CN1928560A - Immune analysis reagent for magnetic separation alkali phosphatase enzyme mark and analytical detection method for same - Google Patents
Immune analysis reagent for magnetic separation alkali phosphatase enzyme mark and analytical detection method for same Download PDFInfo
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- CN1928560A CN1928560A CN 200610060896 CN200610060896A CN1928560A CN 1928560 A CN1928560 A CN 1928560A CN 200610060896 CN200610060896 CN 200610060896 CN 200610060896 A CN200610060896 A CN 200610060896A CN 1928560 A CN1928560 A CN 1928560A
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Abstract
The magnetic separated AKP-labeled immunity analysis reagent comprises: the AKP label, the magnetic separation reagent as nano magnetic micro-ball covered by FITC, the FITC-antibody label, and the standard antigen or antibody and substrate. Wherein, the substrate can be PMP, AMPPD or APS-5. This invention is reliable and nun-polluted, and can detect fast and precisely.
Description
Technical field
The present invention relates to a kind of immunological assay reagents and method of testing thereof, especially the immunological assay reagents of magnetic separation alkali phosphatase enzyme mark and analysis test method thereof.
Background technology
At present, the method for measuring these trace activity substances clinically in China mainly contains the direct chemical luminescent method of radioimmunoassay method, enzyme immunoassay method, enzyme-catalyzed chemical luminescence or acridinium ester mark, the electrochemical luminescence method of tris (bipyridine) ruthenium mark etc.; The radioimmunology analysis method is to adopt radioactive isotope I
125As tracer agent, be marked on antigen or the corresponding antibodies, on gamma-ray detector, by to I
125Though the mensuration of intensity of radiation is determined this method of content of test substance and has solved the quantitative test purpose of trace activity substance preferably that the defective of its existence is clearly: radioactive isotope I
125As tracer technique, in the manufacturing of reagent, storage, application operating process, environment is brought pollution, caused serious injury to human body; Simultaneously, because I
125The restriction of half life period has determined the term of validity of radiommunoassay reagent to have only one month; The non-specific accurate rate variance that causes the result of detachment process can't be done the emergency treatment sample; Enzyme immunoassay method is to adopt the HRP horseradish peroxidase as luminous marker, and the plastic microporous plate is as carrier and isolation technics, and OPD o-phenylenediamine etc. are as chromogenic substrate, 1MH
2SO
4Stop bath as the enzyme chromogenic reaction, analyze the concentration of test substance at the absorbance at 492nm place by measuring the enzyme reaction product wavelength, this method is mainly used to carry out human serum sample's in enormous quantities qualitatively screening, its defective is that the color intensity of colour developing product changes over time, can not immobilize, chromogenic substrate OPD etc. is strong carcinogenic substance, simultaneously to the insufficient sensitivity of quite a few micro-hormone, reaction time is oversize, is difficult to standardization in the operating process; In addition, defectives such as the electrochemical luminescence method of the direct chemical luminescent method master tris (bipyridine) ruthenium mark of enzyme-catalyzed chemical luminescence or acridinium ester mark etc. brings the reagent instability in analytic process, the analysis result accuracy rate is not high and test speed is fast have inadequately brought obstruction for the amount of accurately analyzing trace activity substance.
Summary of the invention
The objective of the invention is to solve and have the immunoreagent instability in the prior art, pollution problems is arranged, a kind of stable reagent is provided, does not have the immunological assay reagents of the magnetic separation alkali phosphatase enzyme mark that pollutes.
The present invention also provides accurate, the fireballing magnetic of a kind of analysis to separate the EIA enzyme immunoassay method of testing.
For achieving the above object, technical scheme of the present invention is:
A kind of immunological assay reagents of magnetic separation alkali phosphatase enzyme mark is provided, comprise label, separation agent, FITC-antibody labeling thing, standard antigen or antibody and substrate, wherein: described separation agent is the nano-magnetic microballon of goat-anti isosulfocyanic acid fluorescence sulphur FITC bag quilt, and described nano-magnetic microballon is that the inside is coated with Fe
3O
4Or Fe
2O
3The outside contain-OH ,-COOH or-NH
2The microballon of reactive group.
The content that described nano-magnetic bead surface contains reactive group is 0.05-0.5eqm/g.
