CN102628866B - Liquid double reagent kit for determining ferritin in serum by utilization of IGY ferritin antibody through latex enhanced turbidimetric immunoassay - Google Patents
Liquid double reagent kit for determining ferritin in serum by utilization of IGY ferritin antibody through latex enhanced turbidimetric immunoassay Download PDFInfo
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Abstract
The invention discloses a liquid double reagent kit for determining ferritin in serum by the utilization of IGY ferritin antibody through latex enhanced turbidimetric immunoassay. The kit provided by the invention contains a reagent R1, a reagent R2 and a ferritin standard, wherein the reagent R1 contains a high-molecular accelerator, an antiseptic, a surfactant, an electrolyte and a buffer; the reagent R2 contains anti-human ferritin antibody bonded polystyrene latex particles, the diameter of which is 100-200nm, and also contains an antiseptic, a surfactant, an electrolyte and a buffer; and the ferritin standard contains antiseptic, purified human ferritin, an electrolyte and a buffer. The kit provided by the invention has advantages of strong singularity, high sensitivity, wide linear range and easy operation, and is applicable to various automatic biochemical analyzers.
Description
Technical field
The invention belongs to medical immunology in-vitro diagnosis field, relate to a kind of immunologic function test reagent, further, the present invention relates to a kind of IGY of utilization sections protein antibodies, pass through the liquid double reagent kit of latex enhancing immune turbidimetry for Determination Iron in Serum albumen.
Background technology
In animal and plant body, extensively there is the albumen of a class storage iron, in the liver of mammal and spleen, content is maximum, approximately 12～14nm of its external diameter, footpath, empty pocket chamber is about 6nm, shell (being apoferritin) is made up of 24 subunits, each subunit is approximately containing 163 amino acid residues, and each molecule at most can be in conjunction with 4500 iron atoms.Molecular weight is about 900,000.Ferritin in conjunction with iron be " molten " in water, the iron storing in the concentration of Plasma Rerritin and body is directly proportional.
Ferritin is a kind of solvable histone of storing iron in body.In normal human serum, contain a small amount of ferritin, but different detection methods there is different normal values.The general about 80-130 μ of normal mean value male sex g/L (80-130ng/ml) women about 35-55 μ g/L (35-55ng/ml).Serum levels of iron level reduces in the time of the gestational period and acute anemia, when acute and chronic liver damage and liver cancer, raises, and domestic report liver cancer patient positive rate is up to 90%.Discovered in recent years liver cancer also contains a kind of isoferritin of acidity, is called cancer embryo isoferritin, may contribute to early diagnosis.Liver cancer patient has the reason that ferritin increases to be: 1. ferritin or isoferritin can be synthesized and secrete to hepatoma carcinoma cell; 2. picked-up and the removing of liver cancer tissue to ferritin is affected; 3. hepatocellular damage necrosis, the ferritin being stored in liver cytoplasm overflows in blood.Though serum ferritin is non-specific, except liver cancer, cancer of pancreas moderate raise, other tumor in digestive tract does not all raise as the cancer of the esophagus, cancer of the stomach, straight colon cancer.Liver cancer patient treatment responder serum ferritin declines, and raises and worsen and do not send out person again, continues to increase prognosis mala, therefore Determination of Serum Ferritin can be used as one of curative effect monitoring means, particularly especially meaningful to the patient of AFP feminine gender.
Summary of the invention
The object of the present invention is to provide a kind of ferritin to detect the improvement of reagent, have the advantages that sample (serum) is wide without dilution, highly sensitive, high specificity, the range of linearity, be applicable to various types of automatic clinical chemistry analyzers.
On the one hand, the invention provides and utilize IGY sections protein antibodies, pass through the liquid double reagent kit of latex enhancing immune turbidimetry for Determination Iron in Serum albumen, this kit comprises the ferritin antigen calibration object of reagent R1, reagent R2 and concentration known, wherein,
Reagent R1 comprises macromolecule accelerator, antiseptic, surfactant, electrolyte and damping fluid; Reagent R2 comprises the polystyrene latex particle in conjunction with anti-ferritin antibody, and latex particle diameter is 100-200nm, also comprises antiseptic, surfactant, electrolyte and damping fluid; The ferritin antigen standard items of concentration known comprise antiseptic, Purification of Human ferritin, electrolyte and damping fluid.
Further, in an embodiment of the present invention, utilize IGY sections protein antibodies, comprise by the liquid double reagent kit of latex enhancing immune turbidimetry for Determination Iron in Serum albumen: the ferritin antigen calibration object of reagent R1, reagent R2 and concentration known.Wherein:
In described reagent R1, the mass percent of macromolecule accelerator is 2%-6%, the mass percent of antiseptic is 0.05%-0.5%, the mass percent of surfactant is 0.1%-1.0%, electrolytical mass percent is 0.1%-10.0%, and the concentration of damping fluid is 10mmol/L-200mmol/L.
