CN115032382A - Ferritin detection kit and preparation method thereof - Google Patents
Ferritin detection kit and preparation method thereof Download PDFInfo
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- 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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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
Abstract
The invention provides a ferritin detection kit, which comprises a first reagent, a second reagent and a ferritin calibrator, wherein the first reagent comprises a first solution and a first preservative, the second reagent comprises a second solution, a second preservative and latex microspheres coupled with an anti-human ferritin antibody, and the anti-human ferritin antibody can be an anti-human ferritin monoclonal antibody or an anti-human ferritin polyclonal antibody; the preparation process of the latex microsphere coupled with the anti-human ferritin antibody only needs one-time centrifugation, the latex microsphere is free of the influence of a surfactant or protein during heavy suspension centrifugal precipitation, the operation is greatly convenient, the batch difference is small and easy to control, the anti-human ferritin antibody used by the kit can be a monoclonal antibody or a polyclonal antibody due to the special reagent proportion, one or more strains can be used, the use scene of the kit is greatly expanded, and the kit is good in clinical relevance, high in accuracy and high in linearity.
Description
Technical Field
The invention belongs to the field of in-vitro diagnosis in medical detection, and particularly relates to a ferritin detection kit and a preparation method thereof.
Background
Ferritin is an iron storage protein widely existing in living body, has a molecular weight of about 450kDa, has a hydrated iron oxide core and a protein shell surrounding the core, and is composed of 24 subunits each containing about 163 amino acid residues. As a main storage form of iron in the body, the level of ferritin content in the body is positively correlated with the storage amount of iron in the body, and therefore ferritin becomes an important biomarker most commonly used in medical examination for evaluating the iron storage condition in the body and diagnosing iron-deficiency anemia.
Ferritin is widely found in liver, spleen, kidney and bone marrow, especially in liver and spleen. The normal reference values determined by different detection methods are different, the normal reference value of ferritin in a male adult is about 15-280ng/ml, the normal reference value of ferritin in a female adult is about 10-150ng/ml, the ferritin level in the body is reduced in anemia or pregnancy, and the iron reserve level of the body can be screened out through detecting the ferritin content clinically, so that corresponding measures can be taken as soon as possible in the early stage of iron deficiency to prevent the occurrence of iron deficiency anemia. When the liver is inflamed, the ferritin metabolism in the body is obstructed, the ferritin content is changed, and the more serious the stem cell damage is, the higher the ferritin content in the body is. The positive rate of liver cancer patients is up to 90 percent in China, ferritin in the patients with liver cancer effectively treated is reduced, the patients with liver cancer worsen and the patients with liver cancer recurred are increased, the prognosis is poor if the patients with liver cancer continuously increase, in addition, when the organisms suffer from lung cancer, pancreatic cancer or leukemia, ferritin synthesized by cancer cells is increased, and the ferritin-protein-containing liver cancer treatment marker can be used as a non-specific tumor marker and one of the treatment effect monitoring means, and the clinical monitoring of the ferritin content has important significance.
At present, the methods for clinically and quantitatively detecting ferritin mainly comprise an enzyme-linked immunosorbent assay, a chromatography method, a chemiluminescence method and a latex turbidimetric method. The enzyme-linked immunosorbent assay has high sensitivity, but is complicated to operate, long in time consumption and limited in application scene; the chromatographic method has quick detection but poor repeatability; the chemiluminescence method has the advantages of high detection speed, high flux, high sensitivity, high detection cost, strong independence of product platforms of companies, and respective barriers to technologies and products; the technical platforms of various companies of the latex turbidimetry are compatible, the operation is simple, the stability and the repeatability are good, and the adaptive instrument is widely applied to various levels of medical institutions and has wide application scenes; however, the existing latex turbidimetry technology is limited by raw material supply (polyclonal antibody or pretreated monoclonal antibody is needed), the technological process needs multiple centrifugal operations, and the influence of surfactant or protein is generated during heavy suspension centrifugal precipitation, so that large batch difference is easily caused, the process is complicated, and the batch difference is difficult to control.
