CN101329340A - Colloidal gold reagent for testing tuberculosis mycobacteria antibody and preparation method thereof - Google Patents
Colloidal gold reagent for testing tuberculosis mycobacteria antibody and preparation method thereof Download PDFInfo
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- CN101329340A CN101329340A CNA2008100410405A CN200810041040A CN101329340A CN 101329340 A CN101329340 A CN 101329340A CN A2008100410405 A CNA2008100410405 A CN A2008100410405A CN 200810041040 A CN200810041040 A CN 200810041040A CN 101329340 A CN101329340 A CN 101329340A
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Abstract
The invention relates to a colloidal gold reagent and a preparation method used for detecting mycobacterium tuberculosis (TB) antibody. The reagent of the invention comprises TB antigen coated colloidal gold, saccharose and 2, 2'-[(1- methylethylidene) bis (4, 1-phenyleneoxymethylene)] double-epoxy alkane polyethyleneglycol derivatives; the preparation method comprises the following steps: TB antigen coated colloidal gold is deposited after the separation of combination/dissociation by a centrifuging way; 10-20mM phosphate buffer solution with 1/5 of original volume is suspended under the pH of 7.4; wherein, the buffer solution contains 5-7% (mass percentage) of saccharose and 2mg/ml of 2, 2'-[(1- methylethylidene) bis (4, 1-phenyleneoxymethylene)] double-epoxy alkane polyethyleneglycol derivative, and is pre-frozen under the temperature of minus 40 DEG C. Therefore, the colloidal gold reagent of the invention is a colloidal gold reagent which has good stability and can detect the mycobacterium tuberculosis (TB).
Description
Technical field
The invention belongs to clinical medicine immunodiagnosis colloid gold reagent and preparation method thereof, be specifically related to a kind of colloid gold reagent that detects tuberculosis mycobacteria antibody and preparation method thereof.
Background technology
Tuberculosis is first killer in the global infectious diseases, the incidence of disease lungy in recent years has the trend of rising, in time find, accurately diagnosis, to cut off the infection sources be effectively preventing means, so laboratory diagnosis lungy becomes clinician's diagnosis of tuberculosis, judge curative effect and assessment more indispensable foundation in back and means.
1971, Faulk and Taylor (Immunochem, 8:1081,1971) at first used the immune colloid gold thing that serves as a mark to be used for immunoelectronmicroscopy, and after this immune colloidal gold technique has obtained using widely as a kind of new immunological method.The colloid gold particle that can prepare various different-grain diameters with reducing process easily from gold chloride, the method that Frens introduces [Nature (Phys.Sci.) 241:20 the most commonly used, 1973], with the trisodium citrate is that 10～70nm colloid gold particle that reductive agent obtains is a magnitude range the most frequently used in the reagent for clinical diagnosis, and particle size is determined by the addition of trisodium citrate.Colloid gold particle has very strong adsorptive power to protein, can with non-covalent combinations such as staphylococcal protein A, immunoglobulin (Ig), toxin, glycoprotein, enzyme, hormone, virus or bacterial antigens, form the immune colloid gold conjugate, be used for immune detection.
Immune colloidal gold technique is medical colleges such as ministry of Health of China height planning teaching material " immunology and immunological test " (Tao Yixun chief editor, second edition, Beijing, the People's Health Publisher, 1997) classified chapters and sections in as, these chapters and sections have specifically been introduced immune colloid gold two methods of normal use in medical test:
(1) immune colloid gold spot percolation test
(2) immune colloidal gold chromatography test.
These chapters and sections have been emphasized the importance of stabilizing agent to immune colloid gold, have introduced the normal various stabilizing agents of using both at home and abroad: bovine serum albumin(BSA) (BSA), glucosan (Dextran), Macrogol 2000 0 (PEG), gelatin (gelatin).Generally use Macrogol 2000 0 (PEG) as stabilizing agent at present.
At present, U.S. Sigama company supply 2,2 '-[(1-methyl ethylidene) two (4,1-phenylene formaldehyde)] bisoxirane polyglycol derivatization compound [Poly (ethylene glycol), compound with 2,2 '-[(1-methylethylidene) bis (4,1-phenyleneoxumethylene)] bisoxirane production code member P2263, mp59 ℃, the white plates crystallization, water-soluble, molecular weight 15000～20000].
