CN104360067B - Treponema pallidum specific antibody immue quantitative detection reagent box and preparation method thereof - Google Patents
Treponema pallidum specific antibody immue quantitative detection reagent box and preparation method thereof Download PDFInfo
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Abstract
Treponema pallidum specific antibody immue quantitative detection reagent box and preparation method thereof, relates to treponema pallidum.Test kit is provided with external packing box, alkali phosphatase enzyme mark monoclonal antibody bottle, luminous substrate bottle, the Treponema pallidum specific antibody calibration object bottle of 6 kinds of variable concentrations, Washing liquid bottle, recombinant antigen are coated microwell plate；Alkali phosphatase enzyme mark monoclonal antibody bottle, luminous substrate bottle, the Treponema pallidum specific antibody calibration object bottle of 6 kinds of variable concentrations, Washing liquid bottle, recombinant antigen are coated microwell plate and are located in external packing box.Preparation method: successively prepare treponema pallidum specificity recombinant antigen, recombinant antigen is coated microwell plate, natural membranes protein monoclonal antibody, the alkali phosphatase enzyme mark of monoclonal antibody, luminous substrate, cleaning mixture, calibration object, finally assembles Treponema pallidum specific antibody immue quantitative detection reagent box.Can be used for the detection by quantitative of syphilis specific in clinical samples, can be as syphilis therapeutic effect and the objective indicator of prognosis.
Description
Technical field
The present invention relates to treponema pallidum, especially relate to Treponema pallidum specific antibody immue quantitative detection reagent box and preparation side thereof
Method.
Background technology
Syphilis is the sexually transmitted disease caused by treponema pallidum, and sickness rate the most at home rises year by year, preventing of syphilis
One of control main task being classified as China's public health service.
Treponema pallidum can not In vitro culture, and directly nosetiology dark-field microscopy positive rate is the highest, Serologic test bag
Including cardiolipin antibody and detection of specific antibody, the false positive rate of cardiolipin antibody is high, and sensitivity is low.And syphilis specific antibody
Detection method includes fluorescent treponemal antibody absorbed test (FTA-ABS), immunoblotting (Western-blot) etc.,
Its specificity is the highest, but can not carry out detection by quantitative, it is impossible to judge the Fluctuation of antibody in the patient, it is impossible to provide disease
Therapeutic effect and the objective indicator of prognosis.
Chinese patent CN101881772A discloses the examination of a kind of treponema pallidum based on flow microsphere carrier technique (TP) antibody test
Agent box and preparation thereof and detection method, specifically include and be coated high dimeric molecule microsphere with TP recombinant antigen, seal with bovine serum albumin
Close blank binding site, make specificity T P probe-high dimeric molecule microsphere；Capture TP antibody, washing is co-cultured with specimen to be measured
It is centrifuged off unconjugated TP antibody, adds fluorescently-labeled anti-human igg or IgM antibody；Use flow cytomery
The fluorescence intensity of microsphere, carries out qualitative or quantitative analysis to tested antibodies.
Summary of the invention
It is an object of the invention to provide can realize detect treponema pallidum specific quantitatively, for syphilis specific antibody test provide
Treponema pallidum specific antibody immue quantitative detection reagent box of detection by quantitative and preparation method thereof.Specifically use double antibody unexpectedly to strive to exempt from
It is quantitative that epidemic disease analytical model carries out Treponema pallidum specific antibody.
