CN105759051B - A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3 and preparation method thereof - Google Patents
A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3 and preparation method thereof Download PDFInfo
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- CN105759051B CN105759051B CN201610104825.7A CN201610104825A CN105759051B CN 105759051 B CN105759051 B CN 105759051B CN 201610104825 A CN201610104825 A CN 201610104825A CN 105759051 B CN105759051 B CN 105759051B
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G01N2333/4722—Proteoglycans, e.g. aggreccan
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Abstract
A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3 has the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody including GPC3 standard items, the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label, coupling.The present invention also provides the production methods of the kit.The present invention is using acridinium ester as the GPC3 of luminous marker nanometer magnetic microsphere chemical luminescence immune assay determination reagent kit, not only have the characteristics that high sensitivity, the range of linearity are wide, specificity is good, marker is stable, at low cost, and its detection program is also easy, quick, only trace sample is needed to can be detected.
Description
Technical field
The present invention relates to the kits that a kind of detection kit more particularly to a kind of GPC3 detect.
Background technique
Monophosphoinositideproteoglycans proteoglycans-3 (glypican-3, GPC3) is a kind of heparin sulfate proteoglycan, by albumen
The complicated saccharide complex that matter, lipid and sugared three are covalently attached.Formation and development of the GPC3 in embryo and fetal tissue organ
Stage plays key player, mainly plays negativity regulating and controlling effect, prevents histoorgan overgrowth and adjust body on the whole
Size；Hepatic parenchymal cells have obvious expression in entire foetal period, do not express in adult human liver, and liver cancer early stage occurs can be by
Reactivation and high expression, GPC3 is not expressed or in low table in benign hepatopathy (hepatitis, fatty liver), cirrhosis and hepatobiliary cancer
It reaches, therefore soluble g PC3 can be used as the Specific marker of primary carcinoma of liver in serum, is played in the antidiastole of liver cancer
Effect.
The chemiluminometry of presently used detection GPC3 is mainly enzymatic chemiluminometry, is with enzyme mark
Remember object specificity junction mixture to be checked, form the compound of object and enzyme to be checked, luminous substrate is then acted on enzyme, is tried in signal
Agent effect is lower to shine, and carries out luminescence assays with luminous signal analyzer.Since enzymatic chemiluminometry is by enzyme and hair
The limitation of light substrate leads to the enhancing of its background luminescence, measurement cost height etc., thus limit this method sensitivity and it
Using.Therefore the kit of detection GPC3 that can be quick, accurate, at low cost a kind of is needed to meet the needs of detection.
Summary of the invention
For effective solution technical problem set forth above, the present invention provides a kind of Monophosphoinositideproteoglycans proteoglycans-3s
Quantification assay kit and preparation method thereof.
A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3
(1) GPC3 standard items, the GPC3 are Monophosphoinositideproteoglycans proteoglycans-3；
(2) the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label；
(3) coupling has the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody.
Preferably, the GPC3 standard items be solution state, concentration be respectively as follows: 0ng/mL, 0.5ng/mL, 2.0ng/mL,
5.0ng/mL、20.0ng/mL、100.0ng/mL。
Preferably, the kit further includes luminescent solution, and the luminescent solution is H2O2Solution.
Preferably, the coupling has the nanometer magnetic microsphere in Monophosphoinositideproteoglycans proteoglycans-3 antibody nano magnetic microspheres solution
It is 3 μm for diameter, the nanometer magnetic microsphere of superparamagnetism of the surface with carboxyl.