Antigen and antibody widespread use abroad over nearly 10 years in the legal property of the immune colloid gold detection body fluid, because of its method quick, easy and simple to handle and very popular.Both at home and abroad quality preferably colloidal gold method tuberculosis antibody (TB-Ab) qualitative detection positive rate more than 90% can be arranged in the positive tubercular of phlegm smear, positive rate about 60% is arranged in the tuberculosis patient of sputum smear negative, false positive is about 5% in the normal population, and the U.S. also only reaches similar accuracy rate with PCR method (polymerase chain molecular cloning technology) in tuberculosis infection patient's sample directly detects at present.Above-mentioned generally acknowledged result shows that fast and convenient gold mark method qualitative detection tuberculosis antibody is the important auxiliary diagnosis or the screening indexes of m tuberculosis infection, and the level of prompting tuberculosis patient tuberculosis antibody is relevant with the discharge of bacteria amount.The nearest result of study of Japan scientist has confirmed that also the tuberculosis antibody level is relevant with the discharge of bacteria amount.Being determined at dynamic observing of clinical treatment a significant index also can be provided of tuberculosis antibody.
The stability of colloid gold reagent is crucial, and the stability of label is the most important guarantee in measuring.The screening of stabilizing agent, immune colloid gold put together technology and lyophilized form can prolong its term of validity.But the colloidal property of immune colloid gold makes it in freeze-drying demulsifying phenomenon can take place, and serious breakdown of emulsion can cause macroscopic particle aggregation.The micro-particle aggregation that slight breakdown of emulsion causes can't pass microporous fiber membranes because of diameter increases in testing process, the red flocculated particle that is trapped on the film is false-positive result with regard to wrong report.
Summary of the invention
The technical problem to be solved in the present invention is that conjugation techniques characteristic when antigen coated collaurum and freeze drying technology overcome the demulsifying phenomenon in liquid phase immune colloid gold poor stability and the colloid gold reagent preparation, a kind of good stability that has is provided, can detects the colloid gold reagent of tuberculosis mycobacteria antibody.
The colloid gold reagent of a kind of detection Mycobacterium tuberculosis of the present invention (TB) antibody comprises:
(1) the antigen coated collaurum of TB, antigen coated concentration is 0.03～0.07mg/ml;
(2) mass percent is 5～7% sucrose;
(3) 2mg/ml 2,2 '-[(1-methyl ethylidene) two (4,1-phenylene formaldehyde)] bis-epoxy alkoxy polyalkylene glycol derivative compound (Poly (ethylene glycol), compoundwith2,2 '-[(1-methylethylldene) bis (4,1-phenyleneoxumethylene)] bisoxirane).
Described TB antigen is Much's bacillus type strain (H37Rv) memebrane protein antigen, should be about 38Kda through SDS-PAGE analyzing molecules amount, and purity is greater than more than 85%, measures its antigen valence＞1: 10 with the ELISA method, more than 000.
The preparation method of the colloid gold reagent of detection tuberculosis mycobacteria antibody of the present invention comprises the steps:
(1) preparation TB antigen
After 36～37 ℃ of cultivations of bacillus H37Rv, be 5% formalin-inactivated through percent by volume, ultrasonic degradation after the surfactant extracting, gets memebrane protein antigen through Sephadex G-200 molecular sieve purification;
(2) the antigen coated collaurum of preparation TB
Prepare blank collaurum routinely, with the antigen coated collaurum of TB, antigen coated concentration is 0.03～0.07mg/ml, and centrifugal do combination/free the separation gets the antigen coated collaurum of TB (being the colloidal gold antigen conjugate);
(3) preparation colloidal gold solution
The antigen coated collaurum precipitation of TB after centrifugal do combination/free separation, 10～20mM phosphate buffer pH 7.4 with original volume 1/5 suspends, wherein, contain mass percent in this damping fluid and be 5～7% sucrose and 2mg/ml 2,2 '-[(1-methyl ethylidene) two (4,1-phenylene formaldehyde)] bis-epoxy alkoxy polyalkylene glycol derivative compound;
(4) preparation colloid gold reagent
Colloidal gold solution-40 ℃ of pre-freezes, is got final product.