The nano-magnetic microballon of described goat-anti FITC bag quilt is that the nano-magnetic microballon connects with the chemistry of goat-anti FITC.
Described label is alkaline phosphatase AKP.
Described substrate comprises chromogenic substrate list phosphoric acid phenolphthalein PMP, luminous substrate diamantane and derivant AMPPD or APS-5.
The invention provides a kind of immunoassay method of testing of magnetic separation alkali phosphatase enzyme mark, test according to the following steps:
A, the monoclonal antibody of alkaline phosphatase, FITC and patient's to be measured serum mixing incubation will be marked with respectively;
B, add goat-anti FITC bag by the immunity magnetic micropearls of polyclonal antibody again;
The specific isolation of C, antigen-antibody complex;
After D, the cleaning, add substrate list phosphoric acid phenolphthalein or diamantane and derivative solutions thereof such as AMPPD, APS-5;
E. in the incubation process, add alkaline stop bath after, the alkaline phosphatase enzymatic impels chromogenic substrate list phosphoric acid phenolphthalein pinkiness; Or the alkaline phosphatase enzymatic impels luminous substrate to decompose, and continues constant luminous;
F, measure the color intensity or the relative light intensity of reaction product, draw the result of determinand again according to the concentration of standard antigen or antibody with analyzer.
Described anti-FITC antibody is to be carrier with the nano-magnetic microballon.
Described luminous substrate is diamantane and derivant AMPPD or APS-5.
The separation of described Ag-Ab specific complex is mark FITC on monoclonal antibody or antigen, combines with the nano-magnetic microballon by it, is adding realization separation under the action of a magnetic field.
Magnetic separating immune luminescence reagent of the present invention and manufacture method thereof, its technique effect has following several respects:
1, adopt that antibody combines with corresponding antigen in the sample to be measured in the magnetic micro-beads, under the effect of magnetic field force, component to be measured is fully separated with other material, need not centrifuging, and be the fast and uniform liquid phase reactor of speed, highly sensitive, analytical test is accurate; Because nanometer magnetic bead is to use its coated antibody, because of its surface area is big, rapid capture antigen, required specimen amount is few, and incubation time reduces greatly, and minute reduces, and simultaneously because of its selective adsorption, can reduce pollution, reduces the cross pollution probability;
2, adopt alkaline phosphatase as luminous marker, no radiologic hazard, the enzyme-catalytic chromogenic reaction result is stable, does not contain carcinogen, and no radiocontamination and harm are low to the Laboratory Request cost, occupy little space; Being valid up to 6 months of reagent can be saved the detection cost;
3, magnetic separates in the EIA enzyme immunoassay Color Appearance System, adopts single phosphoric acid phenolphthalein as substrate, and under alkali condition, color stability has overcome the shortcoming of ELISA (enzyme is exempted from);
4, magnetic separates alkali enzyme labeling luminesceence analysis system and adopts diamantane such as AMPPD, APS-5 and derivant thereof as luminous substrate, its continous-stable luminous, the utmost point is the range of linearity widely, thoroughly changed the inevitable luminous instability of traditional luminous marker, in the luminous fission of course of reaction, cause defectives such as unstable result.
Specific embodiment
The used analyser of analysis test method among the present invention, comprise power circuit, automatic syringe pump 1 and 2, measuring chamber, illuminated chamber, photomultiplier counter and output system, also dispose the Windows Control Software of computing machine and Chinese interface simultaneously, can carry out that data typing, result gather, quality control, the result stores and the result looks into functions such as news, can finish the programming of multiple analytical model, quantitative or qualitative reporting the result, automatically generation and storage, update functions are revised typical curve at 2 automatically.
Embodiment 1
A kind of immunological assay reagents of magnetic separation alkali phosphatase enzyme mark, comprise label, magnetic separation agent, FITC-antibody labeling thing, standard antigen or antibody and substrate, its described separation agent is the nano-magnetic microballon of goat-anti isosulfocyanic acid fluorescence sulphur FITC bag quilt, and described nano-magnetic microballon is that the inside is coated with Fe
3O
4Or Fe
2O
3, the outside contains-OH ,-COOH or-NH
2The microballon of reactive group.This example is to contain-NH
2The magnetic bead of group be example.