The invention also includes a kind of preparation methods of the quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3, including such as
Lower step:
(1) GPC3: being configured to the GPC3 solution of ladder concentration with standard items buffer by the preparation of GPC3 standard items, described
Standard items buffer include mass ratio be 1~5%BSA, 0.5~1.0%NaCl, 0.05~0.1%TritonX-100 or
The phosphate buffer of Tween20,0.05~0.1%ProClin 300 and surplus；The phosphate buffer density be 0.1~
0.2mol/L；
(2) preparation of the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label:
A, GPC3 antibody is taken to be dissolved in, pH is that 6.8~7.3 concentration are in the phosphate buffer of 0.2mol/L, until final concentration of 2
~8mg/L；
B, acridinium ester is added to the final concentration of 0.1~0.5mg/L of acridinium ester to step a treated GPC3 antibody-solutions,
It is protected from light standing 8~16 hours, collects the GPC3 antibody-solutions for being marked with acridinium ester；
(3) coupling has the preparation of the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody:
A, GPC3 antibody is taken to be dissolved in, pH is that 4.5~5.5 concentration are in the MES solution of 0.2mol/L, until final concentration of 2~
8mg/L；The MES is 2- (N- morpholine) ethanesulfonic acid monohydrate；
B, 1 parts by weight nanometer magnetic microsphere is taken, the coupling agent one and 2~5 parts by weight coupling agents two of 1~4 parts by weight is added, sets
It is rotated 20~40 minutes in magnetic microsphere mixer, removes supernatant；It adds and connects buffer washing nanometer magnetic microsphere, in removing
Clearly；The coupling agent one is 1~2%NHS including mass ratio and the pH of surplus is the MES that 4.5~5.5 concentration are 0.2mol/L；
The coupling agent two is 1~2%EDC including mass ratio and the pH of surplus is the MES that 4.5~5.5 concentration are 0.2mol/L；It is described
Connection buffer is the 0.2mol/L MES solution that pH is 4.5~5.5；The NHS is N- hydroxysuccinimide, the EDC
For 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride；
C, by step A treated GPC3 antibody-solutions and step B treated nanometer magnetic microsphere 1:25 in mass ratio~
100 mixings form GPC3 antibody nanometer magnetic microsphere mixed liquor, place magnetic microsphere mixer and rotate 10~14 hours, then with 1~5 times
The coating buffer exchange of the volume of GPC3 antibody nanometer magnetic microsphere mixed liquor connects buffer；Obtaining coupling has phosphatidylinositols
The nano magnetic microspheres solution of -3 antibody of proteoglycans；The coating buffer include mass ratio be 1%~3%BSA, 0.1%~
0.5%NaCl, 0.1%~0.5%TritonX-100 or Tween20,0.05%~0.1%Proclin-300 and surplus
40~50mmol/L Tris-HCl, the pH of the coating buffer is 6.8~7.4.
Preferably, the preparation method, includes the following steps:
(1) GPC3 the preparation of GPC3 standard items: is configured to 0ng/mL, 0.5ng/mL, 2.0ng/ with standard items buffer
ML, 5.0ng/mL, 20.0ng/mL, 100.0ng/mL series of concentrations；The standard items buffer include mass ratio be 3%BSA,
0.6%NaCl, 0.1%TritonX-100 or Tween20,0.05%Proclin-300, surplus 0.2mol/L phosphoric acid buffer
Liquid, the pH of the standard items buffer are 7.0；
(2) preparation of the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label:
1., take GPC3 antibody to be dissolved in, pH is that 7.0 concentration are in the phosphate buffer of 0.2mol/L, until final concentration of 5mg/
L；
2., to step 1. treated GPC3 antibody-solutions acridinium ester is added to the final concentration of 0.25mg/L of acridinium ester, keep away
Light stands 10 hours, collects the GPC3 antibody-solutions for being marked with acridinium ester；
(3) coupling has the preparation of the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody:
I takes GPC3 antibody to be dissolved in, and PH is that 5.0 concentration are in the MES solution of 0.2mol/L, until final concentration of 5mg/L；
II takes 1 parts by weight nanometer magnetic microsphere, and the coupling agent one and 3 parts by weight coupling agents two of 2 parts by weight is added, is placed in magnetic
Microballoon mixer rotates 30 minutes, removes supernatant；Connection buffer washing nanometer magnetic microsphere is added, supernatant is removed；The idol
Join the MES that agent one is 5.0 including the pH that mass ratio is 1% NHS and the concentration of surplus is 0.2mol/L；The coupling agent two wraps
The MES solution for the 0.2mol/L that the pH for including the mass ratio EDC for being 1% and surplus is 5.0；It is 5.0 that the connection buffer, which is pH,
0.2mol/L MES solution；
III, by the GPC3 antibody-solutions after I step process and the nanometer magnetic microsphere 1:40 in mass ratio after II step process
Mixing forms GPC3 antibody nanometer magnetic microsphere mixed liquor, places magnetic microsphere mixer and rotates 12 hours, then is received with 3 times of GPC3 antibody
The coating buffer exchange of the volume of rice magnetic microsphere mixed liquor connects buffer；Obtaining coupling has Monophosphoinositideproteoglycans proteoglycans-3
The nano magnetic microspheres solution of antibody；The coating buffer includes that mass ratio is 1%BSA, 0.5%NaCl, 0.1%TritonX-
The 30mmol/L Tris-HCl of 100 or Tween20,0.05%Proclin-300 and surplus, the coating pH of buffer are
7.0。
Compared with prior art, existing to Monophosphoinositideproteoglycans proteoglycans-3 detection the beneficial effects of the present invention are overcoming
The problems such as the at high cost of method, background interference are big and needs additionally enhance the substrate of signal, and solve prior art serum
It learns detection GPC3 and needs the big halfway deficiency of reaction of specimen amount.The present invention is using acridinium ester as the GPC3 nano magnetic of luminous marker
Microballoon chemical luminescence immune assay determination reagent kit, not only have high sensitivity, the range of linearity is wide, specificity is good, marker is steady
The features such as fixed, at low cost, and its detection program is also easy, quick, only trace sample is needed to can be detected.
