CN113419069A - Kit and method for detecting anti-cyclic citrullinated peptide antibody - Google Patents
Kit and method for detecting anti-cyclic citrullinated peptide antibody Download PDFInfo
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- CN113419069A CN113419069A CN202110664574.9A CN202110664574A CN113419069A CN 113419069 A CN113419069 A CN 113419069A CN 202110664574 A CN202110664574 A CN 202110664574A CN 113419069 A CN113419069 A CN 113419069A
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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
Abstract
The invention discloses an anti-cyclic citrullinated peptide antibody detection kit provided by the invention, which comprises pre-coating liquid, wherein the pre-coating liquid is provided with a streptavidin and carbon nanotube combination. Firstly, adding pre-coating liquid into coating holes for pre-coating, and then adding the coating liquid into the holes of a coated plate; obtaining a coated plate coated with the anti-cyclic citrullinated peptide antigen-streptavidin-carbon nano tube; then adding a horse radish peroxidase goat anti-rabbit IgG reagent; then adding o-phenylenediamine substrate display reagent, adding sulfuric acid termination reagent, and finally recording the reading by using an enzyme-linked immunosorbent assay instrument. The streptavidin is introduced to enhance the loading effect of the pre-coating liquid on the coated plate, so that the pre-coating liquid can be loaded on the plate body in a more uniform and dispersed manner, the binding rate and the effect of the pre-coating liquid on the coated plate are improved, the time of the subsequent coating step is shortened, and the sensitivity is correspondingly improved.
Description
Technical Field
The invention particularly relates to an anti-cyclic citrullinated peptide antibody detection kit and a method.
Background
An anti-cyclic citrullinated peptide antibody is a biological antibody which can react and synthesize a Cyclic Citrullinated Polypeptide (CCP) as an antigen, and the antibody can well indicate and judge the specificity of the disease, namely rheumatoid arthritis, so the anti-cyclic citrullinated peptide antibody can be used as one of important index substances for detecting and diagnosing the disease at an early stage. In the clinical practice, enzyme-linked immunosorbent assay (ELISA) is generally used for the detection of such antibodies.
When the enzyme-linked immunosorbent assay (ELISA) is actually used for detecting the anti-cyclic citrullinated peptide antibody, one problem needs to be particularly noted, namely the detection sensitivity is insufficient, and a part of positive samples are easy to be detected, and in addition, the time needed for coating the anti-cyclic citrullinated peptide antigen on the polystyrene plate is too long and generally needs to reach 20 hours, so that the working efficiency is obviously reduced. The reason for this is that the antigen has an insufficient molecular weight, a slow binding rate to a polystyrene carrier and insufficient dispersibility, and thus cannot be bound or is difficult to bind at some sites, which causes the above-mentioned technical problems.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a technical scheme for improving the combination rate and effect of coating liquid and polystyrene board.
The invention provides an anti-cyclic citrullinated peptide antibody detection kit, which comprises pre-coating liquid, wherein the pre-coating liquid is provided with a streptavidin and carbon nanotube combination.
The loading effect of the pre-coating liquid on the coated plate is enhanced through the introduction of streptavidin, and the dispersibility of the pre-coating liquid is enhanced through the composite effect of the carbon nano tubes, so that the pre-coating liquid is loaded on the plate body in a more uniform and dispersed manner, the combination rate and effect of the pre-coating liquid on the coated plate are improved, the time of the subsequent coating step is shortened, and the sensitivity is correspondingly improved.
In a preferred embodiment, the carbon nanotube is a hydrophobic carbon nanotube, and the hydrophobic carbon nanotube is a polyvinylidene chloride-compounded carbon nanotube. Through the hydrophobic effect, the combination rate and effect of the carbon nano tube and the coated plate are further improved, namely, the combination rate of the carbon nano tube and the coated plate is accelerated after the macromolecule hydrophobic polymer is introduced, so that a better effect is achieved.
In a preferred scheme, the kit further comprises an anti-cyclic citrullinated peptide antigen coating reagent, wherein the coating reagent contains carbonate buffer with the concentration of 0.05mol/L, the pH of the coating reagent is 9.6, and the protein content is 1 mu g/ml to 10 mu g/ml.
In a preferred scheme, the kit further comprises a horseradish peroxidase goat anti-rabbit IgG reagent, an o-phenylenediamine substrate display reagent and a 2mol/L sulfuric acid termination reagent.