(FSH, LH, E2, P, T, PRL, HCG, β-HCG) are example to this example with eight of sexual glands
1, nano-magnetic bead surface bag is by goat-anti FITC Polyclonal Antibody Preparation:
The preparation method of nano-magnetic microballon is by the Chinese patent preparation, and the patent No. is CN92105584.6, and that the magnetic bead the inside coats is Fe
2O
3The surface is for containing-NH
2Group, its content is 0.17eq/g; Get the 10ml magnetic bead, concentration is 30mg/ml, uses the PBS0.01M of P7.4 to clean two times, is suspended at last among the PBS of 10m10.01MPH7.4, add 50% glutaraldehyde solution 10-100 μ l, making the glutaraldehyde final concentration is 0.01-0.5%, adds the goat-anti FITC IgG antibody 100 μ g-1000 μ g of purifying, and 37 ℃ were reacted 2 hours, at the magnet supernatant that gets on, PBS with the 0.01MPH7.4 that contains 0.01-1%BSA cleans three times, is suspended at last in this solution, adds the NaN of 0.01-0.5%
3
2, FITC flag F SH, LH, E2, P, T, PRL, HCG, β-HCG monoclonal antibody
Get 10mg determinand monoclonal antibody, to 1ml, add FITC100ug, put room temperature reaction 20 hours, cross G-25 gel column purifying with PH9.5 carbon slow-readjustment volume.
3, preparation of the immunological assay reagents of magnetic separation alkali phosphatase enzyme mark and interpretation of result
Get FSH, LH, E2, P, T, PRL, HCG and β-HCG standard items, each 20 μ l of serum specimen add each 40 μ l of label alkaline phosphatase and FITC monoclonal antibody, behind the mixing under 37 ℃ temperature incubation, water-bath 15 minutes; Treat that the immune nanometer magnetic bead separation agent 40 μ l that the back adds pan coating goat-anti FITC polyclonal antibody take place in immune response, mixing, water-bath 5 minutes, under the effect of externally-applied magnetic field, last separation vessel separated 4 minutes, removed supernatant; Add washing lotion 400 μ l again, mixing separated 4 minutes, removed supernatant; After repeating above-mentioned steps separation once, directly test tube is put into the measuring chamber of analyser, automatic pump pumps into and excites substrate A MPPD or APS-5 substrate solution, and the analyser analysis is also printed the result.
The present invention is as the thing that serves as a mark with alkaline phosphatase, AMPPD, APS-5 etc. are as substrate, under the effect of alkaline phosphatase, remove phosphatase rapidly, generate unsettled intermediate A PMD, produce single line excited state product, produce luminous by it, with the relative light intensity of analyzer measurement reaction product, draw the result of determinand again according to the concentration of standard antigen or antibody.
Select patient's 796 examples to be measured to carry out hormone determination, through clinical diagnosis secondary amenorrhea 232 examples; Primary amenorrhea 8 examples; Oligomenorrhea 130 examples; Amenorrhea galactorrhea syndrome 51 examples; The breast companion menstrual disorder of overflowing 32 examples; Polycystic ovary syndrome 79 examples; Primary infertility 78 examples; Other case 186 examples such as uterine hemorrhage, sexual abnormality, crinosity, sex premature after the menopause adopt mentioned reagent and method of testing to test.
Testing result is divided following several types:
1, prolactin increases: (PRL normal reference value 66-499uIU/m1; Be 3.3-24.5ng/ml)
Excessive newborn 51 examples account for 17.58% (51/291) in the 291 routine amenorrhoeas; PRL increases 30 examples in excessive newborn 51 examples of amenorrhoea, accounts for 58.9% (30/51); In amenorrhoea 291 examples, PRL increases 39 examples and accounts for sum 13.44% (39/291); No amenorrhea patients 38 examples of breast 89 examples of overflowing account for 42.7% (38/89); Amenorrhoea does not have that PRL increases 9 examples in newborn person's 239 examples of overflowing, and accounts for amenorrhea patients 3.76% (9/239); PRL increases among the loyal person, FSH measurement range 0.2-6.09mIU/ml; LH0.2-5.24mIU/ml; E
25.0-33.92pg/ml prompting FSH, LH, E are low value, hyperprolactinemia has the person's of following up a case by regular visits to 9 examples, clothes bromocriptine, periodic review PRL.