In described reagent R2, the percentage composition of latex particle is 0.05%-0.5%, the content of IGY sections protein polyclone antibody is 0.05mg/mL-0.5mg/mL, the mass percent of antiseptic is 0.05%-0.5%, the mass percent of surfactant is 0.1%-1.0%, electrolytical mass percent is 0.1%-10.0%, and the concentration of damping fluid is 10mmol/L-200mmol/L.
The ferritin antigen standard items of concentration known, the content of the ferritin antigen of purifying is 100ng/mL-1000ng/mL, the mass percent of antiseptic is 0.05%-0.5%, and electrolytical mass percent is 0.1%-10.0%, and the concentration of damping fluid is 10mmol/L-200mmol/L.
Of the present inventionly utilizing IGY sections protein antibodies, by the liquid double reagent kit of latex enhancing immune turbidimetry for Determination Iron in Serum albumen, described IGY sections protein antibodies, from chicken or duck, is preferably and comes from chicken.This polyclonal antibody can be identified the multiple antigen sites of ferritin specifically, therefore can specific bond ferritin monomer and condensate, also eliminate in conventional I GG type antibody the impacts of factor on measured value such as rheumatoid factor simultaneously.Therefore, this polyclonal antibody both can ensure the efficient affinity of antigen-antibody, had also ensured the specificity of kit, had improved antijamming capability.
The damping fluid of reagent R1 of the present invention, R2 is TRIS buffer or glycine buffer, is preferably TRIS buffer.The pH value of described reagent R1, R2 damping fluid is 6.0-8.5, is preferably 7.0-8.0.
In reagent of the present invention, macromolecule accelerator is selected from PEG2000, PEG6000, PEG8000, is preferably PEG6000.
In reagent of the present invention, antiseptic is selected from Sodium azide or ethyl-para-hydroxybenzoate, is preferably Sodium azide.
In reagent of the present invention, surfactant is selected from Tween series or polyoxyethylene phenyl ether, is preferably Tween.
In reagent of the present invention, electrolyte is sodium chloride or lime chloride, is preferably sodium chloride.
The present invention adopts latex particle to strengthen transmission immunological turbidimetry method, its principle is the latex particle that has been coated with antibody with high specificity, with corresponding antigen generation specific binding in sample, form the compound of Ag-Ab-latex particle, in specific damping fluid, this compound forms certain turbidity, antigen concentration in increase degree and the sample of turbidity is proportional, carries out turbidimetric analysis turbidimetry under certain wavelength, can record the content of tested antigen in sample.
In the present invention, ferritin antigen in serum is combined with the anti-human ferritin polyclonal antibody of the specificity chicken that is combined in latex particle surface, there is antigen-antibody reaction, form immune complex, form certain turbidity, measure the absorbance of this turbidity at 570nm wavelength place, reference standard curve can be obtained the content of ferritin in sample.
Adopt the IGY type antibody that utilizes of the present invention, while mensuration by the liquid double reagent kit of latex enhancing immune turbidimetry for Determination Iron in Serum albumen, first sample and reagent R1 are mixed, read absorbance A 1, add afterwards reagent R2, read absorbance A 2, calculate reaction absorbance changing value, reference standard curve draws the content of ferritin in sample.
Technological core of the present invention is: use specificity IGY sections protein antibodies, can identify the multiple antigen sites of ferritin, eliminated in conventional I GG type antibody the impacts of factor on measured value such as rheumatoid factor simultaneously.Therefore mensuration sensitivity and the antijamming capability of kit have been improved.Use method of the present invention can ensure to detect extremely low-level ferritin, there is good clinical value.
Liquid double reagent kit of the present invention, has that accuracy is high, specificity good, an advantage such as highly sensitive and antijamming capability is strong, can be used for detecting the concentration of Iron in Serum albumen, is applicable to clinical various automatic clinical chemistry analyzer.
Brief description of the drawings
Fig. 1: ferritin assay method schematic diagram.
Fig. 2: reagent of the present invention and contrast agent correlativity.