Disclosure of Invention
The invention aims to provide a ferritin detection kit which has the advantages of simple process, wide raw material application, small batch difference and easy control, and a preparation method thereof.
The purpose of the invention can be realized by the following technical scheme: a ferritin detection kit comprises a first reagent, a second reagent and a ferritin calibrator;
the first reagent comprises: a first solution and a first preservative;
the second reagent comprises: a second solution, a second preservative and latex microspheres coupled with anti-human ferritin antibodies;
the anti-human ferritin antibody can be an anti-human ferritin monoclonal antibody or an anti-human ferritin polyclonal antibody;
preferably, the first solution in the first reagent comprises the following components: 50-100mM of glycine, 1.8-4.5% of sodium chloride, 0.5-2.0% of protein protective agent, 0.25-2.0% of bovine serum albumin, 0.05-0.5% of surfactant and 7.5-8.0 of pH; the first preservative is selected from at least one of sodium azide and Proclin series, preferably Proclin 300, and the concentration is 0.1-0.5%;
as still another preferable aspect, the second solution in the second reagent has the following components: 12.5mM of MES, 50-100mM of glycine, 0.5-1.5% of bovine serum albumin and 2.0-4.0% of protein protective agent;
the second preservative is selected from at least one of sodium azide and Proclin series, preferably Proclin 300, and the concentration is 0.1-0.5%;
the latex microspheres are carboxyl modified polystyrene microspheres, the particle size of the microspheres is 200-350nm, preferably 300nm, and the concentration is 0.1-0.2%.
Further, the ferritin calibrator is prepared by diluting ferritin to 0-1000ng/ml with calibrator diluent;
further, the calibrator diluent comprises the following components: 25-50mM of HEPES, 0.9-2.7% of sodium chloride, 0.5-1.0% of surfactant, 0.5-1.0% of protein protective agent, 1.5-3.0% of bovine serum albumin and a third preservative;
the protein protective agent is selected from one of sucrose and trehalose, preferably trehalose;
the surfactant is selected from at least one of Brij-35, Tween 20 and Triton X100, preferably Triton X100;
the third corrosion inhibitor is selected from at least one of sodium azide and Proclin series, preferably Proclin 300, and the concentration is 0.1-0.5%;
the invention also provides a preparation process of the ferritin detection kit, which comprises the following steps:
dissolving latex microspheres in 25mM MES solution with pH of 6.5, adding EDC, stirring, centrifuging, removing supernatant, resuspending precipitate, ultrasonically dispersing, adding anti-human ferritin antibody, stirring, adding storage solution, ultrasonically dispersing, and standing at 37 deg.C for 10-16 h.
Preferably, the preserving fluid comprises the following components: 100mM of glycine, 1.0-3.0 percent of bovine serum albumin, 4.0-8.0 percent of protein protective agent, 0.2-1.0 percent of second preservative and pH 7.1.
Furthermore, the amount of EDC is 15-35 times of the molar amount of the surface carboxyl of the latex microspheres.
Further, the setting conditions of the centrifugation were 9000rpm, 10 min.
Further, the setting condition of the ultrasound is 30% -40% of power for 10 min.
Furthermore, the dosage of the anti-human ferritin antibody is 0.035 to 0.045 times of the mass of the latex microspheres by mass.
The preparation process only needs one-time centrifugation, and the influence of a surfactant or protein is avoided during the resuspension and centrifugal precipitation, so that the preparation method is greatly convenient to operate, small in batch-to-batch difference and easy to control; the anti-human ferritin antibody can be a monoclonal antibody or a polyclonal antibody, and can be used by one or more strains, so that the application scene of the invention is greatly expanded.
Drawings
FIG. 1 is a correlation analysis of the kit of example 1 of the present invention and the kit of comparative example 1;
FIG. 2 is a comparison of the calibration curve of the kit of example 1 and the calibration curve of the kit of example 2.