2, prolactin is normal
Normal reference value
Luteal phase onset of ovulation follicular phase
FSH 3.2-10mIU/ml 7.5-20mIU/ml 1.3-11mIU/ml
LH 1.2-12.5mIU/m 12-82mIU/ml 0.4-19mIU/ml
E
212-48pg/ml 153-237pg/ml 48-172pg/ml
(1) amenorrhoea:
A, FSH rise, LH rises or normal: 33 examples, age 20-42 year, average 31.48 years old.
FSH 40.44-118.1mIU/ml
E
25.0-52.95pg/ml
LH 12.60-31.71mIU/ml
Prompting FSH, LH rise E
2Reduce primary amenorrhea 2 examples wherein, Turners syndrome 1 example.2 examples except that FSH, LH rise, E
2136.7-164.2pg/ml, the prompting hormonal readiness onset of ovulation.
B, FSH, LH value all reduce: totally 61 examples, wherein uncommon Chinese syndrome 4 examples, spiritual apositia 4 examples, primary amenorrhea 3 examples
FSH 0.2-3.76mIU/ml
E
25.0-61.95pg/ml
LH 0.2-3.76mIU/ml
Prompting FSH, LH are low value.
C, LH rise, FSH rises reduction: 31 examples
LH 10.07-23.14mIU/ml
E
217.71-86.93pg/ml
FSH 0.95-9.5mIU/ml
4 routine E wherein
20.08-304.5pg/m; P12.76-21.07ng/ml, 3 examples prompt for hormonal readiness luteal phase, and 1 example turns out to be gestation.
D, FSH, LH are all in normal range
E
216.9-156.3pg/ml
E
2Fluctuation is level before extremely ovulating follicular phase.
(2) polycystic ovary syndrome (pcos): totally 79 examples, age 18-to 34-year-old, average 25.6 years old.68 examples:
FSH 0.42-7.99mIU/ml; Companion T increases 9 examples
LH 2.3-20.19mIU/ml (normal reference value: 10.7-132.2ng/dl)
Prompting LH rising or FSH, LH are in normal range
8 examples:
FSH 0.34-5.72mIU/ml
E
2 105.1-368.9pg/ml
LH 0.2-16.3mIU/ml
P 6.09-19.73ng/ml
The P normal reference value, be 7.66-25.6ng/ml luteal phase, prompting hormonal readiness luteal phase.
3 examples:
FSH 4.54-9.7mIU/ml
E
2223.7-312.4pg/ml
LH 9.7-40.29mIU/ml
P 2.54-4.03ng/ml
Prompting ovulation prohormone level.
(3) oligomenorrhea: 130 examples
93 examples:
FSH 0.27-9.35mIU/ml
E
25.0-83.75pg/ml
LH 2.22-33.03mIU/ml
Prompting LH rising or FSH, LH are in normal range.
15 examples: E, P all increase
FSH 0.32-7.57mIU/ml
E
292.91-345.2pg/ml
LH 0.2-16.63mIU/ml
P 6.36-23.74ng/ml
1 example turns out to be early pregnancy, and all the other prompt for hormonal readiness luteal phase.
19 examples: E
2Raise or the LH rising
FSH 1.17-7.57mIU/ml
E
288.87-308.5pg/ml
LH 1.6-42.96mIU/ml
P 0.1-4.28ng/ml
The prompting onset of ovulation before---the onset of ovulation hormonal readiness
3 examples:
FSH 37.23-83.09mIU/ml
E
25.0-33.10pg/ml
LH 15.6-43.44mIU/ml
The decline of prompting ovarian function
From above-mentioned data analysis, the result who is drawn with this bright described magnetic enzyme immunoluminescence reagent and analysis test method has the good clinical compatibility, and test speed is fast.
Embodiment 2
With eight in thyroid gland is example:
Nano-magnetic bead surface bag is identical with embodiment 1 by goat-anti FITC Polyclonal Antibody Preparation method, the surface of magnetic bead is to contain-group of COOH, its content is 0.3eqm/g, chromogenic substrate is single phosphoric acid phenolphthalein, in the incubation process, add alkaline stop bath after, the alkaline phosphatase enzymatic impels chromogenic substrate list phosphoric acid phenolphthalein pinkiness, with the color intensity of analyzer measurement reaction product, draw the result of determinand again according to the concentration of standard antigen or antibody.