Described Treponema pallidum specific antibody immue quantitative detection reagent box is provided with external packing box, alkali phosphatase enzyme mark monoclonal antibody
Bottle, luminous substrate bottle, Treponema pallidum specific antibody 1000mIU/mL calibration object bottle, 500mIU/mL calibration object bottle, 200
MIU/mL calibration object bottle, 50mIU/mL calibration object bottle, 10mIU/mL calibration object bottle, 0mIU/mL calibration object bottle, washing
Liquid bottle, recombinant antigen are coated microwell plate；Alkali phosphatase enzyme mark monoclonal antibody bottle, luminous substrate bottle, treponema pallidum are special
Property antibody 1000mIU/mL calibration object bottle, 500mIU/mL calibration object bottle, 200mIU/mL calibration object bottle, 50mIU/mL
Calibration object bottle, 10mIU/mL calibration object bottle, 0mIU/mL calibration object bottle, Washing liquid bottle, recombinant antigen are coated microwell plate and are located at
In external packing box；Alkali phosphatase enzyme mark monoclonal antibody bottle is built with alkali phosphatase enzyme mark monoclonal antibody, luminous substrate bottle
Built with luminous substrate, Treponema pallidum specific antibody 1000mIU/mL calibration object bottle resists built with treponema pallidum specificity
Body 1000mIU/mL calibration object, 500mIU/mL calibration object bottle is built with 500mIU/mL calibration object, 200mIU/mL school
Quasi-product bottle is built with 200mIU/mL calibration object, and 50mIU/mL calibration object bottle is built with 50mIU/mL calibration object, 10mIU/mL
Calibration object bottle is built with 10mIU/mL calibration object, and 0mIU/mL calibration object bottle is built with 0mIU/mL calibration object, Washing liquid bottle
Built with Washing liquid bottle.
The preparation method of described Treponema pallidum specific antibody immue quantitative detection reagent box, comprises the following steps:
1) treponema pallidum specificity recombinant antigen is prepared:
Use gene clone technology, the DNA of PCR amplification coding TP antigen, and insert and escherichia coli make it express,
Obtain treponema pallidum specific antigen TPN17 and TPN47.
2) prepare recombinant antigen and be coated microwell plate
By step 1) in treponemal recombinant antigen TPN17 and TPN47 be diluted to 20 μ g/mL with being coated buffer, with
Every hole 0.1mL adds in microwell plate, and 4 DEG C are coated 24h；Take out antigen plate, air-dry；With 0.01mmol/L pH7.4 phosphate
1.0% defatted milk powder of buffer, every hole 0.2mL, close 24h for 4 DEG C, taking-up phosphate buffer washs 5 times, room
Warm air is dry, sterilization, prepares recombinant antigen and is coated microwell plate, seals standby.
3) natural membranes protein monoclonal antibody is prepared
Extract the memebrane protein of treponema pallidum type strain Nichols strain, immunity Babl/c mice, utilize hybridoma cell technology,
The monoclonal antibody of Screening Syphilis spirillum specific proteins TPN17 and TPN47.
In step 3) in, described treponema pallidum type strain Nichols strain is Treponema pallidum ssp.pallidum strain
Nichol。
4) alkali phosphatase enzyme mark of monoclonal antibody
Alkali phosphatase activates through sodium periodate, respectively with step 3)-NH2 the coupling of monoclonal antibody molecule, form alkalescence phosphorus
Acid enzyme labelling monoclonal antibody.
5) prepared by luminous substrate
Synthetic diamantane (obsolete) amine luminous substrate (AMPPD) and reinforcing agent thereof, all through aseptic filtration, prepare luminous substrate work
Make liquid.
6) prepared by cleaning mixture
Cleaning mixture is the phosphate buffer dissolved with Tween-20, wherein final concentration of the 0.01% of Tween-20.
7) calibration object
By high level Treponema pallidum specific antibody calibration object diluted, with national standard (purchased from Products in China
Calibrating institute) repeat to demarcate three times.Three calibration values, take average as the Treponema pallidum specific antibody concentration demarcated.Described
Calibration object diluent is by the disodium hydrogen phosphate-sodium dihydrogen phosphate of 0.05mmol/L, 3% Ox blood serum of the sodium chloride of 0.01mmol/L
Albumin forms, pH value 7.4.
Treponema pallidum specific antibody standard concentration series 1000mIU/mL, 500mIU/mL, 200mIU/mL, 50
MIU/mL, 10mIU/mL, 0mIU/mL, be distributed into 0.5mL/ bottle, and 2～8 DEG C (12 months) or-20 DEG C (4 years) deposit standby
With.