The working method of the anti-cyclic citrullinated peptide antibody detection kit provided by the invention comprises the following steps:
s1: firstly, adding the pre-coating solution into a coating hole for pre-coating, and incubating for 1 hour at 37 ℃; then adding the coating liquid into the holes of the coated plate; after incubation at 37 ℃ for 5 to 6 hours at 4 ℃;
s2: pouring out the liquid in the coated plate, adding a washing reagent, and repeating the pouring out and washing processes for three times to obtain the coated plate coated with the anti-cyclic citrullinated peptide antigen-streptavidin-carbon nano tube;
s3: placing a sample to be detected into the coating plate, setting a control group, and reacting for 2-3 hours at 37 ℃;
s4: repeating the process of pouring out and washing for three times by using the washing reagent again, then adding a horse radish peroxidase goat anti-rabbit IgG reagent, and reacting for 1 hour at 37 ℃;
s5: repeating the processes of pouring out and washing for three times by using the washing reagent again, then adding the o-phenylenediamine substrate displaying reagent, and reacting for 15 minutes at 37 ℃ in a dark environment;
s6: adding a sulfuric acid termination reagent, and finally recording the reading by using an enzyme-linked immunosorbent assay.
The working method of the anti-cyclic citrullinated peptide antibody detection kit provided by the invention comprises the following steps:
s1: adding the carboxylated carbon nano tube particles into a buffer solution, and then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide for activation;
s2: collecting activated carbon nano tube particles, adding the carbon nano tube particles into streptavidin, mixing and stirring to obtain a combination of the streptavidin and the carbon nano tube;
s3: performing ultrasonic dispersion on the combination of the streptavidin and the carbon nano tube in absolute ethyl alcohol, then adding polyvinylidene chloride, and performing the ultrasonic dispersion step again to obtain uniformly dispersed liquid correspondingly; and pouring out the liquid, drying, and drying to obtain powder which is a combination of streptavidin-carbon nano tube-polyvinylidene chloride.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
The first embodiment:
the invention provides an anti-cyclic citrullinated peptide antibody detection kit, which comprises pre-coating liquid, wherein the pre-coating liquid is provided with a streptavidin and carbon nanotube combination.
The loading effect of the coating liquid on the coated plate is enhanced through the introduction of streptavidin, and the dispersibility of the coating liquid is enhanced through the composite effect of the carbon nano tubes, so that the coating liquid is loaded on the coated plate body in a more uniform and dispersed manner, the combination rate and effect of the coating liquid on the coated plate are improved, the time of the subsequent coating step is shortened, and the sensitivity is correspondingly improved.
The kit also comprises an anti-cyclic citrullinated peptide antigen coating reagent, wherein the coating reagent contains carbonate buffer solution with the concentration of 0.05mol/L, the pH of the coating reagent is 9.6, and the protein content is 1 mu g/ml to 10 mu g/ml.
The kit also comprises a horseradish peroxidase goat anti-rabbit IgG reagent, an o-phenylenediamine display substrate reagent and a 2mol/L sulfuric acid termination reagent.
The working method of the anti-cyclic citrullinated peptide antibody detection kit provided by the invention comprises the following steps:
s1: firstly, adding the pre-coating solution into a coating hole for pre-coating, and incubating for 1 hour at 37 ℃; then adding the coating liquid into the holes of the coated plate; after incubation at 37 ℃ for 5 to 6 hours at 4 ℃;
s2: pouring out the liquid in the coated plate, adding a washing reagent, and repeating the pouring out and washing processes for three times to obtain the coated plate coated with the anti-cyclic citrullinated peptide antigen-streptavidin-carbon nano tube;
s3: placing a sample to be detected into the coating plate, setting a control group, and reacting for 2-3 hours at 37 ℃;
s4: repeating the process of pouring out and washing for three times by using the washing reagent again, then adding a horse radish peroxidase goat anti-rabbit IgG reagent, and reacting for 1 hour at 37 ℃;
s5: repeating the processes of pouring out and washing for three times by using the washing reagent again, then adding the o-phenylenediamine substrate displaying reagent, and reacting for 15 minutes at 37 ℃ in a dark environment;
s6: adding a sulfuric acid termination reagent, and finally recording the reading by using an enzyme-linked immunosorbent assay.
Second embodiment:
preferably, the carbon nanotube of this embodiment is a hydrophobic carbon nanotube, and the hydrophobic carbon nanotube is a polyvinylidene chloride-compounded carbon nanotube. Through the hydrophobic effect, the combination rate and effect of the carbon nano tube and the coated plate are further improved, namely, the combination rate of the carbon nano tube and the coated plate is accelerated after the macromolecule hydrophobic polymer is introduced, so that a better effect is achieved.