Select 40 parts of clinical samples, adopt said method to carry out analytical test, with radioimmunoassay method 40 parts of clinical samples are compared the result such as the table 1 of analytical test simultaneously:
| T3 | T4 | FT3 | FT4 | TSH | TGA | TMA | TRAb | |||||||||
| The present invention | Put and exempt from | The present invention | Put and exempt from | The present invention | Put and exempt from | The present invention | Put and exempt from | The present invention | Put and exempt from | The present invention | Put and exempt from | The present invention | Put and exempt from | The present invention | Put and exempt from | |
| ng/ml | ng/ml | pg/ml | pg/ml | uIU/ml | IU/ml | IU/ml | IU/ml | |||||||||
| 1 | 0.78 | 0.801 | 53.1 | 44.5 | 1.532 | 1.873 | 7.531 | 7.821 | 3.270 | 3.450 | 8.12 | 15％ | 4.33 | 10％ | 3.12 | 3.53 |
| 2 | 4.26 | 4.73 | 195.2 | 187.4 | 9.644 | 10.33 | 25.34 | 26.45 | 0.112 | 0.095 | 12.56 | 40％ | 9.38 | 25％ | 65.1 | 69.8 |
| 3 | 2.58 | 2.01 | 125.5 | 130.6 | 4.05 | 5.53 | 18.36 | 17.78 | 0.598 | 0.476 | 10.70 | 25％ | 6.74 | 12％ | 4.98 | 5.34 |
| 4 | 6.341 | 6.09 | 285.1 | ＞240 | 19.65 | 18.32 | 32.20 | 39.53 | 0.078 | 0.130 | 29.31 | 50％ | 18.35 | 25％ | 112.5 | 121.3 |
| 5 | 8.155 | 9.89 | 247.7 | ＞240 | 15.39 | 17.52 | 39.44 | 42.53 | ＜0.03 | 0.101 | 45.3 | 59％ | 25.7 | 35％ | 89.7 | 93.5 |
| 6 | 7.763 | 7.75 | ＞300 | ＞240 | 19.56 | 25.16 | 60.95 | ＞55 | ＜0.03 | 0.07 | 8.9 | 30％ | 3.2 | 12％ | 4.52 | 6.13 |
| 7 | 8.414 | 8.32 | ＞300 | ＞240 | 18.67 | 20.11 | 42.6 | 48.33 | 0.058 | 0.01 | 85.6 | 70％ | 24.3 | 43％ | 182.3 | 189.9 |
| 8 | 1.56 | 1.87 | 85.6 | 88.2 | 2.17 | 2.35 | 11.21 | 13.47 | 3.25 | 4.16 | 11.30 | 29％ | 7.53 | 13％ | 0.98 | 1.12 |
| 9 | 1.740 | 1.81 | 79.65 | 84.38 | 3.003 | 3.202 | 12.49 | 13.18 | 0.782 | 0.72 | 4.56 | 16％ | 3.25 | 10％ | 2.35 | 3.12 |
| 10 | 1.169 | 1.03 | 4O.23 | 37.38 | 3.996 | 4.14 | 6.395 | 6.081 | 23.25 | 29.27 | 3.54 | 15％ | 2.71 | 8％ | 5.01 | 6.13 |
| 11 | ＞10 | 12.44 | ＞300 | ＞240 | ＞25 | ＞27 | 60 | ＞55 | ＜0.03 | 0.01 | 150 | 65％ | 96 | 49％ | 132.8 | 139.7 |
| 12 | 0.233 | 0.345 | 5.6 | 7.8 | 1.073 | 1.259 | 3.521 | 4.029 | 36.22 | 39.48 | 7.82 | 27％ | 4.78 | 11％ | 4.75 | 5.13 |
| 13 | 1.195 | 1.02 | 18.74 | 20.48 | 2.857 | 3.029 | 1.05 | 0.59 | ＞50 | ＞45 | 6.81 | 17％ | 2.95 | 8％ | 5.18 | 5.24 |