Specific embodiment
In the following, being described further in conjunction with specific embodiment to the present invention:
Embodiment one
A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3 includes the following:
(1) GPC3 standard items；
(2) the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label；
(3) coupling has the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody；
The kit is made by following steps:
(1) GPC3: being configured to the GPC3 solution of ladder concentration with standard items buffer by the preparation of GPC3 standard items, described
Standard items buffer includes that mass ratio is 1%BSA, 0.5%NaCl, 0.05%TritonX-100 or Tween20,0.05%
The phosphate buffer of ProClin 300 and surplus；The phosphate buffer density is 0.1mol/L.
(2) preparation of the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label:
A, GPC3 antibody is taken to be dissolved in, pH is that 6.8 concentration are in the phosphate buffer of 0.2mol/L, until final concentration of 2mg/L.
B, acridinium ester is added to the final concentration of 0.1mg/L of acridinium ester to step a treated GPC3 antibody-solutions, is protected from light quiet
It sets 8 hours, collects the GPC3 antibody-solutions for being marked with acridinium ester.
(3) coupling has the preparation of the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody:
A, GPC3 antibody is taken to be dissolved in, pH is that 4.5 concentration are in the MES solution of 0.2mol/L, until final concentration of 2mg/L.
B, 1 parts by weight nanometer magnetic microsphere is taken, the coupling agent one and 2 parts by weight coupling agents two of 1 parts by weight is added, it is micro- to be placed in magnetic
Ball mixer rotates 20 minutes, removes supernatant；Connection buffer washing nanometer magnetic microsphere is added, supernatant is removed；The coupling
Agent one is 1%NHS including mass ratio and the pH of surplus is the MES that 4.5 concentration are 0.2mol/L；The coupling agent two includes quality
Than being MES that 4.5 concentration are 0.2mol/L for the pH of 1%EDC and surplus；It is 4.5 that the connection buffer, which is pH,
0.2mol/L MES solution；The nanometer magnetic microsphere be diameter be 3 μm, the nanometer magnetic microsphere of superparamagnetism of the surface with carboxyl.
C, by step A treated GPC3 antibody-solutions and step B, treated that nanometer magnetic microsphere 1:25 in mass ratio is mixed
Even formation GPC3 antibody nanometer magnetic microsphere mixed liquor is placed magnetic microsphere mixer and is rotated 10 hours, then with 1 times of GPC3 antibody nanometer
The coating buffer exchange of the volume of magnetic microsphere mixed liquor connects buffer；Obtaining coupling has Monophosphoinositideproteoglycans proteoglycans-3 anti-
The nano magnetic microspheres solution of body；The coating buffer includes that mass ratio is 1%BSA, 0.1%NaCl, 0.1%TritonX-
100 or the 40mmol/L Tris-HCl, the pH of the coating buffer of Tween20,0.05%Proclin-300 and surplus be
6.8。
Embodiment two
A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3 includes the following:
(1) GPC3 standard items；
(2) the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label；
(3) coupling has the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody；
The kit is made by following steps:
(1) GPC3: being configured to the GPC3 solution of ladder concentration with standard items buffer by the preparation of GPC3 standard items, described
Standard items buffer includes that mass ratio is 5%BSA, 1.0%NaCl, 0.1%TritonX-100 or Tween20,0.1%
The phosphate buffer of Proclin-300 and surplus；The phosphate buffer density is 0.2mol/L.
