CN101639476B - Visualized tuberculosis antibody detection protein chip, preparation method and application - Google Patents
Visualized tuberculosis antibody detection protein chip, preparation method and application Download PDFInfo
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Abstract
The invention discloses a visualized multi-target tuberculosis antibody detection protein chip prepared by using seven mycobacterium tuberculosis specific dominant epitope antigens of cloning expression, and also discloses a preparation method for the protein chip and application thereof. By dotting the seven mycobacterium tuberculosis specific dominant epitope antigens on a modified substrate, a multi-target protein microarray which can detect seven tuberculosis antibodies is prepared, and an immune gold-silver staining detection system is established to replace the prior fluorescent marker of the chip. Through the application of the detection system, a fluorescent scanner of the chip does not need to be used, the reaction result of the chip can be judged through visual inspection and simple image scanning, and the aim of detecting tuberculosis diseases is achieved. The sensitivities of the visualized multi-target tuberculosis antibody detection protein chip are 98.5 percent and 96.6 percent respectively, and the specificity is 93.3 percent and is remarkably higher than that of a sputum smear and a sputum bacteria cultivation method; and the protein chip remarkably improves the detection rates of the sputum smear and sputum bacteria cultivation negative patients, can be used for clinical auxiliary diagnosis of the tuberculosis diseases, and has certain clinical application prospect.
Description
Technical field
The present invention relates to a kind of visual many target spots tuberculosis antibody detection protein chip, be specifically related to the protein chip that a kind of many target spots tuberculosis antibody detects, also relate to this chip production method and medical application.
Background of invention
In recent years, tuberculosis is revivable, and the population in the whole world about 1/3 infects tubercle bacillus, dies from about 3,000,000 people of number lungy every year.The 4th tuberculosis epidemiological random sampling survey report of China shows that China has active tuberculosis patient 4,510,000 now, bacterium sun lunger 1,960,000.And China's tuberculosis infection number is ascendant trend gradually in recent years, and its quantity occupies the infectious disease first place.Therefore, discovery morning lungy, diagnosis morning, early treatment seem particularly important.The traditional bacteria diagnostic techniques is diagnosis of tuberculosis " goldstandard ", is used till today, but owing to shortcomings such as there are detection length consuming time in its, susceptibility is low, specificity is relatively poor, is difficult to satisfy the needs of clinical diagnosis always.The serology immune diagnostic technique remains the important tool of tuberculosis auxiliary diagnosis because Inherent advantages such as it is easy and simple to handle, quick, good stabilities.Yet at present the tuberculosis antibody detectable is the requirement that the antigen that adopted can not satisfy high special susceptibility diagnostic reagent still having some problems, main cause aspect specificity and the susceptibility.In the research formerly; Our clonal expression seven species specificity strong and have a complementary negre antigen (38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and Mtb16.3); In this research we further through bioinformatics software analysis screening advantage epi-position, and successful clonal expression the advantage epitope antigen.Utilize these advantage epitope antigens to be developed into the visual antibody detection protein chip that detects tuberculosis antibody.This method is compared with phlegm bacterium cultural method with traditional phlegm smear, has improved susceptibility and specificity that tuberculosis detects greatly, is expected to be used for the tuberculosis auxiliary diagnosis.
38kD antigen is a kind of lipoprotein and main immunogens, is one of diagnosis of tuberculosis antigen the most commonly used at present.The WilKinson report is with 38kD Detection of antigen pulmonary tuberculosis human blood, and sensitivity and specificity is respectively 65.6% and 95.8% (J Clin Microbiol, 35 (3): 553-557 (1997)).
Early stage secreted antigenicity target (ESAT-6) and culturing filtrate protein 10 (CFP10) are two up-and-coming antigens in the tuberculosis immunodiagnosis, and it is active to have stronger cellular immunity, in the immune anamnedstic response of resisting tuberculosis infection, plays an important role.These two antigens are by RD1 district coding, and this district only is present in the pathogenic tubercle bacillus gene group, and all lacking in all BCG strain gene groups should the zone.Combined application ESAT-6 and CFP10 diagnosis of tuberculosis are found hybrid antigen susceptibility than the former increase of monoclonal antibody, and do not reduce its specificity.So ESAT-6 and CFP10 can be expected to become the specific antigen of the pathogenic mycobacterium tuberculosis infection of diagnosis animal and human.
MPT64 is the major protein that growth of bacillus tubercle is secreted the earliest, only in tubercle bacillus and ox type bacillus, exists.Though the result of study of Lyashchenko etc. shows; Is lower as the positive rate of Detection of antigen approach antibody with MPT64 albumen in various tubercle bacillus secreted proteins; If but form fusion with other albumen then might have stability (Clin Diagn Lab Immunol, 7 (2): 155-160 (2000)) preferably.Therefore, MPT64 albumen promises to be one of the candidate of the combined antigen of tuberculosis hematology diagnosis.