| 14 | 4.074 | 4.53 | 244.1 | ＞240 | ＞25 | ＞27 | 51.46 | 51.68 | 0.063 | 0.02 | 72 | 82％ | 28 | 46％ | 75.1 | 79.8 |
| 15 | 1.469 | 1.617 | 121.7 | 136.5 | 1.897 | 2.319 | 10.98 | 12.18 | 1.773 | 1.559 | 5.01 | 22％ | 2.17 | 9％ | 6.32 | 7.01 |
| 16 | 1.935 | 1.887 | 98 | 104.5 | 3.986 | 4.67 | 9.272 | 10.02 | 1.063 | 0.83 | 4.05 | 11％ | 1.23 | 8％ | 6.11 | 6.98 |
| 17 | 5.74 | 6.82 | 296.5 | ＞240 | 13.84 | 14.47 | 28.96 | 30.65 | 0.058 | 0.033 | 25.6 | 21％ | 15.1 | 7％ | 42.5 | 45.6 |
| 18 | 1.545 | 1.540 | 150 | 168 | 11.97 | 10.09 | 34.69 | 33.35 | ＜0.03 | 0.012 | 79.6 | 45％ | 28.7 | 31％ | 6.23 | 7.45 |
| 19 | 0.520 | 0.783 | 49.8 | 50.32 | 1.031 | 1.540 | 6.983 | 7.829 | 5.43 | 6.08 | 6.59 | 23％ | 1.87 | 7％ | 4.25 | 4.98 |
| 20 | 1.635 | 1.822 | 116.4 | 126.5 | 2.607 | 2.54 | 9.891 | 10.08 | 1.726 | 1.630 | 6.19 | 22％ | 1.97 | 8％ | 3.96 | 5.12 |
| 21 | 9.156 | 10.99 | 247.7 | ＞240 | 9.461 | 9.53 | 40.74 | 41.81 | 0.129 | 0.081 | 7.29 | 18％ | 4.65 | 10％ | 5.09 | 6.28 |
| 22 | 2.393 | 2.496 | 129.0 | 132.9 | 7.159 | 7.697 | 55.54 | 50.83 | ＜0.03 | 0.012 | 89 | 24％ | 47 | 12％ | 78.6 | 82.4 |
| 23 | 1.781 | 1.882 | 117.6 | 120.3 | 1.919 | 1.993 | 10.92 | 10.41 | 3.405 | 4.08 | 7.2 | 16％ | 3.8 | 12％ | 4.32 | 5.18 |
| 24 | 0.917 | 1.006 | 99.23 | 92.9 | 2.766 | 3.08 | 13.43 | 14.38 | 1.421 | 1.29 | 4.3 | 10％ | 1.2 | 7％ | 4.95 | 5.23 |
| 25 | 4.943 | 5.061 | 237.8 | 231.4 | 6.12 | 6.20 | 21.7 | 23.5 | 0.58 | 0.24 | 11.0 | 35％ | 7.6 | 18％ | 1.02 | 2.53 |
| 26 | 9.53 | 9.01 | 198.1 | 200.3 | 10.81 | 12.95 | 31.0 | 29.4 | 0.04 | 0.02 | 4.52 | 28％ | 3.71 | 16％ | 0.95 | 0.82 |
| 27 | ＞10 | ＞10 | 245.6 | ＞240 | 18.91 | 20.23 | 50.6 | 48.3 | ＜0.03 | 0.01 | ＞280 | 60％ | ＞280 | 47％ | ＞280 | 312.3 |
| 28 | ＞10 | ＞10 | ＞300 | ＞240 | ＞25 | 26.7 | 31.3 | 37.9 | ＜0.03 | 0.01 | ＞280 | 72％ | ＞280 | 49％ | ＞280 | ＞405 |
| 29 | 7.56 | 8.06 | 187.2 | 192.5 | 17.3 | 19.6 | 28.5 | 30.9 | 0.237 | 0.458 | 14.0 | 35％ | 8.1 | 22％ | 6.52 | 7.68 |
| 30 | 6.42 | 7.12 | 173.3 | 183.1 | 12.4 | 13.8 | 24.5 | 28.7 | 0.112 | 0.075 | 7.2 | 29％ | 1.8 | 10％ | 1.34 | 2.12 |
| 31 | 5.96 | 6.23 | 181.3 | 181.0 | 16.9 | 18.2 | 27.4 | 38.9 | 1.120 | 0.658 | 4.3 | 15％ | 1.9 | 6％ | 2.78 | 3.01 |
| 32 | 1.12 | 1.10 | 100.2 | 110.1 | 3.41 | 2.87 | 18.2 | 17.4 | 2.13 | 1.89 | 7.3 | 16％ | 2.8 | 7％ | 1.98 | 1.76 |