(2) preparation of the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label:
A, GPC3 antibody is taken to be dissolved in, pH is that 7.3 concentration are in the phosphate buffer of 0.2mol/L, until final concentration of 8mg/
L。
B, acridinium ester is added to the final concentration of 0.5mg/L of acridinium ester to step a treated GPC3 antibody-solutions, is protected from light quiet
It sets 16 hours, collects the GPC3 antibody-solutions for being marked with acridinium ester.
(3) coupling has the preparation of the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody:
A, GPC3 antibody is taken to be dissolved in, pH is that 5.5 concentration are in the MES solution of 0.2mol/L, until final concentration of 8mg/L.
B, 1 parts by weight nanometer magnetic microsphere is taken, the coupling agent one and 5 parts by weight coupling agents two of 4 parts by weight is added, it is micro- to be placed in magnetic
Ball mixer rotates 40 minutes, removes supernatant；Connection buffer washing nanometer magnetic microsphere is added, supernatant is removed；The coupling
Agent one is 2%NHS including mass ratio and the pH of surplus is the MES that 5.5 concentration are 0.2mol/L；The coupling agent two includes quality
Than being MES that 5.5 concentration are 0.2mol/L for the pH of 2%EDC and surplus；The connection buffer is the 0.2mol/ that pH is 5.5
L MES solution；The nanometer magnetic microsphere be diameter be 3 μm, the nanometer magnetic microsphere of superparamagnetism of the surface with carboxyl.
C, by step A treated GPC3 antibody-solutions and step B, treated that nanometer magnetic microsphere 1:100 in mass ratio is mixed
Even formation GPC3 antibody nanometer magnetic microsphere mixed liquor is placed magnetic microsphere mixer and is rotated 14 hours, then with 5 times of GPC3 antibody nanometers
The coating buffer exchange of the volume of magnetic microsphere mixed liquor connects buffer；Obtaining coupling has Monophosphoinositideproteoglycans proteoglycans-3 anti-
The nano magnetic microspheres solution of body；The coating buffer includes that mass ratio is 3%BSA, 0.5%NaCl, 0.5%TritonX-
100 or the 50mmol/L Tris-HCl, the pH of the coating buffer of Tween20,0.1%Proclin-300 and surplus be
7.4。
Embodiment three
A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3 includes the following:
(1) GPC3 standard items；
(2) the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label；
(3) coupling has the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody；
(4) luminescent solution.
The kit is made by following steps:
(1) GPC3 the preparation of GPC3 standard items: is configured to 0ng/mL, 0.5ng/mL, 2.0ng/ with standard items buffer
ML, 5.0ng/mL, 20.0ng/mL, 100.0ng/mL series of concentrations；The standard items buffer include mass ratio be 3%BSA,
0.6%NaCl, 0.1%TritonX-100 or Tween20,0.05%Proclin-300, surplus 0.2mol/L phosphoric acid buffer
Liquid, the pH of the standard items buffer are 7.0.
(2) preparation of the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label:
1., take GPC3 antibody to be dissolved in, PH is that 7.0 concentration are in the phosphate buffer of 0.2mol/L, until final concentration of 5mg/
L。
2., to step 1. treated GPC3 antibody-solutions acridinium ester is added to the final concentration of 0.25mg/L of acridinium ester, keep away
Light stands 10 hours, collects the GPC3 antibody-solutions for being marked with acridinium ester.
(3) coupling has the preparation of the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody:
I takes GPC3 antibody to be dissolved in, and PH is that 5.0 concentration are in the MES solution of 0.2mol/L, until final concentration of 5mg/L.
II takes 1 parts by weight nanometer magnetic microsphere, and the coupling agent one and 3 parts by weight coupling agents two of 2 parts by weight is added, is placed in magnetic
Microballoon mixer rotates 30 minutes, removes supernatant；Connection buffer washing nanometer magnetic microsphere is added, supernatant is removed；The idol
Join the MES that agent one is 5.0 including the pH that mass ratio is 1%NHS and the concentration of surplus is 0.2mol/L；The coupling agent two includes
The MES solution for the 0.2mol/L that the pH of EDC and surplus that mass ratio is 1% are 5.0；It is 5.0 that the connection buffer, which is PH,
0.2mol/L MES solution；The nanometer magnetic microsphere be diameter be 3 μm, the nanometer magnetic microsphere of superparamagnetism of the surface with carboxyl.