8) Treponema pallidum specific antibody immue quantitative detection reagent box is prepared
Alkali phosphatase enzyme mark monoclonal antibody, luminous substrate, cleaning mixture, calibration object are loaded in bottle, then by alkali phosphatase
Labeled monoclonal antibody bottle, luminous substrate bottle, Washing liquid bottle, calibration object bottle and recombinant antigen are coated microwell plate and load external packing box
In, form Treponema pallidum specific antibody immue quantitative detection reagent box, described calibration object include 1000mIU/mL, 500mIU/mL,
200mIU/mL, 50mIU/mL, 10mIU/mL, 0mIU/mL totally 6 concentration.
In step 8) in, described bottle can use polyethylene bottle.
The invention provides a kind of Treponema pallidum specific antibody immue quantitative detection reagent box, can be used for syphilis in clinical samples special
Property detection by quantitative, can be simple to operate as syphilis therapeutic effect and the objective indicator of prognosis, result is accurate, environment friendly and pollution-free,
There is long-range benefit.
The present invention uses treponema pallidum recombinant antigen TPN15, TPN17 as controller used in syphilis diagnosis antigen, it is achieved treponema pallidum is special
Opposite sex detection by quantitative, can be as syphilis therapeutic effect and the objective indicator of prognosis.The product of the present invention not only can be to syphilitic's body
Interior helicoid antibody is carried out quantitatively, and simple to operate, and result is accurate, environment friendly and pollution-free, has long-range benefit.
Accompanying drawing explanation
Fig. 1 is the structure composition schematic diagram of Treponema pallidum specific antibody immue quantitative detection reagent box of the present invention.
Fig. 2 is standard substance testing result schematic diagrams.
Detailed description of the invention
Following example will the present invention is further illustrated in conjunction with accompanying drawing.
Embodiment 1
Seeing Fig. 1, described Treponema pallidum specific antibody immue quantitative detection reagent box embodiment is provided with external packing box 1, alkaline phosphatase
Enzyme labelling monoclonal antibody bottle 2, luminous substrate bottle 3, Treponema pallidum specific antibody 1000mIU/mL calibration object bottle 4,500
MIU/mL calibration object bottle 5,200mIU/mL calibration object bottle 6,50mIU/mL calibration object bottle 7,10mIU/mL calibration object bottle
8,0mIU/mL calibration object bottle 9, Washing liquid bottle 10, recombinant antigen are coated microwell plate 11；Alkali phosphatase enzyme mark monoclonal anti
Body bottle 2, luminous substrate bottle 3, Treponema pallidum specific antibody 1000mIU/mL calibration object bottle 4,500mIU/mL calibrate
Product bottle 5,200mIU/mL calibration object bottle 6,50mIU/mL calibration object bottle 7,10mIU/mL calibration object bottle 8,0mIU/mL
Calibration object bottle 9, Washing liquid bottle 10, recombinant antigen are coated microwell plate 11 and are located in external packing box 1；Alkali phosphatase enzyme mark list
Clonal antibody bottle 2 is built with alkali phosphatase enzyme mark monoclonal antibody, and luminous substrate bottle 3 is built with luminous substrate, treponemal
Body specific antibody 1000mIU/mL calibration object bottle 4 built with Treponema pallidum specific antibody 1000mIU/mL calibration object,
500mIU/mL calibration object bottle 5 is built with 500mIU/mL calibration object, and 200mIU/mL calibration object bottle 6 is built with 200
MIU/mL calibration object, 50mIU/mL calibration object bottle 7 is built with 50mIU/mL calibration object, 10mIU/mL calibration object bottle 8
Built with 10mIU/mL calibration object, 0mIU/mL calibration object bottle 9 is built with 0mIU/mL calibration object, in Washing liquid bottle 10
Equipped with Washing liquid bottle.
