CN111239419A - Glycosylated hemoglobin detection reagent and method for coupling latex microspheres and polylysine - Google Patents

Glycosylated hemoglobin detection reagent and method for coupling latex microspheres and polylysine Download PDF

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Publication number
CN111239419A
CN111239419A CN202010055042.0A CN202010055042A CN111239419A CN 111239419 A CN111239419 A CN 111239419A CN 202010055042 A CN202010055042 A CN 202010055042A CN 111239419 A CN111239419 A CN 111239419A
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reagent
polylysine
latex microspheres
latex
concentration
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赵年福
张辉
贾引军
王明伟
金君玉
吴一凡
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Suzhou Diagvita Biotechnology Co ltd
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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
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G01N33/723—Glycosylated haemoglobin
    • 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
    • G01N33/54346—Nanoparticles
    • 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

Abstract

The invention discloses a glycosylated hemoglobin detection reagent, which comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein the R2 reagent comprises a primary antibody and a secondary antibody, the primary antibody is a mouse anti-human HbA1c monoclonal antibody, the secondary antibody is a goat anti-mouse IgG polyclonal antibody, the R3 reagent comprises a hemolytic agent, R1 is a polystyrene latex reagent, latex microspheres of the R1 reagent are coupled with polylysine, the coupling of the polylysine and the latex microspheres increases the positions of the surface of the latex microspheres and glycosylated hemoglobin, and the coupling efficiency is improved. According to the glycosylated hemoglobin detection reagent, polylysine is coupled to the latex microspheres of the R1 reagent, the coupling of polylysine and the latex microspheres increases the positions of the surface of the latex microspheres, which are combined and adsorbed with glycosylated hemoglobin, so that the efficiency of combining the surface of the latex microspheres with the glycosylated hemoglobin can be improved, the detection sensitivity of the glycosylated hemoglobin detection reagent is obviously improved, the reaction detection time is shortened, and the detection efficiency is improved.