III, by the GPC3 antibody-solutions after I step process and the nanometer magnetic microsphere 1:40 in mass ratio after II step process
Mixing forms GPC3 antibody nanometer magnetic microsphere mixed liquor, places magnetic microsphere mixer and rotates 12 hours, then is received with 3 times of GPC3 antibody
The coating buffer exchange of the volume of rice magnetic microsphere mixed liquor connects buffer；Obtaining coupling has Monophosphoinositideproteoglycans proteoglycans-3
The nano magnetic microspheres solution of antibody；The coating buffer includes that mass ratio is 1%BSA, 0.5%NaCl, 0.1%TritonX-
The 30mmol/L Tris-HCl of 100 or Tween20,0.05%Proclin-300 and surplus, the coating pH of buffer are
7.0。
Example IV
A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3 includes the following:
(1) GPC3 standard items；
(2) the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label；
(3) coupling has the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody.
The kit the production method is as follows:
(1) GPC3 the preparation of GPC3 standard items: is configured to 0ng/mL, 0.5ng/mL, 2.0ng/ with standard items buffer
ML, 5.0ng/mL, 20.0ng/mL, 100.0ng/mL series of concentrations；The standard items buffer include mass ratio be 3%BSA,
0.6%NaCl, 0.1%TritonX-100 or Tween20,0.05%Proclin-300, surplus 0.2mol/L phosphoric acid buffer
Liquid, the pH of the standard items buffer are 7.0.
(2) preparation of the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label:
1., take GPC3 antibody to be dissolved in, pH is that 7.0 concentration are in the phosphate buffer of 0.2mol/L, until final concentration of 3mg/
L。
2., to step 1. treated GPC3 antibody-solutions acridinium ester is added to the final concentration of 0.35mg/L of acridinium ester, keep away
Light stands 10 hours, collects the GPC3 antibody-solutions for being marked with acridinium ester；
(3) coupling has the preparation of the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody:
I takes GPC3 antibody to be dissolved in, and pH is that 5.0 concentration are in the MES solution of 0.2mol/L, until final concentration of 3mg/L.
II takes 1 parts by weight nanometer magnetic microsphere, and the coupling agent one and 3 parts by weight coupling agents two of 2 parts by weight is added, is placed in magnetic
Microballoon mixer rotates 30 minutes, removes supernatant；Connection buffer washing nanometer magnetic microsphere is added, supernatant is removed；The idol
Join the MES that agent one is 5.0 including the pH that mass ratio is 1%NHS and the concentration of surplus is 0.2mol/L；The coupling agent two includes
The MES solution for the 0.2mol/L that the pH of EDC and surplus that mass ratio is 1% are 5.0；It is 5.0 that the connection buffer, which is pH,
0.2mol/L MES solution；The nanometer magnetic microsphere be diameter be 3 μm, the nanometer magnetic microsphere of superparamagnetism of the surface with carboxyl.
III, by the GPC3 antibody-solutions after I step process and the nanometer magnetic microsphere 1:60 in mass ratio after II step process
Mixing forms GPC3 antibody nanometer magnetic microsphere mixed liquor, places magnetic microsphere mixer and rotates 12 hours, then is received with 3 times of GPC3 antibody
The coating buffer exchange of the volume of rice magnetic microsphere mixed liquor connects buffer；Obtaining coupling has glypican-
The nano magnetic microspheres solution of 3 antibody；The coating buffer includes that mass ratio is 1%BSA, 0.5%NaCl, 0.1%
The 30mmol/L Tris-HCl of TritonX-100 or Tween20,0.05%Proclin-300 and surplus, the coating are slow
Fliud flushing pH is 7.0.
Embodiment five
A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3 includes the following:
(1) GPC3 standard items；
(2) the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label；
(3) coupling has the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody.
The kit the production method is as follows:
(1) GPC3 the preparation of GPC3 standard items: is configured to 0ng/mL, 0.5ng/mL, 2.0ng/ with standard items buffer
ML, 5.0ng/mL, 20.0ng/mL, 100.0ng/mL series of concentrations；The standard items buffer include mass ratio be 3%BSA,
0.6%NaCl, 0.1%TritonX-100 or Tween20,0.05%Proclin-300, surplus 0.2mol/L phosphoric acid buffer
Liquid, the pH of the standard items buffer are 7.0.