The preparation method of described Treponema pallidum specific antibody immue quantitative detection reagent box given below, comprises the following steps:
(1) treponema pallidum specificity recombinant antigen is prepared:
Use gene clone technology, the DNA of PCR amplification coding TP antigen, and insert and escherichia coli make it express,
Obtain treponema pallidum specific antigen TPN17 and TPN47.
(2) prepare recombinant antigen and be coated microwell plate
Treponemal recombinant antigen TPN17 and TPN47 in step (1) is diluted to 20 μ g/mL with being coated buffer,
Adding in microwell plate with every hole 0.1mL, 4 DEG C are coated 24h；Take out antigen plate, air-dry；With 0.01mmol/L pH7.4 phosphoric acid
1.0% defatted milk powder of salt buffer preparation, every hole 0.2mL, close 24h for 4 DEG C, taking-up phosphate buffer washs 5 times,
Room temperature air-dries, sterilization, prepares recombinant antigen and is coated microwell plate, seals standby.
(3) natural membranes protein monoclonal antibody is prepared
Extract the memebrane protein of treponema pallidum type strain Nichols strain, immunity Babl/c mice, utilize hybridoma cell technology,
The monoclonal antibody of Screening Syphilis spirillum specific proteins TPN17 and TPN47.
The type strain Nichols strain of step (3) described treponema pallidum is Treponema pallidum ssp.pallidum strain
Nichol。
(4) alkali phosphatase enzyme mark of monoclonal antibody
Alkali phosphatase activates through sodium periodate, respectively with the-NH2 coupling of step (3) monoclonal antibody molecule, forms alkalescence
Phosphatase enzyme mark monoclonal antibody 3.
(5) prepared by luminous substrate
Synthetic diamantane (obsolete) amine luminous substrate (AMPPD) and reinforcing agent thereof, all through aseptic filtration, prepare luminous substrate work
Make liquid.
(6) prepared by cleaning mixture
Cleaning mixture is the phosphate buffer dissolved with Tween-20, wherein final concentration of the 0.01% of Tween-20.
(7) calibration object
By high level Treponema pallidum specific antibody calibration object diluted, with national standard (purchased from Products in China
Calibrating institute) repeat to demarcate three times.Three calibration values, take average as the Treponema pallidum specific antibody concentration demarcated.Described
Calibration object diluent is by the disodium hydrogen phosphate-sodium dihydrogen phosphate of 0.05mmol/L, 3% Ox blood serum of the sodium chloride of 0.01mmol/L
Albumin forms, pH value 7.4.
Treponema pallidum specific antibody standard concentration series 1000mIU/mL, 500mIU/mL, 200mIU/mL, 50
MIU/mL, 10mIU/mL, 0mIU/mL, be distributed into 0.5mL/ bottle, and 2～8 DEG C (12 months) or-20 DEG C (4 years) deposit standby
With.
(8) syphilis helicoid antibody high throughput testing test kit is prepared
Recombinant antigen is coated microwell plate, alkali phosphatase enzyme mark monoclonal antibody, luminous substrate, cleaning mixture, calibration object and outsourcing
The Treponema pallidum specific antibody immue quantitative detection reagent box that mounted box collectively constitutes.
Described calibration object include 1000mIU/mL, 500mIU/mL, 200mIU/mL, 50mIU/mL, 10mIU/mL,
0mIU/mL totally 6 concentration.
Step (8) described alkali phosphatase enzyme mark monoclonal antibody, luminous substrate, cleaning mixture, calibration object are all contained in corresponding poly-
In ethylene bottle.
Embodiment 2
Treponemal in the clinical samples of employing Treponema pallidum specific antibody immue quantitative detection reagent box given below detection patient
Body specific antibody:
1, sample disposal: serum: venous blood 5mL, puts 37 DEG C of water-bath 30min, and 3000g is centrifuged 10min, and supernatant is inspection
Test sample product are standby.