Detailed Description
The following is a detailed description of the present invention, and the technical solutions of the present invention are further described with reference to the accompanying drawings, but the following examples should not limit the application of the present invention.
The conditions for detecting the content of human ferritin by using the kit are as follows:
the instrument uses a Hitachi 7180 full-automatic biochemical analyzer, the detection wavelength is 570nm, the reaction direction is increased, and the calibration method is 6-point Spline;
and mixing the sample with the first reagent, incubating for 5min, adding the second reagent, taking an absorbance A1 after 18 seconds, taking an absorbance A2 after reacting for 5min, and calculating the reactivity delta A which is A2-A1. Taking the concentration of each calibrator as an X axis, taking the Delta A measured by each calibrator as a Y axis to draw a calibration curve, and substituting the Delta A of the sample to be measured into the calibration curve when the sample to be measured is detected so as to calculate the concentration of the ferritin in the sample to be measured;
reagent ratio, first reagent: a second reagent: sample 150:50: 10.
Example 1:
the detection kit of the embodiment consists of a first reagent, a second reagent and a calibration substance matched with ferritin;
wherein, the first reagent comprises the following components: 50mM of glycine, 1.8% of sodium chloride, 0.5% of trehalose, 0.5% of bovine serum albumin, 0.2% of surfactant, 3000.2% of Proclin and pH 7.5;
the second reagent comprises the following components: MES 12.5mM, glycine 50mM, bovine serum albumin 0.5%, trehalose 2.5%, Proclin 3000.2%, coupled with anti-human ferritin monoclonal antibody latex microspheres 0.12%;
the ferritin calibrator comprises the following components: HEPES 50mM, sodium chloride 0.9%, surfactant 1.0%, trehalose 0.5%, bovine serum albumin 3.0%, Proclin 3000.2%, and recombinant ferritin antigen obtained by gradient dilution of calibrator diluent to 20/50/250/500/1000 ng/ml.
The preparation process of the latex microsphere coupled with the anti-human ferritin monoclonal antibody comprises the following steps:
dissolving carboxyl activated polystyrene latex microspheres with the particle size of 300nm in 25mM MES solution with the pH of 6.5, wherein the concentration of the latex microspheres is 1.0%, adding EDC with the molar weight of 35 times of the carboxyl of the latex microspheres, stirring for 30min, centrifuging at 9000rpm for 10min, discarding supernatant, carrying out heavy suspension precipitation by using 25mM MES solution with the pH of 6.5 until the concentration is 0.5%, carrying out 35% power ultrasonic 10min for dispersion, adding an anti-human ferritin monoclonal antibody with the mass of 0.04 time of the latex microspheres, stirring for 2h, adding a preservative fluid with the same dosage as the MES solution used in the heavy suspension precipitation, carrying out 35% power ultrasonic 10min for dispersion, and placing at 37 ℃ for 12h to obtain the modified polyethylene glycol methacrylate copolymer latex microsphere.
Example 2:
the detection kit comprises a first reagent, a second reagent and a matched ferritin calibrator;
wherein, the first reagent comprises the following components: glycine 50mM, sodium chloride 0.9%, trehalose 0.5%, bovine serum albumin 0.5%, surfactant 0.2%, Proclin 3000.2%, pH 7.5;
the second reagent comprises the following components: MES 12.5mM, glycine 50mM, bovine serum albumin 0.5%, trehalose 2.5%, Proclin 3000.2%, and latex microsphere coupled with anti-human ferritin polyclonal antibody 0.12%;
the ferritin calibrator comprises the following components: HEPES 50mM, sodium chloride 0.9%, surfactant 1.0%, trehalose 0.5%, bovine serum albumin 3.0%, Proclin 3000.2%, and recombinant ferritin antigen obtained by gradient dilution of calibrator diluent to 20/50/250/500/1000 ng/ml.