(2) preparation of the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label:
1., take GPC3 antibody to be dissolved in, pH is that 7.0 concentration are in the phosphate buffer of 0.2mol/L, until final concentration of 7mg/
L。
2., to step 1. treated GPC3 antibody-solutions acridinium ester is added to the final concentration of 0.45mg/L of acridinium ester, keep away
Light stands 10 hours, collects the GPC3 antibody-solutions for being marked with acridinium ester；
(3) coupling has the preparation of the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody:
I takes GPC3 antibody to be dissolved in, and pH is that 5.0 concentration are in the MES solution of 0.2mol/L, until final concentration of 7mg/L.
II takes 1 parts by weight nanometer magnetic microsphere, and the coupling agent one and 3 parts by weight coupling agents two of 2 parts by weight is added, is placed in magnetic
Microballoon mixer rotates 30 minutes, removes supernatant；Connection buffer washing nanometer magnetic microsphere is added, supernatant is removed；The idol
Join the MES that agent one is 5.0 including the pH that mass ratio is 1%NHS and the concentration of surplus is 0.2mol/L；The coupling agent two includes
The MES solution for the 0.2mol/L that the pH of EDC and surplus that mass ratio is 1% are 5.0；It is 5.0 that the connection buffer, which is pH,
0.2mol/L MES solution；The nanometer magnetic microsphere be diameter be 3 μm, the nanometer magnetic microsphere of superparamagnetism of the surface with carboxyl.
III, by the GPC3 antibody-solutions after I step process and the nanometer magnetic microsphere 1:80 in mass ratio after II step process
Mixing forms GPC3 antibody nanometer magnetic microsphere mixed liquor, places magnetic microsphere mixer and rotates 12 hours, then is received with 3 times of GPC3 antibody
The coating buffer exchange of the volume of rice magnetic microsphere mixed liquor connects buffer；Obtaining coupling has Monophosphoinositideproteoglycans proteoglycans-3
The nano magnetic microspheres solution of antibody；The coating buffer includes that mass ratio is 1%BSA, 0.5%NaCl, 0.1%TritonX-
The 30mmol/L Tris-HCl of 100 or Tween20,0.05%Proclin-300 and surplus, the coating pH of buffer are
7.0。
Embodiment six
(1) measurement of Pearson correlation coefficient r:
The quantification assay kit of the Monophosphoinositideproteoglycans proteoglycans-3 described in embodiment one to embodiment five, to concentration
It is quantified for the GPC3 standard items of 0ng/mL, 0.5ng/mL, 2.0ng/mL, 5.0ng/mL, 20.0ng/mL, 100.0ng/mL
Measurement, is repeated 5 times, makes standard curve according to luminous intensity and standard concentration, and acquire Pearson correlation coefficient r, as a result
As shown in Table 1；The specific test method is as follows:
There are the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody and each 50ul of GPC3 standard items to add coupling
Enter into reaction tube, 37 DEG C of vibrations are incubated for 20min, and reaction tube is then placed on 30s on Magneto separate frame, is carefully removed
Clearly, 500ul cleaning solution (the PBS cleaning buffer solution of pH 7.2, the X-100 containing 0.15%Triton) is added, and piping and druming keeps it mixed repeatedly
It is even, supernatant is removed after repeating above step 3 times；It is subsequently added into the Monophosphoinositideproteoglycans proteoglycans-3 antibody of 100ul acridinium ester label
Solution mixes, and 37 DEG C of vibrations are incubated for 20min, and supernatant is removed after then cleaning 5 times repeatedly with cleaning solution, then on detector plus pre-
Detection and tracer signal value after exciting liquid and exciting liquid.
(2) sensitivity for analysis measures:
The quantification assay kit of the Monophosphoinositideproteoglycans proteoglycans-3 described in embodiment one to embodiment five respectively, it is right
The GPC3 standard items that 20 concentration are 0ng/mL are measured, and are calculated its mean value (M) and standard deviation (SD), are obtained M+2SD, M+
Its corresponding concentration on standard curve of 2SD is sensitivity for analysis.As a result as shown in Table 1.