Description

Glycosylated hemoglobin detection reagent and method for coupling latex microspheres and polylysine
Technical Field
The invention relates to the technical field of biochemical analysis, in particular to a glycosylated hemoglobin detection reagent and a method for coupling latex microspheres and polylysine.
Background
At present, the detection methods of glycated hemoglobin which are widely used clinically mainly include high performance liquid chromatography, affinity chromatography, electrophoresis and the like, the detection methods are complex and time-consuming to operate, require special instruments and are high in cost, and other detection methods exist in the market, but the defects of low measurement accuracy generally exist.
The latex particle enhanced turbidimetry is a relatively stable and accurate body fluid protein homogeneous phase immune turbidimetry detection technology which appears in recent years. The specific principle is that the antibody is crosslinked or physically adsorbed on the surface of the polymer latex microsphere, and when the microsphere with the crosslinked antibody is combined with the glycosylated hemoglobin HbA1c, the microspheres can be rapidly gathered together in a short time, so that the light scattering performance and the light transmission performance of the reaction liquid are changed.
However, 3 reagents (R1, R2, and R3) are commonly used in currently available latex turbidimetry kits, and in the prior art, the R1 reagent is a polystyrene latex microsphere reagent, which binds to glycated hemoglobin HbA1c by physical adsorption, and thus has low binding efficiency, resulting in low detection sensitivity, and thus needs to be improved.
Disclosure of Invention
The present invention has been made in view of the problems of the conventional glycated hemoglobin measurement reagent and the like described in the background art, and an object of the present invention is to provide a glycated hemoglobin measurement reagent that can solve the problems described above.
In order to achieve the purpose, the invention is realized by the following technical scheme: the glycosylated hemoglobin detection reagent is characterized in that: the reagent comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein the R2 reagent comprises a primary antibody and a secondary antibody, the primary antibody is a mouse anti-human HbA1c monoclonal antibody, the secondary antibody is a goat anti-mouse IgG polyclonal antibody, the R3 reagent comprises a hemolytic agent, the R1 reagent is a polystyrene latex microsphere reagent, the latex microsphere of the R1 reagent is coupled with polylysine, and the coupling of the polylysine and the latex microsphere increases the positions of the surface of the latex microsphere and glycosylated hemoglobin for binding and adsorption, so that the binding efficiency is improved.
In the R1 reagent, the particle size of the polystyrene latex microsphere is 50nm-500nm, the concentration is 0.05% -0.5% (mass/volume ratio), the concentration of polylysine is 0.1g/L-5g/L, and the molecular weight is 1000-30000.
In a further scheme, in the R2 reagent, the concentration of the mouse anti-human HbA1c monoclonal antibody is 50mg/L-100mg/L, and the concentration of the goat anti-mouse IgG polyclonal antibody is 10mg/L-20 mg/L.
In a further embodiment, in the reagent R3, the hemolytic agent is triton X-100, and the concentration thereof is 0.1-1% (volume/volume ratio).
It is another object of the present invention to provide a method for coupling latex microspheres to polylysine, comprising the steps of:
1) measuring a certain amount of latex microspheres, placing the latex microspheres in a beaker, and magnetically stirring the latex microspheres;
2) slowly adding a quantity of MES BUFFER;
3) respectively weighing a certain amount of EDAC, Sulfo-NHS and polylysine, adding into a beaker, and magnetically stirring uniformly at room temperature;
4) transferring the uniformly stirred solution in the step 3 from the beaker to a centrifuge tube, flushing the beaker with deionized water, and transferring the beaker and the centrifuge tube together;
5) centrifuging after balancing, setting the centrifugal rotating speed at 13000 and 18000rpm, setting the temperature at 8 ℃, and centrifuging for 60 minutes;
6) and after the centrifugation is finished, pouring out the supernatant, adding deionized water, fully resuspending and ultrasonically processing the coupled particles of the latex microspheres and the polylysine to finish the coupling of the latex microspheres and the polylysine.
The invention has the positive effects that: according to the glycosylated hemoglobin detection reagent, polylysine is coupled to polystyrene latex microspheres of an R1 reagent, the coupling of polylysine and the latex microspheres increases the positions of the surface of the latex microspheres, which are combined and adsorbed with glycosylated hemoglobin, so that the efficiency of combining the surface of the latex microspheres with the glycosylated hemoglobin can be improved, the detection sensitivity of the glycosylated hemoglobin detection reagent is obviously improved, the reaction detection time is shortened, and the detection efficiency is improved.
Detailed Description