| 33 | 3.46 | 3.78 | 231.5 | 229.5 | 5.95 | 6.03 | 18.9 | 19.4 | 0.651 | 0.495 | 8.5 | 11％ | 1.1 | 12％ | 14.5 | 15.2 |
| 34 | 2.74 | 3.04 | 141.2 | 145.6 | 6.12 | 5.86 | 14.3 | 16.7 | 1.891 | 0.215 | 1.3 | 10％ | 0.8 | 8％ | 5.43 | 5.98 |
| 35 | 0.32 | 0.59 | 23 | 11 | 0.79 | 0.86 | 4.3 | 2.5 | 12.93 | 15.82 | 1.1 | 12％ | 0.3 | 9％ | 1.23 | 1.98 |
| 36 | 3.94 | 4.02 | 176 | 186 | 12.45 | 14.21 | 14.9 | 16.8 | 0.05 | 0.078 | 5.2 | 16％ | 1.2 | 19％ | 0.92 | 1.21 |
| 37 | 0.931 | 1.19 | 89 | 91 | 2.37 | 3.28 | 12.4 | 14.5 | 4.12 | 2.94 | 3.3 | 19％ | 0.4 | 18％ | 1.68 | 1.88 |
| 38 | 8.74 | 7.96 | 283 | ＞240 | 21.5 | 23.1 | 31.6 | 35.9 | ＜0.03 | 0.01 | 11.5 | 25％ | 6.3 | 30％ | 35.1 | 40.2 |
| 39 | ＞10 | ＞10 | 291.4 | ＞240 | 20.1 | 18.7 | 41.8 | 38.7 | ＜0.03 | 0.01 | 195 | 45％ | 87 | 60％ | 234.5 | 246.7 |
| 40 | 1.98 | 2.43 | 78.6 | 76.9 | 1.92 | 2.37 | 10.74 | 8.21 | 0.431 | 2.74 | 10.1 | 32％ | 0.8 | 5％ | 6.08 | 6.85 |
Table 1
Put and exempt from eight normal values of thyroid gland and be respectively
T3：0.72-2.21ng/ml T4：42-135ng/ml
FT3：1.54-5.01pg/ml FT4：7.38-20.1pg/ml
TSH：0.35-4.4uIU/ml TRAb：＜6IU/ml
TGAb：≤30％ TPO(TM)Ab：≤20％
Eight normal values of thyroid gland of the present invention are respectively
T3：0.69-2.15ng/ml T4：52-127ng/ml
FT3：1.21-4.17pg/ml FT4：7.2-17.2pg/ml
TSH：0.60-4.5uIU/ml TRAb：＜6IU/ml
TGAb：≤9IU/ml TMAb：≤5IU/ml
Putting the curve ranges of exempting from is:
T3：0.5-10ng/ml T4：20-240ng/ml
FT3：1.0-27pg/ml FT4：2-55pg/ml
TSH：0.35-45uIU/ml TRAb：0.5-405IU/ml
Curve ranges of the present invention is:
T3：0.5-10ng/ml T4：10-3000ng/ml
FT3：1.21-4.17pg/ml FT4：4-120pg/ml
TSH：0.5-50uIU/ml TGA：2-280IU/ml
TMA：2-280IU/ml TRAb：0.1-280IU/ml
The thyroid gland discussion of results:
(1) through his-and-hers watches 1 result's statistical computation, gets the linearity of regression equation, X: put the method for exempting from; Y: magnetic enzyme immunoluminescence method of the present invention.
T3：Y＝0.927X+0.08，R＝0.986，P＜0.001
T4：Y＝1.162X-15.49，R＝0.976，P＜0.001
FT3：Y＝0.92X+0.107，R＝0.991，P＜0.001
FT4：Y＝0.993X-0.741，R＝0.980，P＜0.001
TSH：Y＝0.974X-0.0069，R＝0.989，P＜0.001
TRAb：Y＝0.985X-1.982，R＝0.999，P＜0.001
The result shows: to T3, T4, FT3, FT4, TSH and TRAb reagent, and two method height correlations, the P value all＜0.001 represents that two kinds of methods have comparability.