The preparation process of the latex microsphere coupled with the anti-human ferritin monoclonal antibody comprises the following steps:
dissolving carboxyl activated polystyrene latex microspheres with the particle size of 300nm in 25mM MES solution with the pH of 6.5, wherein the concentration of the latex microspheres is 1.0%, adding EDC with the molar weight of 35 times of the carboxyl of the latex microspheres, stirring for 30min, centrifuging at 9000rpm for 10min, discarding supernatant, resuspending and precipitating by using 25mM MES solution with the pH of 6.5 until the concentration is 0.5%, performing 35% power ultrasonic 10min for dispersion, adding an anti-human ferritin polyclonal antibody with the mass of 0.04 time of the latex microspheres, stirring for 2h, adding a preservative fluid with the same dosage as the MES solution during resuspension precipitation, performing 35% power ultrasonic 10min for dispersion, and placing at 37 ℃ for 12h to obtain the product.
Example 3:
the detection kit comprises a first reagent, a second reagent and a matched ferritin calibrator;
wherein, the first reagent comprises the following components: 100mM of glycine, 3.6% of sodium chloride, 1.5% of trehalose, 1.5% of bovine serum albumin, 0.5% of surfactant, 3000.4% of Proclin and pH 8.0;
the second reagent comprises the following components: MES 12.5mM, glycine 100mM, bovine serum albumin 1.5%, trehalose 3.5%, Proclin 3000.4%, and coupled anti-human ferritin polyclonal antibody latex microspheres 0.2%;
the ferritin calibrator comprises the following components: HEPES 25mM, sodium chloride 2.7%, surfactant 1.0%, trehalose 1.0%, bovine serum albumin 2.0%, Proclin 3000.4%, and recombinant ferritin antigen which is respectively diluted by calibrator diluent in a gradient manner to 20/50/250/500/1000 ng/ml.
The preparation process of the latex microsphere coupled with the anti-human ferritin monoclonal antibody comprises the following steps:
dissolving carboxyl activated polystyrene latex microspheres with the particle size of 250nm in 25mM MES solution with the pH of 6.5, wherein the concentration of the latex microspheres is 1.0%, adding EDC with the molar weight 15 times that of the carboxyl of the latex microspheres, stirring for 30min, centrifuging at 9000rpm for 10min, discarding supernatant, resuspending and precipitating by using 25mM MES solution with the pH of 6.5 until the concentration is 0.5%, performing 30% power ultrasonic 10min for dispersion, adding an anti-human ferritin monoclonal antibody with the mass 0.045 times that of the latex microspheres, stirring for 2h, adding a preservative fluid with the same dosage as the MES solution during resuspension precipitation, performing 30% power ultrasonic 10min for dispersion, and placing at 37 ℃ for 15h to obtain the product.
Comparative example 1: the commercial "ferritin detection kit (latex turbidimetry)".
The kit of example 1 was calibrated according to the test method of the present invention, and the kit of comparative example 1 was calibrated according to the instructions thereof. Then, the same serum sample was tested using the above two kits, and a linear analysis was performed using the concentration value measured with the kit of comparative example 1 as the X-axis and the concentration value measured with the kit of example 1 as the Y-axis, and the correlation is shown in fig. 1.
The kits of example 1 and example 2 were used for calibration according to the test method of the present invention, and the calibration curves are shown in FIG. 2. Then, one clinical low-value sample and one clinical high-value sample are respectively detected, each sample is detected three times, and the deviation of each detection result is calculated, as shown in table 1.
Table 1: accuracy detection result of kit of the invention
It can be seen from table 1 that the deviation of the detection results of the ferritin content of the clinical sample is within ± 5%, which indicates that both example 1 using the anti-human ferritin monoclonal antibody and example 2 using the anti-human ferritin polyclonal antibody have good detection accuracy, and the use of the anti-human ferritin antibody by the method of the present invention is not limited to the monoclonal antibody or the polyclonal antibody.