The technical solutions of the present invention are described clearly and completely by the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The reagent for detecting the glycosylated hemoglobin comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein in the R1 reagent, the particle size of a polystyrene latex microsphere is 50nm, the concentration is 0.05 percent (mass/volume ratio), the concentration of polylysine is 0.1g/L, the molecular weight is 1000, and the coupling steps of the latex microsphere and the polylysine are as follows:
1) measuring 0.5mL of 50nm latex microspheres (10%), placing the latex microspheres in a 10mL beaker, and performing magnetic stirring by using a medium stirrer;
2) slowly add 0.1ml 500mM MES BUFFER (pH 6.0);
3) 50mg of EDAC,5mg of Sulfo-NHS and 0.01g of polylysine are weighed respectively and added into a beaker respectively;
4) stirring for 1 hour at room temperature by magnetic force;
5) transferring the latex microspheres into a 50ml centrifuge tube, purging the beaker with 10ml deionized water, and transferring the latex microspheres into the centrifuge tube;
6) centrifuging after balancing, 16000rpm,8 ℃ for 60 minutes;
7) pouring out the supernatant, adding 5ml of deionized water, and fully suspending the latex microspheres;
8) sonicate for 10 minutes, add 0.25g NaH2PO4·2H2O，1.22g Na2HPO4·12H2O,0.1mL of proclin 300, and water is added to make the volume of the solution to be 100 mL.
The R2 reagent comprises a primary antibody and a secondary antibody, wherein the primary antibody is a mouse anti-human HbA1c monoclonal antibody, the secondary antibody is a goat anti-mouse IgG polyclonal antibody, the concentration of the mouse anti-human HbA1c monoclonal antibody is 50mg/L, and the concentration of the goat anti-mouse IgG polyclonal antibody is 10 mg/L; the R3 reagent comprises a hemolytic agent, and in the R3 reagent, the hemolytic agent is triton X-100 with the concentration of 0.1% (volume/volume ratio).
Glycated hemoglobin reagent assay sensitivity test (test concentration of 5% HbA1c quality control for absorbance change within 5 minutes):
Figure BDA0002372519740000031
Figure BDA0002372519740000041
example 2
The glycosylated hemoglobin detection reagent comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein in the R1 reagent, the particle size of a polystyrene latex microsphere is 100nm, the concentration is 0.2 percent (mass/volume ratio), the concentration of polylysine is 1g/L, the molecular weight is 5000, and the latex microsphere is coupled with the polylysine:
a. measuring 2mL of 100nm latex microspheres (10%), placing the latex microspheres in a 10mL beaker, and performing magnetic stirring by using a medium stirrer;
b. slowly add 0.1ml 500mM MES BUFFER (pH 6.0);
c. 50mg of EDAC,5mg of Sulfo-NHS and 0.1g of polylysine are weighed respectively and added into a beaker respectively;
d. stirring for 1 hour at room temperature by magnetic force;
e. transferring the latex microspheres into a 50ml centrifuge tube, purging the beaker with 10ml deionized water, and transferring the latex microspheres into the centrifuge tube;
f. centrifuging after balancing, 16000rpm,8 ℃ for 60 minutes;
g. pouring out the supernatant, adding 5ml of deionized water, and fully suspending the latex microspheres;
h. sonicate for 10 minutes, add 0.25g NaH2PO4·2H2O，1.22g Na2HPO4·12H2O,0.1mL of proclin 300, and water is added to make the volume of the solution to be 100 mL.
The R2 reagent comprises a primary antibody and a secondary antibody, wherein the primary antibody is a mouse anti-human HbA1c monoclonal antibody, the secondary antibody is a goat anti-mouse IgG polyclonal antibody, the concentration of the mouse anti-human HbA1c monoclonal antibody is 60mg/L, and the concentration of the goat anti-mouse IgG polyclonal antibody is 15 mg/L; the R3 reagent comprises a hemolytic agent, and in the R3 reagent, the hemolytic agent is triton X-100 with the concentration of 0.3% (volume/volume ratio).
Glycated hemoglobin reagent assay sensitivity test (test concentration of 5% HbA1c quality control for absorbance change within 5 minutes):
Figure BDA0002372519740000042
Figure BDA0002372519740000051
example 3
The glycosylated hemoglobin detection reagent comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein in the R1 reagent, the particle size of a polystyrene latex microsphere is 300nm, the concentration is 0.3 percent (mass/volume ratio), the concentration of polylysine is 2g/L, the molecular weight is 10000, and the latex microsphere is coupled with the polylysine:
a. measuring 3mL of 300nm latex microspheres (10%), placing the latex microspheres in a 10mL beaker, and performing magnetic stirring by using a medium stirrer;
b. slowly add 0.1ml 500mM MES BUFFER (pH 6.0);
c. 50mg of EDAC,5mg of Sulfo-NHS and 0.2g of polylysine are weighed respectively and added into a beaker respectively;
d. stirring for 1 hour at room temperature by magnetic force;
e. transferring the latex microspheres into a 50ml centrifuge tube, purging the beaker with 10ml deionized water, and transferring the latex microspheres into the centrifuge tube;
f. centrifuging after balancing, 16000rpm,8 ℃ for 60 minutes;
g. pouring out the supernatant, adding 5ml of deionized water, and fully suspending the latex microspheres;
h. sonicate for 10 minutes, add 0.25g NaH2PO4·2H2O，1.22g Na2HPO4·12H2O,0.1mL of proclin 300, and water is added to make the volume of the solution to be 100 mL.
