CN102628866A - 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, the liquid double reagent kit through ferritin in the latex enhancing immune turbidimetry for Determination serum.
Background technology
Extensively there is one type of albumen of storing iron in the animal and plant body; Content is maximum in the liver of mammal and spleen; About 12~the 14nm of its external diameter, the empty pocket chamber directly is about 6nm, and shell (being apoferritin) is made up of 24 subunits; Each subunit contains 163 amino acid residues approximately, and each molecule can combine 4500 iron atoms at most.Molecular weight is about 900,000.Ferritin in conjunction with iron be " dissolving " in water, the iron that stores in the concentration of blood plasma ferritin and the body is directly proportional.
Ferritin is a kind of solvable histone of storing iron in the body.Contain small amounts of iron albumen in the normal human serum, but the different detection method 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).The serum levels of iron level reduces when the gestational period and acute anemia, raises when acute and chronic liver damage and liver cancer, 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, has the early diagnosis of helping.The reason that liver cancer patient has ferritin to increase possibly be: 1. HCC can synthesize and secrete ferritin or isoferritin; 2. liver cancer tissue is affected to the picked-up and the removing of ferritin; 3. hepatocellular damage is downright bad, and the ferritin that is stored in the liver cytoplasm overflows in the blood.Though serum ferritin is non-specific, except that liver cancer, cancer of pancreas moderate raise, other tumor in digestive tract such as the cancer of the esophagus, cancer of the stomach, straight colon cancer did not all raise.Liver cancer patient treatment responder serum ferritin descends, and raises and worsen and do not send out the person again, continues to increase then prognosis mala, can be used as one of curative effect monitoring means so serum ferritin is measured, and is particularly meaningful especially to the AFP negative patients.
Summary of the invention
The object of the present invention is to provide a kind of improvement of ferritin detectable, the characteristics that have that sample (serum) need not to dilute, highly sensitive, high specificity, the range of linearity are wide, are applicable to various types of automatic clinical chemistry analyzers.
On the one hand, the liquid double reagent kit that the present invention provides and utilizes IGY sections protein antibodies, passes through ferritin in the latex enhancing immune turbidimetry for Determination serum, 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 that combines anti-people's ferritin antibody, and the latex particle diameter is 100-200nm, also comprises antiseptic, surfactant, electrolyte and damping fluid; The ferritin antigen standard items of concentration known comprise antiseptic, purifying people ferritin, electrolyte and damping fluid.
Further; In an embodiment of the present invention, utilize IGY sections protein antibodies, the liquid double reagent kit through ferritin in the latex enhancing immune turbidimetry for Determination serum to comprise: the ferritin antigen calibration object of reagent R1, reagent R2 and concentration known.Wherein:
The mass percent of macromolecule accelerator is 2%-6% among the said reagent R1; 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 percentage composition of latex particle is 0.05%-0.5% among the said reagent R2; 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%, and 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.
Utilize in IGY sections protein antibodies, the liquid double reagent kit through ferritin in the latex enhancing immune turbidimetry for Determination serum according to the invention, described IGY sections protein antibodies is from chicken or duck, is preferably to come from chicken.This polyclonal antibody can be discerned a plurality of antigen sites of ferritin specifically, therefore can specific bond ferritin monomer and condensate, also eliminated of the influence of factor such as rheumatoid factor in the conventional I GG type antibody simultaneously to measured value.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 according to the invention, R2 is TRIS buffer or glycine buffer, is preferably TRIS buffer.The pH value of said reagent R1, R2 damping fluid is 6.0-8.5, is preferably 7.0-8.0.
The macromolecule accelerator is selected from PEG2000, PEG6000, PEG8000 in the reagent according to the invention, is preferably PEG6000.
Antiseptic is selected from Sodium azide or ethyl-para-hydroxybenzoate in the reagent according to the invention, is preferably Sodium azide.
Surfactant is selected from Tween series or polyoxyethylene phenyl ether in the reagent according to the invention, is preferably Tween.
Electrolyte is sodium chloride or lime chloride in the reagent according to the invention, is preferably sodium chloride.
The present invention adopts latex particle to strengthen the transmission immunoturbidimetry; Its principle is the latex particle that has encapsulated antibody with high specificity, combines with corresponding antigen generation specificity in the sample, forms the compound of Ag-Ab-latex particle; This compound forms certain turbidity in specific damping fluid; The increase degree and the antigen concentration in the sample of turbidity are proportional, under certain wavelength, carry out turbidimetric analysis turbidimetry, can record the content of seized antigen in the sample.
In the present invention; Ferritin antigen in the serum combines with the anti-people's ferritin of the specificity chicken polyclonal antibody that is combined in the latex particle surface; Antigen-antibody reaction taking place, form immune complex, forms certain turbidity; Measure the absorbance of this turbidity in the 570nm wavelength, the reference standard curve can be obtained the content of ferritin in the sample.
Adopt the IGY type antibody that utilizes of the present invention; When measuring,, read absorbance A 1 earlier with sample and reagent R1 mixing through the liquid double reagent kit of ferritin in the latex enhancing immune turbidimetry for Determination serum; Add reagent R2 afterwards; Read absorbance A 2, calculate reaction absorbance changing value, the reference standard curve draws the content of ferritin in the sample.
Technological core of the present invention is: use specificity IGY sections protein antibodies, can discern a plurality of antigen sites of ferritin, eliminated the influence of factor such as rheumatoid factor in the conventional I GG type antibody to measured value simultaneously.Therefore the mensuration sensitivity and the antijamming capability of kit have been improved.Use method of the present invention can guarantee to detect extremely low-level ferritin, have the good clinical using value.