Surfactant in the described step (1) is SDS or TritonX-100.
Blank collaurum in the described step (2), in pH6.4～6.7 o'clock, particle diameter is 18～25nm, A
520Between nm=0.85～1.30.
The clinical trial result of the tuberculosis mycobacteria antibody gold quick kit of mark (TB-DOT) of use colloid gold reagent is as follows:
Respectively from northern Cai commune hospital, normal person's health check-up serum specimen totally 120 examples are collected by Xuhui District central hospital, collect phlegm smear positive patients serum specimen totally 24 examples from Shanghai No.1 Pulmonary Department Hospital, press the operation of TB TPPA step, under the TB-DOT curve, record and the results are shown in Table 1;
With the upper limit of normal person's health check-up serum specimen test result mean value+2SD as the TB negative antibody, then phlegm is coated with positive tuberculosis patient positive rate 91.7% (22/24), as table 2.
Table 1 normal person health check-up serum specimen test result (n=120)
Beneficial effect:
Utilize spot immune gold percolation test (DIGFA) principle, be used for the detection of human serum mycobacterium tuberculosis antibody, be suitable for auxiliary diagnosis lungy.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The preparation of embodiment 1 TB antigen
Bacterial strain: derive from the Center for Disease Control (CDC), through Nat'l Pharmaceutical ﹠ Biological Products Control Institute's calibrating, bacterial strain is Much's bacillus type strain H37Rv, and standard No. is ATCC27294.
Build the storehouse: with the bacterial strain available from the U.S. is former generation, and freeze-drying is preserved.Propagate time and to cultivate 10 strains, after the freeze-drying for for generations.By propagating 30 for generations, as the strain of storage facility located at processing plant work lot number, freeze-drying is preserved again.Each work lot number strain allows time cultivation continuously to go down to posterity 3 times.
Produce: get one of work lot number strain, bring back to life, be inoculated in the tuberculosis medium slant, collects a certain amount of after, propagate in homemade Agar Plating 37 ℃ of 4 weeks of cultivation more in a large number.After collecting bacterium colony, use 5% formalin-inactivated, centrifugal, taking precipitate, ultrasonic-wave crushing is with surfactant extractings such as SDS, TritonX-100.Supernatant is after Sephadex G-200 molecular sieve purification, and mensuration is tired, and requires ELISA＞1: 10, more than 000; Get supernatant, measure protein concentration, packing 4～5mg/ml ,-20 ℃ of cryopreservation.
The antigen certificate test:
1) the HPLC detection molecules is sifted out peak figure, the sample analysis;
2) Sephadex G-200 purifying tubercle bacillus memebrane protein is got the I peak, sample solution C=12～15mg/ml, applied sample amount 15～20ml;
3) ELISA detects the I peak and tires: ELISA tires should be at 1: 10, more than 000.
4) get above-mentioned purifying I peak, through the SDS-PAGE electrophoretic analysis, molecular weight MW should be about 38kDa, and the Zone electophoresis band is mainly Yi Tiao district band.
5) purity detecting: purity should be greater than more than 85%.
The preparation of embodiment 2 colloid gold reagents
Chlorauride with classics---the trisodium citrate method prepares collaurum, and particle diameter is 20.5nm, and the pH6.6 that surveys, 520nm record absorbance A
520=1.06.
With the antigen coated collaurum of TB, antigen coated concentration 0.03mg/ml, centrifugal do combination/free the separation.The antigen coated collaurum precipitation of TB after the centrifuging, contain 2mg/ml 2 with original volume 1/5,2 '-[(1-methyl ethylidene) two (4,1-phenylene formaldehyde)] bis-epoxy alkoxy polyalkylene glycol derivative compound, mass percent are after 10mM pH 7.4 phosphate buffers of 5% sucrose suspend,-40 ℃ of pre-freezes, get final product.