Embodiment
Embodiment 1: utilize IGY sections protein antibodies, pass through the liquid double reagent kit of latex enhancing immune turbidimetry for Determination Iron in Serum albumen
Principal ingredient and the proportioning of kit are as follows:
Reagent R1:
Reagent R2:
Ferritin standard items:
Kit measurement method as shown in Figure 1.Analytical approach: Two point end assay, reaction time: 10min, read point: 19 o'clock to 31 o'clock, sample size: 7 μ L, reagent R1:140 μ L, reagent R2:70 μ L, predominant wavelength: 570nm, commplementary wave length 800nm, the Direction of Reaction: the reaction of rising, calibrating mode: Spline, calibrating method: 5 calibrations, calibrate first for instrument arrange water spot, second and third, four, five be that concentration is the ferritin standard items of 100ng/mL, 200ng/mL, 500ng/mL and 1000ng/mL.
Embodiment 2: correlativity, sensitivity and the range of linearity of detection kit
(the Britain Ferritin of RANDOX company kit, batch number is: 214284) in experiment, to use reagent of the present invention (specifically filling a prescription referring to embodiment 1) and contrast agent.
1, correlativity experiment
Use respectively reagent of the present invention and contrast agent, adopt Hitachi's 7080 automatic clinical chemistry analyzers to measure (table 1) to 50 parts of human serums (comprising normal and monstrosity) by each autoregressive parameter, reagent parameter of the present invention is shown in embodiment 1 simultaneously.Measured value is carried out to linear regression, obtaining regression equation is y=1.0346x-3.2384, related coefficient is that (y is reagent of the present invention to R2=0.9996, x is contrast agent) (Fig. 2), result show reagent of the present invention and contrast agent correlativity fine, and then explanation reagent of the present invention there is good specificity and accuracy.In addition this reagent is not only applicable to Hitachi's 7080 automatic clinical chemistry analyzers, also can be used for the full-automatic and semi-automatic biochemical analyzer of other series such as Hitachi, Olympus, and design parameter can make the appropriate adjustments according to major parameter.
Table 1: reagent of the present invention and contrast agent correlativity
| Sequence number | Reagent of the present invention | Contrast agent | Sequence number | Reagent of the present invention | Contrast agent |
| 1 | 66.7 | 65.1 | 26 | 101.3 | 99.6 |
| 2 | 88.8 | 89.4 | 27 | 334.0 | 345.2 |
| 3 | 81.9 | 82.0 | 28 | 96.8 | 96.1 |
| 4 | 148.0 | 152.6 | 29 | 136.3 | 135.3 |
| 5 | 89.6 | 90.9 | 30 | 181.9 | 183.7 |
| 6 | 172.7 | 180.2 | 31 | 44.2 | 42.3 |
| 7 | 24.4 | 23.0 | 32 | 107.0 | 107.4 |
| 8 | 201.8 | 203.7 | 33 | 107.2 | 105.7 |
| 9 | 103.2 | 103.0 | 34 | 123.3 | 120.8 |
| 10 | 17.0 | 16.3 | 35 | 81.3 | 81.6 |
| 11 | 150.9 | 152.9 | 36 | 132.8 | 133.7 |
| 12 | 60.4 | 59.9 | 37 | 118.3 | 117.9 |
| 13 | 118.9 | 121.3 | 38 | 7.3 | 5.1 |
| 14 | 494.4 | 515.1 | 39 | 9.9 | 11.6 |
| 15 | 101.9 | 101.3 | 40 | 45.5 | 45.5 |
| 16 | 47.4 | 49.1 | 41 | 169.5 | 170.3 |
| 17 | 259.0 | 265.1 | 42 | 77.1 | 75.1 |
| 18 | 205.4 | 206.1 | 43 | 269.8 | 272.3 |
| 19 | 192.6 | 196.4 | 44 | 172.5 | 172.4 |
| 20 | 416.5 | 423.7 | 45 | 160.5 | 162.3 |
| 21 | 100.9 | 101.8 | 46 | 87.9 | 88.5 |
| 22 | 406.2 | 419.9 | 47 | 122.2 | 122.1 |
| 23 | 108.3 | 106.3 | 48 | 456.9 | 460.7 |
| 24 | 119.8 | 120.8 | 49 | 259.7 | 266.1 |
| 25 | 19.9 | 17.3 | 50 | 506.7 | 526.6 |
2, sensitivity test
What table 2 and table 3 represented respectively is the sensitivity result that the present invention detects reagent and contrast agent.
Result shows that the sensitivity of reagent of the present invention can reach 2.02ng/mL (table 2), is better than the 3.51ng/mL (table 3) of contrast agent.