2, sample-adding: respectively add the specimen of 100 μ L and standard substance 1～6 in Sptting plate, make blank simultaneously；
3, enzyme-added labeled monoclonal antibody: alkali phosphatase enzyme mark monoclonal antibody 100 μ L, hatches 30min for 37 DEG C；
4, washing: after 37 DEG C of reaction 30min, determined syphilis specific antibody and alkali phosphatase enzyme mark monoclonal antibody and
On microwell plate, coated syphilis specific recombinant antigen strives combination unexpectedly, and washing separates unconjugated free composition；
5, add luminous substrate working solution, alkaline phosphatase substrate for enzymatic activity dephosphorylation base, and send the visible ray of 463nm.In
10min measures the relative luminous intensity unit (relative light units, RLU) respectively adding sample well.The RLU of sample and prunus mume (sieb.) sieb.et zucc. to be measured
Poison specific antibody concentrations negative correlation.Testing concentration in sample is set up according to by determinand standard concentration and corresponding RLU
Math equation carry out quantitatively, Fig. 2 show standard substance detection curve, and wherein abscissa is standard concentration value, and vertical coordinate is
RLU value, can calculate the syphilis specific antibody concentration of sample to be tested by sample also corresponding RLU value.
Claims (2)
1. the preparation method of Treponema pallidum specific antibody immue quantitative detection reagent box, it is characterised in that described treponema pallidum is special
Property antibody immue quantitative detection reagent box is provided with external packing box, alkali phosphatase enzyme mark monoclonal antibody bottle, luminous substrate bottle, syphilis spiral shell
Rotation body specific antibody 1000mIU/mL calibration object bottle, 500mIU/mL calibration object bottle, 200mIU/mL calibration object bottle, 50
MIU/mL calibration object bottle, 10mIU/mL calibration object bottle, 0mIU/mL calibration object bottle, Washing liquid bottle, recombinant antigen are coated micro-
Orifice plate；Alkali phosphatase enzyme mark monoclonal antibody bottle, luminous substrate bottle, Treponema pallidum specific antibody 1000mIU/mL school
Quasi-product bottle, 500mIU/mL calibration object bottle, 200mIU/mL calibration object bottle, 50mIU/mL calibration object bottle, 10mIU/mL
Calibration object bottle, 0mIU/mL calibration object bottle, Washing liquid bottle, recombinant antigen are coated microwell plate and are located in external packing box；Alkalescence phosphorus
Acid enzyme labelling monoclonal antibody bottle is built with alkali phosphatase enzyme mark monoclonal antibody, and luminous substrate bottle is built with luminous substrate, prunus mume (sieb.) sieb.et zucc.
Poison spirillum specific antibody 1000mIU/mL calibration object bottle is calibrated built with Treponema pallidum specific antibody 1000mIU/mL
Product, 500mIU/mL calibration object bottle is built with 500mIU/mL calibration object, and 200mIU/mL calibration object bottle is built with 200
MIU/mL calibration object, 50mIU/mL calibration object bottle is built with 50mIU/mL calibration object, and 10mIU/mL calibration object bottle is built-in
Having 10mIU/mL calibration object, 0mIU/mL calibration object bottle is built with 0mIU/mL calibration object, and Washing liquid bottle is built with cleaning mixture；
Described preparation method, comprises the following steps:
1) treponema pallidum specificity recombinant antigen is prepared:
Use gene clone technology, the DNA of PCR amplification coding TP antigen, and insert and escherichia coli make it express,
Obtain treponema pallidum specific antigen TPN17 and TPN47；
2) prepare recombinant antigen and be coated microwell plate
By step 1) in treponema pallidum recombinant antigen TPN17 and TPN47 be diluted to 20 μ g/mL with being coated buffer,