The preparation of embodiment 3 colloid gold reagents
Chlorauride with classics---the trisodium citrate method prepares collaurum, and particle diameter is 22nm, and the pH6.5 that surveys, 520nm record absorbance A
520=1.25.
With the antigen coated collaurum of TB, antigen coated concentration 0.06mg/ml, centrifugal do combination/free the separation.The antigen coated collaurum precipitation of TB after the centrifuging, contain 2mg/ml 2 with original volume 1/5,2 '-[(1-methyl ethylidene) two (4,1-phenylene formaldehyde)] bis-epoxy alkoxy polyalkylene glycol derivative compound, mass percent are after 20mM pH 7.4 phosphate buffers of 7% sucrose suspend,-40 ℃ of pre-freezes, get final product.
Claims (5)
1. colloid gold reagent that detects tuberculosis mycobacteria antibody comprises:
(1) the antigen coated collaurum of TB, antigen coated concentration is 0.03～0.07mg/ml;
(2) mass percent is 5～7% sucrose;
(3) 2mg/ml 2,2 '-[(1-methyl ethylidene) two (4,1-phenylene formaldehyde)] bis-epoxy alkoxy polyalkylene glycol derivative compound.
2. the colloid gold reagent of detection tuberculosis mycobacteria antibody according to claim 1 is characterized in that: described TB antigen is Much's bacillus type strain H37Rv memebrane protein antigen, and molecular weight is 38Kda.
3. detect the preparation method of the colloid gold reagent of tuberculosis mycobacteria antibody, comprise the steps:
(1) preparation TB antigen
After 36～37 ℃ of cultivations of bacillus H37Rv, be 5% formalin-inactivated through percent by volume, ultrasonic degradation after the surfactant extracting, gets memebrane protein antigen through Sephadex G-200 molecular sieve purification;
(2) the antigen coated collaurum of preparation TB
The collaurum of preparation blank routinely, with the antigen coated collaurum of TB, antigen coated concentration is 0.03～0.07mg/ml, centrifugal do combination/free the separation;
(3) preparation colloidal gold solution
The antigen coated collaurum precipitation of TB after centrifugal do combination/free separation, 10～20mM phosphate buffer pH 7.4 with original volume 1/5 suspends, wherein, contain mass percent in this damping fluid and be 5～7% sucrose and 2mg/ml 2,2 '-[(1-methyl ethylidene) two (4,1-phenylene formaldehyde)] bis-epoxy alkoxy polyalkylene glycol derivative compound;
(4) preparation colloid gold reagent
Colloidal gold solution-40 ℃ of pre-freezes, is got final product.
4. the preparation method of the colloid gold reagent of detection tuberculosis mycobacteria antibody according to claim 3 is characterized in that: the surfactant in the described step (1) is SDS or TritonX-100.
5. the preparation method of the colloid gold reagent of detection tuberculosis mycobacteria antibody according to claim 3 is characterized in that: the blank collaurum in the described step (2), in pH6.4～6.7 o'clock, particle diameter is 18～25nm, A
520Between nm=0.85～1.30.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923091A (en) * | 2010-05-18 | 2010-12-22 | 青岛瑞杰生物科技有限公司 | Method for detecting high-sensitivity mycobacterium tuberculosis |
| CN111443198A (en) * | 2020-03-19 | 2020-07-24 | 济南杏恩生物科技有限公司 | Method for rapidly detecting tubercle bacillus secretory protein based on colloidal gold method |
-
2008
- 2008-07-25 CN CNA2008100410405A patent/CN101329340A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923091A (en) * | 2010-05-18 | 2010-12-22 | 青岛瑞杰生物科技有限公司 | Method for detecting high-sensitivity mycobacterium tuberculosis |
| CN101923091B (en) * | 2010-05-18 | 2014-03-19 | 青岛瑞杰生物科技有限公司 | Kit for detecting high-sensitivity mycobacterium tuberculosis |
| CN111443198A (en) * | 2020-03-19 | 2020-07-24 | 济南杏恩生物科技有限公司 | Method for rapidly detecting tubercle bacillus secretory protein based on colloidal gold method |
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Application publication date: 20081224 |