Be qualitative judgement reagent owing to put TGA, the TMA reagent of the method for exempting from, can't calculate the equation of linear regression of two methods, but judge from positive value, in 40 samples, 17#, 22#, three samples of 38# are only arranged and put the agent of being excused from an examination and be not inconsistent, wherein near the deviation the positive value of 17# and 38# only has 22# to differ greatly, and the positive coincidence rate of its two method is 37/40=92.5%.
(2) to T3,3#, 19#, three sample two methods of 40# do not conform to (as known from Table 1) wherein 3# and 40# be near the T3 normal value upper limit difference, and the 19# sample is near the difference the T3 lower limits of normal, two method coincidence rates are 92%.
To T4,3# and 19# are not inconsistent at clinical positive diagnosis, and owing to different the causing of positive critical value of two methods, also because this measured value is in not limitting of T4 normal value just, as calculated, two method coincidence rates are 95%.
The coincidence rate that FT3, FT4 calculate is all 92.5%, and the two method coincidence rates of TSH are 92.5%, except near critical, two methods occur outside the deviation aspect the diagnosis of hyperthyroidism disease, hyperthyroidism and first are subtracted, and normal person's mensuration two methods have good consistance, and its precious breaking conforms to fully with clinical.
Claims (9)
1, a kind of immunological assay reagents of magnetic separation alkali phosphatase enzyme mark, comprise label, magnetic separation agent, FITC-antibody labeling thing, standard antigen or antibody and substrate, it is characterized in that: described separation agent is the nano-magnetic microballon of goat-anti isosulfocyanic acid fluorescence sulphur FITC bag quilt, and described nano-magnetic microballon is that the inside is coated with Fe
3O
4Or Fe
2O
3, the outside contains-OH ,-COOH or-NH
2The microballon of reactive group.
2, the immunological assay reagents of magnetic separation alkali phosphatase enzyme mark according to claim 1 is characterized in that: the activity group content that described nano-magnetic bead surface contains is 0.05-0.5eqm/g.
3, the immunological assay reagents of magnetic separation alkali phosphatase enzyme mark according to claim 2 is characterized in that: the nano-magnetic microballon of described goat-anti FITC bag quilt is that the nano-magnetic microballon connects with the chemistry of goat-anti FITC.
4, the immunological assay reagents of magnetic separation alkali phosphatase enzyme mark according to claim 3 is characterized in that: described label is alkaline phosphatase AKP.
5, the immunological assay reagents of magnetic separation alkali phosphatase enzyme mark according to claim 4 is characterized in that: described substrate comprises chromogenic substrate list phosphoric acid phenolphthalein PMP, luminous substrate diamantane and derivant AMPPD or APS-5.
6, the immunoassay method of testing of magnetic separation alkali phosphatase enzyme mark is characterized in that testing according to the following steps:
A, the monoclonal antibody of alkaline phosphatase, FITC and patient's to be measured serum mixing incubation will be marked with respectively;
B, add goat-anti FITC bag by the immunity magnetic micropearls of polyclonal antibody again;
The specific isolation of C, antigen-antibody complex;
After D, the cleaning, add chromogenic substrate list phosphoric acid phenolphthalein or luminous substrate;
In E, the incubation process, add alkaline stop bath after, the alkaline phosphatase enzymatic impels chromogenic substrate list phosphoric acid phenolphthalein pinkiness; Or the alkaline phosphatase enzymatic impels luminous substrate to decompose, and continues constant luminous;
F, measure the color intensity or the relative light intensity of reaction product, draw the result of determinand again according to the concentration of standard antigen or antibody with analyzer.
7, magnetic according to claim 6 separates the EIA enzyme immunoassay method of testing, it is characterized in that: the specific isolation of described antigen-antibody mediated immunity compound is to be carrier with the nano-magnetic microballon.
8, magnetic according to claim 7 separates the EIA enzyme immunoassay method of testing, and it is characterized in that: described luminous substrate is diamantane and derivant AMPPD or APS-5.
9, magnetic according to claim 8 separates the EIA enzyme immunoassay method of testing, it is characterized in that: the separation of described Ag-Ab specific complex is mark FITC on monoclonal antibody or antigen, combine with the nano-magnetic microballon by it, adding realization separation under the action of a magnetic field.
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