The R2 reagent comprises a primary antibody and a secondary antibody, wherein the primary antibody is a mouse anti-human HbA1c monoclonal antibody, the secondary antibody is a goat anti-mouse IgG polyclonal antibody, the concentration of the mouse anti-human HbA1c monoclonal antibody is 80mg/L, and the concentration of the goat anti-mouse IgG polyclonal antibody is 20 mg/L; the R3 reagent comprises a hemolytic agent, and in the R3 reagent, the hemolytic agent is triton X-100 with the concentration of 0.6% (volume/volume ratio).
Glycated hemoglobin reagent assay sensitivity test (test concentration of 5% HbA1c quality control for absorbance change within 5 minutes):
Figure BDA0002372519740000052
Figure BDA0002372519740000061
example 4
The glycosylated hemoglobin detection reagent comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein in the R1 reagent, the particle size of a polystyrene latex microsphere is 500nm, the concentration is 0.5 percent (mass/volume ratio), the concentration of polylysine is 4g/L, the molecular weight is 20000, and the latex microsphere is coupled with the polylysine:
a. measuring 5mL of 500nm latex microspheres (10%), placing the latex microspheres in a 10mL beaker, and performing magnetic stirring by using a medium stirrer;
b. slowly add 0.1ml 500mM MES BUFFER (pH 6.0);
c. 50mg of EDAC,5mg of Sulfo-NHS and 0.4g of polylysine are weighed respectively and added into a beaker respectively;
d. stirring for 1 hour at room temperature by magnetic force;
e. transferring the latex microspheres into a 50ml centrifuge tube, purging the beaker with 10ml deionized water, and transferring the latex microspheres into the centrifuge tube;
f. centrifuging after balancing, 16000rpm,8 ℃ for 60 minutes;
g. pouring out the supernatant, adding 5ml of deionized water, and fully suspending the latex microspheres;
h. sonicate for 10 minutes, add 0.25g NaH2PO4·2H2O，1.22g Na2HPO4·12H2O,0.1mL of proclin 300, and water is added to make the volume of the solution to be 100 mL.
The R2 reagent comprises a primary antibody and a secondary antibody, wherein the primary antibody is a mouse anti-human HbA1c monoclonal antibody, the secondary antibody is a goat anti-mouse IgG polyclonal antibody, the concentration of the mouse anti-human HbA1c monoclonal antibody is 100mg/L, and the concentration of the goat anti-mouse IgG polyclonal antibody is 20 mg/L; the R3 reagent comprises a hemolytic agent, and in the R3 reagent, the hemolytic agent is triton X-100 with the concentration of 0.8% (volume/volume ratio).
Glycated hemoglobin reagent assay sensitivity test (test concentration of 5% HbA1c quality control for absorbance change within 5 minutes):
Figure BDA0002372519740000062
example 5
The reagent for detecting the glycosylated hemoglobin comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein in the R1 reagent, the particle size of a polystyrene latex microsphere is 500nm, the concentration is 0.5 percent (mass/volume ratio), the concentration of polylysine is 5g/L, the molecular weight is 30000, and the latex microsphere is coupled with the polylysine:
a. measuring 5mL of 500nm latex microspheres (10%), placing the latex microspheres in a 10mL beaker, and performing magnetic stirring by using a medium stirrer;
b. slowly add 0.1ml 500mM MES BUFFER (pH 6.0);
c. 50mg of EDAC,5mg of Sulfo-NHS and 0.5g of polylysine are weighed respectively and added into a beaker respectively;
d. stirring for 1 hour at room temperature by magnetic force;
e. transferring the latex microspheres into a 50ml centrifuge tube, purging the beaker with 10ml deionized water, and transferring the latex microspheres into the centrifuge tube;
f. centrifuging after balancing, 16000rpm,8 ℃ for 60 minutes;
g. pouring out the supernatant, adding 5ml of deionized water, and fully suspending the latex microspheres;
h. sonicate for 10 minutes, add 0.25g NaH2PO4·2H2O，1.22g Na2HPO4·12H2O,0.1mL of proclin 300, and water is added to make the volume of the solution to be 100 mL.
The R2 reagent comprises a primary antibody and a secondary antibody, wherein the primary antibody is a mouse anti-human HbA1c monoclonal antibody, the secondary antibody is a goat anti-mouse IgG polyclonal antibody, the concentration of the mouse anti-human HbA1c monoclonal antibody is 90mg/L, and the concentration of the goat anti-mouse IgG polyclonal antibody is 18 mg/L; the R3 reagent comprises a hemolytic agent, and in the R3 reagent, the hemolytic agent is triton X-100 with the concentration of 1.0% (volume/volume ratio).
Glycated hemoglobin reagent assay sensitivity test (test concentration of 5% HbA1c quality control for absorbance change within 5 minutes):
Figure BDA0002372519740000071
in the schemes of embodiments 1 to 5 of the present invention, polylysine in the R1 reagent is coupled to polystyrene latex microspheres, and coupling of polylysine to latex microspheres increases the number of positions where the surface of latex microspheres is bonded and adsorbed to glycated hemoglobin, which can improve the efficiency of bonding the surface of latex microspheres to glycated hemoglobin, thereby significantly improving the detection sensitivity of the glycated hemoglobin detection reagent, and also facilitating shortening of the reaction detection time and improving the detection efficiency.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (5)

