tested serum is added in reagent 1, is incubated in 5min and adds the formation agglutination of reagent 2, reagent 1
Volume ratio with reagent 2 is 3:1, measures absorbance under 660nm wavelength, calculates sample by the standard curve of lipoprotein (a)
The content of middle lipoprotein (a).
Embodiment 4: the accuracy analysis of detection method
Test apparatus: 7080 automatic clinical chemistry analyzer of Hitachi；
Test sample: 20 examinee's serum samples；
Contrast agent 1: commercially available domestic lipoprotein (a) kit (latex enhancing immune turbidimetry).
20 samples are measured respectively with the detection reagent and contrast agent of embodiment 1 to embodiment 3, and testing result is shown in Table 1-
1~1-3.
The accuracy analysis result of 1 reagent of table 1-1 embodiment
The accuracy analysis result of 2 reagent of table 1-2 embodiment
The accuracy analysis result of 3 reagent of table 1-3 embodiment
The results show that the R of calculated 1 reagent test method of embodiment according to testing result2Value is 0.995, embodiment
2, the R of 3 detection methods2Value shows the method for the present invention testing result with contrast agent box testing result without bright also greater than 0.95
Significant difference is different, has high accuracy (degree of conformity).
Embodiment 5: the Precision Analyze of detection method
Test apparatus: 7080 automatic clinical chemistry analyzer of Hitachi；
Test sample: low value, intermediate value, each portion of high level blood serum sample；Intermediate value serum sample is a；
Detection 10 times, detection are repeated to same sample to be tested using the reagent and detection method of embodiment 1 to embodiment 3
It the results are shown in Table 2.
2 test sample lipoprotein (a) concentration of table (10 times) and standard rate (coefficient of variation)
The results show that 1,2,3 low value of example, intermediate value, high level sample canonical rate is respectively 2.72%, 2.07%,
2.27%；1.45%, 1.07%, 1.12%；1.19%, 0.99%, 1.12%, respectively less than 3%, and it is less than the mark of contrast agent
Quasi- rate shows that the method for the present invention has high precision.
Embodiment 6: the anti-jamming Performance Analysis of the method for the present invention
Test apparatus: 7080 automatic clinical chemistry analyzer of Hitachi；
Test sample: any one blood serum sample:
The rheumatoid factor (50,150,250,500,800IU/mL) of various concentration is added in same a serum sample,
Detection 3 times is repeated to same sample to be tested using the detection method of embodiment 1 to embodiment 3, and rheumatoid factor is not added
Serum compares, and calculates deviation, and testing result is shown in Table 3.In the present embodiment, commercially available domestic lipoprotein (a) kit is chosen
(latex enhancing immune turbidimetry) reagent as a comparison, compares.
The average value and absolute value of the bias of 3 test sample lipoprotein (a) concentration of table
The results show that reagent rheumatoid factor≤800IU/mL of the invention does not influence measured value, and contrast agent box 1
Only measured value is not influenced in rheumatoid factor < 300IU/mL.
The formula of commercially available domestic lipoprotein (a) kit (latex enhancing immune turbidimetry) is as follows in above example:
Glycine buffer of the reagent 1:40mmol/L containing 1.0%NaN3
Reagent 2: the sensitization latex particle suspension of anti-human lipoprotein (a)-IgG
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (7)
1. a kind of detection reagent of lipoprotein (a), which is characterized in that the detection reagent is made of reagent 1 and reagent 2；
The reagent 1 is made of MOPS buffer, Tween80, preservative and water；
The reagent 2 is by being coated with the latex microsphere, glycine buffer, Tween80, anti-corrosion of lipoprotein (a) monoclonal antibody
Agent, glycerol and water composition；
The dosage of each component in the reagent 1 are as follows:
MOPS buffer: 50~200mmol/L；
Tween80:0.5~5vt%；
Preservative: 0.05~0.2wt%；
The pH value of the reagent 1 is 6~8；
The dosage of each component in the reagent 2 are as follows:
It is coated with the latex microsphere of lipoprotein (a) monoclonal antibody: 0.001~0.02g/L；
Glycine buffer: 10~100mmol/L；
Tween80:0.5~5vt%；
Preservative: 0.05~0.2wt%；
Glycerol: 2~10vt%；
The pH value of the reagent 2 is 7~9.
2. detection reagent according to claim 1, which is characterized in that the preservative is Sodium azide.
3. detection reagent according to claim 1, which is characterized in that lipoprotein (a) monoclonal antibody of being coated with
Latex microsphere is to be obtained by lipoprotein (a) monoclonal antibody and latex microsphere using chemical crosslink technique.
4. detection reagent according to claim 3, which is characterized in that lipoprotein (a) monoclonal antibody is that mouse is anti-human
Apolipoprotein (a) monoclonal antibody.
5. detection reagent according to claim 3, which is characterized in that the latex microsphere is band sulfonic group, carboxyl
The latex microsphere of group or one or more groups of aldehyde groups.
6. the detection reagent according to claim 3 or 5, which is characterized in that the average grain diameter of the latex microsphere be 100~
300nm。
7. detection reagent according to claim 3, which is characterized in that lipoprotein (a) monoclonal antibody and the glue
The mass ratio of newborn microballoon is (0.5~1): 100.
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| CN111239422A (en) * | 2018-12-25 | 2020-06-05 | 武汉生之源生物科技股份有限公司 | Serum lipoprotein (a) latex enhanced turbidimetric detection kit |
| CN110082532A (en) * | 2019-05-20 | 2019-08-02 | 吉林瑞特生物科技有限公司 | Lipoprotein a assay kit and preparation method thereof |
| CN112763731B (en) * | 2020-12-29 | 2023-02-28 | 广州市伊川生物科技有限公司 | Lipoprotein (a) determination kit and detection method thereof |
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