The working method of the cyclic citrullinated peptide antibody detection kit provided by the invention comprises the following steps:
s1: adding the carboxylated carbon nano tube particles into a buffer solution, and then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide for activation;
s2: collecting activated carbon nano tube particles, adding the carbon nano tube particles into streptavidin, mixing and stirring to obtain a combination of the streptavidin and the carbon nano tube;
s3: performing ultrasonic dispersion on the combination of the streptavidin and the carbon nano tube in absolute ethyl alcohol, then adding polyvinylidene chloride, and performing the ultrasonic dispersion step again to obtain uniformly dispersed liquid correspondingly; and pouring out the liquid, drying, and drying to obtain powder which is a combination of streptavidin-carbon nano tube-polyvinylidene chloride.
TABLE 1 Performance test of different types of precoating solutions on the Overall binding time of the coated plates
| Group of | Binding time |
| Streptavidin | 16-18 hours |
| Streptavidin-carbon nanotubes | 6 to 7 hours |
| Streptavidin-carbon nanotube-polyvinylidene chloride | 2-3 hours |
The pre-coating liquids used in the above groups are different, and other specific test procedures are the same, and the combination time includes the sum of the pre-coating time and the coating time. In the test of the binding time, different binding times are respectively given to the same group, then the respective positive detection rates are counted, and the binding time is recorded when the detection efficiency is not obviously changed after the binding time is gradually increased. The diagnostic sensitivity of streptavidin was 38%, that of streptavidin-carbon nanotubes was 45%, and that of streptavidin-carbon nanotubes-polyvinylidene chloride was 60% or more. The diagnosis specificity of the three is above 90% without obvious difference. The weight ratio of carbon nanotubes to polyvinylidene chloride is preferably 1: 1. The content of streptavidin in the streptavidin-carbon nanotube-polyvinylidene chloride is 60% to 90%.
It is to be understood that the described embodiments are merely a few embodiments of the 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.
Claims (6)
1. An anti-cyclic citrullinated peptide antibody detection kit is characterized in that: comprises a pre-coating solution, wherein the pre-coating solution is provided with a streptavidin and carbon nanotube combination.
2. The anti-cyclic citrullinated peptide antibody detection kit according to claim 1, characterized in that: the carbon nano tube is a hydrophobic carbon nano tube, and the hydrophobic carbon nano tube is a polyvinylidene chloride compounded carbon nano tube.
3. The anti-cyclic citrullinated peptide antibody detection kit according to claim 2, wherein: the kit also comprises an anti-cyclic citrullinated peptide antigen coating reagent, wherein the coating reagent contains carbonate buffer solution with the concentration of 0.05mol/L, the pH of the coating reagent is 9.6, and the protein content is 1 mu g/ml to 10 mu g/ml.
4. The anti-cyclic citrullinated peptide antibody detection kit according to claim 3, wherein: the kit also comprises a horseradish peroxidase goat anti-rabbit IgG reagent, an o-phenylenediamine display substrate reagent and a 2mol/L sulfuric acid termination reagent.
5. The method for operating the anti-cyclic citrullinated peptide antibody detection kit according to claim 4, characterized in that it comprises the following steps:
s1: firstly, adding the pre-coating solution into a coating hole for pre-coating, and incubating for 1 hour at 37 ℃; then adding the coating liquid into the holes of the coated plate; after incubation at 37 ℃ for 5 to 6 hours at 4 ℃;
s2: pouring out the liquid in the coated plate, adding a washing reagent, and repeating the pouring out and washing processes for three times to obtain the coated plate coated with the anti-cyclic citrullinated peptide antigen-streptavidin-carbon nano tube;
s3: placing a sample to be detected into the coating plate, setting a control group, and reacting for 2-3 hours at 37 ℃;
s4: repeating the process of pouring out and washing for three times by using the washing reagent again, then adding a horse radish peroxidase goat anti-rabbit IgG reagent, and reacting for 1 hour at 37 ℃;
s5: repeating the processes of pouring out and washing for three times by using the washing reagent again, then adding the o-phenylenediamine substrate displaying reagent, and reacting for 15 minutes at 37 ℃ in a dark environment;
s6: adding a sulfuric acid termination reagent, and finally recording the reading by using an enzyme-linked immunosorbent assay.
6. The method for operating the anti-cyclic citrullinated peptide antibody detection kit according to claim 2, characterized in that it comprises the following steps:
s1: adding the carboxylated carbon nano tube particles into a buffer solution, and then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide for activation;
s2: collecting activated carbon nano tube particles, adding the carbon nano tube particles into streptavidin, mixing and stirring to obtain a combination of the streptavidin and the carbon nano tube;
s3: performing ultrasonic dispersion on the combination of the streptavidin and the carbon nano tube in absolute ethyl alcohol, then adding polyvinylidene chloride, and performing the ultrasonic dispersion step again to obtain uniformly dispersed liquid correspondingly; and pouring out the liquid, drying, and drying to obtain powder which is a combination of streptavidin-carbon nano tube-polyvinylidene chloride.
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