1. A glycated hemoglobin measurement reagent, comprising: the reagent comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein the R2 reagent comprises a primary antibody and a secondary antibody, the primary antibody is a mouse anti-human HbA1c monoclonal antibody, the secondary antibody is a goat anti-mouse IgG polyclonal antibody, the R3 reagent comprises a hemolytic agent, the R1 is a polystyrene latex reagent, a latex microsphere of the R1 reagent is coupled with polylysine, and the coupling of the polylysine and the latex microsphere increases the positions of the surface of the latex microsphere and glycosylated hemoglobin for binding and adsorption, so that the binding efficiency is improved.
2. The glycated hemoglobin measurement reagent according to claim 1, wherein: in the R1 reagent, the particle size of the polystyrene latex microspheres is 50nm-500nm, the concentration is 0.05-0.5% (mass/volume ratio), the concentration of polylysine is 0.1-5 g/L, and the molecular weight is 1000-30000.
3. The glycated hemoglobin measurement reagent according to claim 1, wherein: in the R2 reagent, the concentration of the mouse anti-human HbA1c monoclonal antibody is 50mg/L-100mg/L, and the concentration of the goat anti-mouse IgG polyclonal antibody is 10mg/L-20 mg/L.
4. The glycated hemoglobin measurement reagent according to claim 1, wherein: in the R3 reagent, the hemolytic agent is triton X-100, and the concentration of the hemolytic agent is 0.1-1% (volume/volume ratio).
5. The coupling method of the latex microspheres and the polylysine is characterized in that: which comprises the following steps:
1) measuring a certain amount of latex microspheres, placing the latex microspheres in a beaker, and magnetically stirring the latex microspheres;
2) slowly adding a quantity of MES BUFFER;
3) respectively weighing a certain amount of EDAC, Sulfo-NHS and polylysine, adding into a beaker, and magnetically stirring uniformly at room temperature;
4) transferring the uniformly stirred solution in the step 3 from the beaker to a centrifuge tube, flushing the beaker with deionized water, and transferring the beaker and the centrifuge tube together;
5) centrifuging after balancing, setting the centrifugal rotating speed at 13000 and 18000rpm, setting the temperature at 8 ℃, and centrifuging for 60 minutes;
6) and after the centrifugation is finished, pouring out the supernatant, adding deionized water, fully resuspending and ultrasonically processing the coupled particles of the latex microspheres and the polylysine to finish the coupling of the latex microspheres and the polylysine.
CN202010055042.0A 2020-01-17 2020-01-17 Glycosylated hemoglobin detection reagent and method for coupling latex microspheres and polylysine Pending CN111239419A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023142398A1 (en) * 2022-01-26 2023-08-03 丹娜(天津)生物科技股份有限公司 Coronavirus rbd protein-fluorescent microsphere complex preparation

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023142398A1 (en) * 2022-01-26 2023-08-03 丹娜(天津)生物科技股份有限公司 Coronavirus rbd protein-fluorescent microsphere complex preparation

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