The kit of example 1 and example 2 was used to calibrate according to the test method of the present invention, and then two clinical samples with different ferritin contents were tested ten times, and the average value, standard deviation SD and coefficient of variation CV of the test results of ten times were calculated, and the results are shown in table 2.
Table 2: precision detection result of kit of the invention
As can be seen from Table 2, the coefficient of variation CV of both serum samples is within 4.5%, which indicates that the kit of the embodiment of the invention has higher precision.
Various embodiments of the present invention have many experimental data, and it is not suitable to list the embodiments one by one, and various changes and modifications made by those skilled in the art without departing from the principle of the present invention should be within the claims of the present invention.
Claims (11)
1. A ferritin detection kit is characterized in that the kit consists of a first reagent, a second reagent and a ferritin calibrator,
the first reagent comprises: a first solution and a first preservative;
the second reagent comprises: a second solution, a second preservative and latex microspheres coupled with anti-human ferritin antibodies; the anti-human ferritin antibody is an anti-human ferritin monoclonal antibody or an anti-human ferritin polyclonal antibody.
2. The ferritin detection kit of claim 1 wherein the first solution in the first reagent comprises glycine 50-100mM, sodium chloride 1.8-4.5%, protein protectant 0.5-2.0%, bovine serum albumin 0.25-2.0%, surfactant 0.05-0.5%, and the pH of the first solution is 7.5-8.0; the first preservative in the first reagent is at least one of sodium azide and Proclin series, and the concentration of the first preservative is 0.1-0.5%.
3. The ferritin detection kit of claim 1 wherein the second solution in the second reagent comprises: 12.5mM MES, 50-100mM glycine, 0.5-1.5% bovine serum albumin, 2.0-4.0% protein protective agent;
the second preservative is selected from at least one of sodium azide and Proclin series, and the concentration is 0.1-0.5%;
the latex microspheres are carboxyl modified polystyrene microspheres, the particle size of the microspheres is 200-350nm, and the concentration of the microspheres is 0.1-0.2%.
4. The ferritin detection kit of claim 1 wherein the ferritin calibrator is diluted to 0-1000ng/ml with calibrator diluent.
5. The ferritin detection kit according to claim 4 wherein the calibrator diluent comprises: 25-50mM of HEPES, 0.9-2.7% of sodium chloride, 0.5-1.0% of surfactant, 0.5-1.0% of protein protective agent, 1.5-3.0% of bovine serum albumin and a third preservative;
the protein protective agent is selected from one of sucrose and trehalose;
the surfactant is selected from at least one of Brij-35, Tween 20 and Triton X100;
the third corrosion inhibitor is at least one of sodium azide and Proclin series, and the concentration is 0.1-0.5%.
6. A method for preparing the ferritin detection kit according to claims 1-5, comprising the following steps: dissolving latex microspheres in 25mM MES solution with pH of 6.5, adding EDC, stirring, centrifuging, removing supernatant, suspending precipitate, ultrasonically dispersing, adding anti-human ferritin antibody, stirring, adding preservative solution, ultrasonically dispersing, and standing at 37 deg.C for 10-16 h.
7. The method for preparing a ferritin detection kit according to claim 6 wherein the storage solution comprises the following components: 100mM of glycine, 1.0-3.0 percent of bovine serum albumin, 4.0-8.0 percent of protein protective agent, 0.2-1.0 percent of second preservative and pH 7.1.
8. The method for preparing the ferritin detection kit according to claim 6 wherein the molar amount of EDC is 15-35 times the molar amount of carboxyl groups on the surface of latex microspheres.
9. The method for preparing the ferritin detection kit according to claim 6 wherein the centrifugation is set at 9000rpm for 10 min.
10. The method for preparing the ferritin detection kit according to claim 6 wherein the setting condition of ultrasound is 30% -40% power for 10 min.
11. The method for preparing the ferritin detection kit according to claim 6 wherein the amount of anti-human ferritin antibody is 0.035-0.045 times the amount of latex microspheres by weight.
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