Adding in microwell plate with every hole 0.1mL, 4 DEG C are coated 24h；Take out antigen plate, air-dry；With 0.01mmol/L pH7.4 phosphoric acid
1.0% defatted milk powder of salt buffer preparation, every hole 0.2mL, close 24h for 4 DEG C, taking-up phosphate buffer washs 5 times,
Room temperature air-dries, sterilization, prepares recombinant antigen and is coated microwell plate, seals standby；
3) natural membranes protein monoclonal antibody is prepared
Extract the memebrane protein of treponema pallidum type strain Nichols strain, immunity Babl/c mice, utilize hybridoma cell technology,
The monoclonal antibody of Screening Syphilis spirillum specific proteins TPN17 and TPN47；Described treponema pallidum type strain Nichols
Strain is Treponema pallidum ssp.pallidum strain Nichol；
4) alkali phosphatase enzyme mark of monoclonal antibody
Alkali phosphatase activates through sodium periodate, respectively with step 3)-NH2 the coupling of monoclonal antibody molecule, form alkalescence phosphorus
Acid enzyme labelling monoclonal antibody；
5) prepared by luminous substrate
Synthetic diamantane (obsolete) amine luminous substrate and reinforcing agent thereof, all through aseptic filtration, prepare luminous substrate working solution；
6) prepared by cleaning mixture
Cleaning mixture is the phosphate buffer dissolved with Tween-20, wherein final concentration of the 0.01% of Tween-20；
7) calibration object
By high level Treponema pallidum specific antibody calibration object diluted, repeat to demarcate three times with national standard, three times
Calibration value, takes average as the Treponema pallidum specific antibody concentration demarcated；Described calibration object diluent is by 0.05mmol/L's
Disodium hydrogen phosphate-sodium dihydrogen phosphate, the sodium chloride of 0.01mmol/L and 3% bovine serum albumin composition, pH value 7.4；
Treponema pallidum specific antibody standard concentration series 1000mIU/mL, 500mIU/mL, 200mIU/mL, 50
MIU/mL, 10mIU/mL, 0mIU/mL, be distributed into 0.5mL/ bottle, deposit standby for 2～8 DEG C or-20 DEG C；
8) Treponema pallidum specific antibody immue quantitative detection reagent box is prepared
Alkali phosphatase enzyme mark monoclonal antibody, luminous substrate, cleaning mixture, calibration object are loaded in bottle, then by alkali phosphatase
Labeled monoclonal antibody bottle, luminous substrate bottle, Washing liquid bottle, calibration object bottle and recombinant antigen are coated microwell plate and load external packing box
In, form Treponema pallidum specific antibody immue quantitative detection reagent box, described calibration object include 1000mIU/mL, 500mIU/mL,
200mIU/mL, 50mIU/mL, 10mIU/mL, 0mIU/mL totally 6 concentration.
2. the preparation method of Treponema pallidum specific antibody immue quantitative detection reagent box as claimed in claim 1, it is characterised in that
Step 8) in, described bottle uses polyethylene bottle.
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| JPS5151515A (en) * | 1974-11-01 | 1976-05-07 | Nippon Toketsu Kanso Kenkyusho | JUKAKUSETSUKETSUKYUO TANTAITOSURU BAIDOKUSETSUKETSUKYUGYOSHUHANNOJINSOKUHOYOKOGENNO SEIZOHO |
| DE3911361A1 (en) * | 1989-04-07 | 1990-10-11 | Behringwerke Ag | METHOD FOR DETERMINING ANTIBODIES AGAINST EXHIBITORS OF INFECTIOUS DISEASES IN BODY LIQUIDS, MEANS THEREFOR AND THEIR USE IN THIS METHOD |
| CN101363860A (en) * | 2007-08-06 | 2009-02-11 | 北京科美东雅生物技术有限公司 | Syphilis helicoid antibody chemiluminescence immune assay determination kit and method for preparing same |
| CN101738473B (en) * | 2008-11-13 | 2013-06-05 | 威海威高生物科技有限公司 | Treponema pallidum antibody diagnostic kit and preparation method thereof |
| CA2794710C (en) * | 2010-03-31 | 2018-05-08 | Sekisui Medical Co., Ltd. | Reagent for assaying anti-treponema pallidum antibody |
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