Table one, Pearson correlation coefficient r and sensitivity for analysis
It will be apparent to those skilled in the art that can make various other according to the above description of the technical scheme and ideas
Corresponding change and deformation, and all these changes and deformation all should belong to the protection scope of the claims in the present invention
Within.
Claims (1)
1. a kind of preparation method of the quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3, the kit include the following:
(1) GPC3 standard items；
(2) the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label；
(3) coupling has the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody；
(4) luminescent solution；
The kit is made by following steps:
(1) preparation of GPC3 standard items: with standard items buffer by GPC3 be configured to 0ng/mL, 0.5ng/mL, 2.0ng/mL,
5.0ng/mL, 20.0ng/mL, 100.0ng/mL series of concentrations；The standard items buffer include mass ratio be 3%BSA,
0.6%NaCl, 0.1%TritonX-100 or Tween20,0.05%Proclin-300, surplus 0.2mol/L phosphoric acid buffer
Liquid, the pH of the standard items buffer are 7.0；
(2) preparation of the Monophosphoinositideproteoglycans proteoglycans-3 antibody-solutions of acridinium ester label:
1., take GPC3 antibody to be dissolved in, pH is that 7.0 concentration are in the phosphate buffer of 0.2mol/L, until final concentration of 5mg/L；
2., to step 1. treated GPC3 antibody-solutions acridinium ester is added to the final concentration of 0.25mg/L of acridinium ester, be protected from light quiet
It sets 10 hours, collects the GPC3 antibody-solutions for being marked with acridinium ester；
(3) coupling has the preparation of the nano magnetic microspheres solution of Monophosphoinositideproteoglycans proteoglycans-3 antibody:
I takes GPC3 antibody to be dissolved in, and pH is that 5.0 concentration are in the MES solution of 0.2mol/L, until final concentration of 5mg/L；
II takes 1 parts by weight nanometer magnetic microsphere, and the coupling agent one and 3 parts by weight coupling agents two of 2 parts by weight is added, is placed in magnetic microsphere
Mixer rotates 30 minutes, removes supernatant；Connection buffer washing nanometer magnetic microsphere is added, supernatant is removed；The coupling agent
One includes the MES that the pH that mass ratio is 1%NHS and the concentration of surplus is 0.2mol/L is 5.0；The coupling agent two includes quality
The MES solution for the 0.2mol/L that pH than EDC and surplus for 1% is 5.0；It is 5.0 that the connection buffer, which is pH,
0.2mol/L MES solution；The nanometer magnetic microsphere be diameter be 3 μm, the nanometer magnetic microsphere of superparamagnetism of the surface with carboxyl；
III mixes the GPC3 antibody-solutions after I step process and the nanometer magnetic microsphere 1:40 in mass ratio after II step process
GPC3 antibody nanometer magnetic microsphere mixed liquor is formed, magnetic microsphere mixer is placed and rotates 12 hours, then with 3 times of GPC3 antibody nano magnetics
The coating buffer exchange of the volume of microballoon mixed liquor connects buffer；Obtaining coupling has Monophosphoinositideproteoglycans proteoglycans-3 antibody
Nano magnetic microspheres solution；The coating buffer includes that mass ratio is 1%BSA, 0.5%NaCl, 0.1%TritonX-100
Or the 30mmol/L Tris-HCl of Tween20,0.05%Proclin-300 and surplus, the coating pH of buffer are 7.0.
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| CN109633173B (en) * | 2019-01-04 | 2022-02-22 | 东北农业大学 | Immunomagnetic bead indirect ELISA kit for detecting IBDV antibody and application thereof |
| CN111381034A (en) * | 2020-03-03 | 2020-07-07 | 武汉生之源生物科技股份有限公司 | GPC-3 antibody coated magnetic bead, and preparation method and kit thereof |
| CN113960306B (en) * | 2020-07-20 | 2023-04-28 | 菲鹏生物股份有限公司 | Reagent for stabilizing acridinium ester marker protein and application thereof |
| CN112014577B (en) * | 2020-09-04 | 2023-07-18 | 武汉生之源生物科技股份有限公司 | Kit for improving GPC3 detection sensitivity and preparation method thereof |
| CN116359506A (en) * | 2023-03-21 | 2023-06-30 | 深圳市巨东生物医学工程有限公司 | Kit for detecting nasopharyngeal carcinoma |
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