Liquid double reagent kit according to the invention has the accuracy height, an advantage such as specificity is good, highly sensitive and antijamming capability is strong, can be used for detecting the concentration of ferritin in the serum, is applicable to clinical various automatic clinical chemistry analyzer.
Description of drawings
Fig. 1: ferritin assay method synoptic diagram.
Fig. 2: reagent of the present invention and contrast agent correlativity.
Embodiment
Embodiment 1: utilize IGY sections protein antibodies, the liquid double reagent kit through ferritin in the latex enhancing immune turbidimetry for Determination serum
The principal ingredient and the proportioning of kit are following:
Reagent R1:
Reagent R2:
The ferritin standard items:
The kit measurement method is as shown in Figure 1.Analytical approach: 2 end-point methods, reaction time: 10min, read a little: 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 water spot that is provided with for instrument, 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: the correlativity of detection kit, sensitivity and the range of linearity
(the Britain Ferritin of RANDOX company kit, batch number is: 214284) to use reagent of the present invention (specifically filling a prescription referring to embodiment 1) and contrast agent in the experiment.
1, correlativity experiment
Use reagent of the present invention and contrast agent respectively, adopt Hitachi's 7080 automatic clinical chemistry analyzers that 50 parts of human serums (comprising normal and monstrosity) are measured (table 1) simultaneously by each autoregressive parameter, reagent parameter of the present invention is seen embodiment 1.Measured value is carried out linear regression; Getting regression equation is y=1.0346x-3.2384; Related coefficient is that (y is a reagent of the present invention to R2=0.9996; X is a contrast agent) (Fig. 2), the result shows that reagent of the present invention and contrast agent correlativity are fine, and then explanation reagent of the present invention has excellent specificity and accuracy.This reagent is not only applicable to Hitachi's 7080 automatic clinical chemistry analyzers in addition, also can be used for the full-automatic and semi-automatic biochemical analyzer of other series such as Hitachi, Olympus, and concrete parameter can be done suitable adjustment 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 |
[0039]?
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
That table 2 and table 3 are represented respectively is the sensitivity result of detectable of the present invention and contrast agent.
The result shows that the sensitivity of reagent of the present invention can reach 2.02ng/mL (table 2), is superior to the 3.51ng/mL (table 3) of contrast agent.
Table 2: reagent sensitivity of the present invention
This reagent LDL (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 LDL (LLD)=13.75+3*3.22=23.41ng/mL
Sensitivity: 0.9 * 23.41/6.01=3.51ng/mL
3, the range of linearity
Used sample is the high value serum of a ferritin physiological 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 the result shows the reagent range of linearity of the present invention wideer (table 4).
Table 4: the range of linearity of reagent of the present invention and contrast agent contrast relatively
Above embodiment is to explanation of the present invention and further explanation, rather than limitation of the present invention, and any modification of in spirit of the present invention and rights protection scope, being made all falls into protection scope of the present invention.
Claims (11)
- One kind utilize IGY sections protein antibodies, through the liquid double reagent kit of ferritin in the latex enhancing immune turbidimetry for Determination serum, kit comprises the ferritin antigen standard items of reagent R1, reagent R2 and concentration known, wherein,Said reagent R1 comprises macromolecule accelerator, antiseptic, surfactant, electrolyte and damping fluid;Said reagent R2 comprises the polystyrene latex particle that combines anti-people's ferritin antibody, and the latex particle diameter is 100-200nm, also comprises antiseptic, surfactant, electrolyte and damping fluid;The ferritin antigen standard items of said concentration known comprise antiseptic, purifying people ferritin, electrolyte and damping fluid.
- 2. kit according to claim 1, wherein IGY sections protein antibodies is preferably from chicken from chicken or duck.
- 3. kit according to claim 1, this kit comprise the ferritin antigen standard items of reagent R1, reagent R2 and concentration known, wherein:The mass percent of macromolecule accelerator is 2%-6% among the said reagent R1; 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 percentage composition of latex particle is 0.05%-0.5% among the said reagent R2; 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%, and 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 said 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.
- 4. according to claim 1 or 3 described kits, wherein damping fluid is TRIS buffer or glycine buffer, is preferably TRIS buffer.
- 5. according to claim 1 or 3 described kits, wherein the pH of buffer value is 6.0-8.5, is preferably 7.0-8.0.
- 6. according to claim 1 or 3 described kits, wherein the macromolecule accelerator among the reagent R1 is selected from PEG2000, PEG6000, PEG8000, is preferably PEG6000.
- 7. according to claim 1 or 3 described kits, wherein antiseptic is selected from Sodium azide or ethyl-para-hydroxybenzoate, is preferably Sodium azide.
- 8. according to claim 1 or 3 described kits, wherein the surfactant among reagent R1, the R2 is selected from Tween series or polyoxyethylene phenyl ether, is preferably Tween.
- 9. according to claim 1 or 3 described kits, wherein electrolyte is sodium chloride or lime chloride, is preferably sodium chloride.
- 10. according to claim 1 or 3 described kits, wherein the ferritin concentration of the ferritin antigen standard items of concentration known be respectively 100,200,500,1000ng/mL.
- 11. according to claim 1 or 3 described kits, wherein ferritin antigen standard items are the human serum of the pure article of interpolation recombinant human iron albumen or the liquid of other similar serum matrix.
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