Table 2: reagent sensitivity of the present invention
This reagent lowest detectable limit (LLD)=10.65+3*1.60=15.45ng/mL
Sensitivity: 0.9 × 15.45/6.89=2.02ng/mL
Table 3: contrast agent sensitivity
Contrast agent lowest detectable limit (LLD)=13.75+3*3.22=23.41ng/mL
Sensitivity: 0.9 × 23.41/6.01=3.51ng/mL
3, the range of linearity
Sample used is the high value serum of ferritin normal saline dilution gained, and linear error 10% is respectively with the interior range of linearity: reagent of the present invention can reach 519mg/L; Contrast agent is 501mg/L, and result shows the reagent range of linearity of the present invention wider (table 4).
Table 4: the range of linearity comparison that reagent of the present invention contrasts with contrast agent
Above embodiment is to explanation of the present invention and further explains, instead of limitation of the present invention, and any amendment of making within the scope of spirit of the present invention and rights protection, all falls into protection scope of the present invention.
Claims (18)
1. utilize IGY sections protein antibodies, pass through a liquid double reagent kit for latex enhancing immune turbidimetry for Determination Iron in Serum albumen, kit comprises:
Reagent R1,
Reagent R2 and
The ferritin antigen standard items of concentration known,
Wherein:
In described reagent R1, the mass percent of macromolecule accelerator is 2%-6%, the mass percent of antiseptic is 0.05%-0.5%, the mass percent of surfactant is 0.1%-1.0%, electrolytical mass percent is 0.1%-10.0%, and the concentration of damping fluid is 10mmol/L-200mmol/L;
Described reagent R2 comprises the polystyrene latex particle in conjunction with anti-ferritin antibody, latex particle diameter is 100-200nm, in described reagent R2, the percentage composition of latex particle is 0.05%-0.5%, the content of IGY sections protein polyclone antibody is 0.05mg/mL-0.5mg/mL, the mass percent of antiseptic is 0.05%-0.5%, the mass percent of surfactant is 0.1%-1.0%, electrolytical mass percent is 0.1%-10.0%, and the concentration of damping fluid is 10mmol/L-200mmol/L;
The ferritin antigen standard items of described concentration known, the content of the ferritin antigen of purifying is 100ng/mL-1000ng/mL, the mass percent of antiseptic is 0.05%-0.5%, and electrolytical mass percent is 0.1%-10.0%, and the concentration of damping fluid is 10mmol/L-200mmol/L.
2. kit according to claim 1, wherein IGY sections protein antibodies is from chicken or duck.
3. kit according to claim 2, wherein IGY sections protein antibodies is from chicken.
4. kit according to claim 1, wherein damping fluid is TRIS buffer or glycine buffer.
5. kit according to claim 4, wherein damping fluid is TRIS buffer.
6. kit according to claim 1, wherein pH of cushioning fluid is 6.0-8.5.
7. kit according to claim 6, wherein pH of cushioning fluid is 7.0-8.0.
8. kit according to claim 1, wherein the macromolecule accelerator in reagent R1 is selected from PEG2000, PEG6000, PEG8000.
9. kit according to claim 8, wherein the macromolecule accelerator in reagent R1 is PEG6000.
10. kit according to claim 1, wherein antiseptic is selected from Sodium azide or ethyl-para-hydroxybenzoate.
11. kits according to claim 10, wherein antiseptic is Sodium azide.
12. kits according to claim 1, wherein the surfactant in reagent R1, R2 is selected from Tween series or polyoxyethylene phenyl ether.
13. kits according to claim 12, wherein the surfactant in reagent R1, R2 is Tween.
14. kits according to claim 1, wherein electrolyte is sodium chloride or lime chloride.
15. kits according to claim 14, wherein electrolyte is sodium chloride.
16. kits according to claim 1, wherein the ferritin concentration of the ferritin antigen standard items of concentration known be respectively 100,200,500,1000ng/mL.
17. kits according to claim 1, wherein ferritin antigen standard items are the human serum that adds recombinant human iron albumen sterling.
18. kits according to claim 1, it comprises:
Reagent R1,
Reagent R2 and
The ferritin antigen standard items of concentration known,
Wherein:
Reagent R1 comprises:
Reagent R2 comprises:
Ferritin antigen standard items comprise:
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| CN105137089B (en) * | 2015-08-28 | 2017-03-22 | 宁波瑞源生物科技有限公司 | Serum ferritin content detection kit |
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| CN111089974A (en) * | 2019-12-26 | 2020-05-01 | 迪瑞医疗科技股份有限公司 | Ferritin detect reagent box |
| CN112129934A (en) * | 2020-09-25 | 2020-12-25 | 上海捷门生物技术有限公司 | Ferritin detection kit and preparation method thereof |
| CN114217071B (en) * | 2021-12-01 | 2023-11-14 | 柏荣诊断产品（上海）有限公司 | High-specificity integrated transmission and scattering ferritin latex turbidimetry detection kit |
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