Mtb8.4 (Rv1174c), Mtb8 (Rv0379) and Mtb16.3 (Rv2185c) extensively are present in the tubercle bacillus, and its meaning aspect diagnosis of tuberculosis is still unclear.Mtb8.4 is a kind of LMWP antigen that purifies and separates obtains from combine bacillus culture filtrating recently; Scholars' such as Coler research further discloses Mtb8.4 and suffers from important immunocompetence T cellular antigens in the individuality that combines bacillus infection of hiding; In confirming pathogenic combination bacillus infection, has vital role (J Immunol., 161:2356-2364 (1998)).Mtb8 possibly be a transport protein matter; Only in the research of Houghton, Mtb8 and other several kinds of antigens (Mtb11, Mtb48,38kD etc.) composition fused antigen are used to detect tuberculosis; Show reactivity (Clin Diagno Lab Immun, 9 (4): 883-891 (2002)) that Mtb8 might strengthen 38kD antigen with other several kinds of antigens.And Mtb16.3 is a kind of unknown function protein, there are some researches show Mtb16.3 and Mtb9.7 combination help to improve to the recall rate of the tuberculosis patient of HIV mixed infection.
Different negre antigens detect same patient blood, and its reactivity is different, and has certain complementarity between each antigen.Its reason possibly be that negre antigen is many and complicated, maybe be with patient's individual immunity background and the course of disease and different on quantity, kind or the opportunity of host's expression in vivo, show different antibody repertoires.Therefore, multiple antigen preparation is become protein chip, joint-detection can guarantee to help to improve detection sensitivity on the specific basis, is the developing direction of development high specific and hypersensitivity diagnostic reagent.
3 kinds of tuberculosis antigens of application such as Wang Haibo are set up mycobacterium tuberculosis antibody and are detected protein chip, and compare with ELISA, and susceptibility and specificity are respectively 69.9% and 84.9%.We are prepared into many target point proteins chip with 7 kinds of advantage epitope antigens of tubercle bacillus, and the visual antibody detection protein chip of the immune gold and silver of foundation detects tuberculosis patient blood sample technology, and this technology is simple and easy to do, susceptibility is high, high specificity.We analyze PRELIMINARY RESULTS, and the visual antibody detection protein chip of this tubercle bacillus is coated with chip detection method with phlegm and compares, and susceptibility is 98.5%, is significantly higher than the susceptibility 35.4% that phlegm is coated with chip detection method; Cultivate detection method with the phlegm bacterium and compare, susceptibility is 96.6%, is significantly higher than the susceptibility 48.3% that the phlegm bacterium is cultivated detection method.Its reason possibly be two aspects, is that we have selected seven kinds of negre antigens for use on the one hand, has complementarity between the antigen, helps to improve recall rate; On the other hand; Immunogold silver staining visible protein chip detection technique ultimate principle is to utilize the catalytic action of silver-colored developer solution and colloid gold particle; As long as have 2 gold atoms to have just the redox reaction of effectively catalysis silver in theory, have very strong signal enlarge-effect.Therefore, this visual many target spots tuberculosis antibody detection protein chip can be used for clinical assistant diagnosis lungy, improves phlegm smear and phlegm bacterium negative culture results patient's recall rate.In addition, because the advantage epitope antigen of tubercle bacillus differential is adopted in this research, guaranteed the specificity that detects to a certain extent.Through the detection to blood donor's blood sample, the specificity of calculating this visual many target spots tuberculosis antibody detection protein chip is 93.3%, and also proof can satisfy clinical demand.
Biochip technology has been one of the most far-reaching great science and technology progress of influence since the mid-90; Be that to melt microelectronics, biology, physics, chemistry, computer science be the new technology that the height of one intersects; Have great fundamental research and be worth, have tangible industrialization prospect again.Protein chip is the biochip that is used for protein function research and transactional analysis thereof, adopts that original position is synthetic, methods such as mechanical deposition or covalent bond are fixed in substrate with polypeptide, albumen, enzyme, antigen, antibody and form molecular lattice.Utilize the probe molecule of biomolecule and protein chip hybridization to take place or interact a technology of detection and analysing protein.Detection to the protein chip reaction signal at present mainly contains fluorescence method, enzyme development process and immune Jin-Yin development process.Fluorescence method is the signal detection system that protein chip adopts usually; Mainly utilize the laser confocal scanning appearance that hybridization signal is carried out high throughput testing and analysis; Have automaticity height, highly sensitive characteristics; But need to buy expensive chip scanner and professional, can't promote at different medical unit.The enzyme development process mainly utilizes horseradish enzyme or other biochemical enzymes and the specific colour developing liquid phase interaction of mark, the process of reaction result being analyzed according to the change color of chip matrix, but sensitivity and specificity are low.Immunity Jin-Yin development process mainly utilizes collaurum to carry out protein labeling and directly develops the color through silver; This method had both had high sensitivity and specificity; Simultaneously need not to increase equipment again, is the important model of visual chip, has application potential and prospect widely.But still do not have application visualizes immunity gold and silver protein chip at present both at home and abroad and detect relevant report lungy.
Summary of the invention
Present tuberculosis detection recall rate is low in order to solve, the shortcoming of poor specificity; The invention provides a kind of seven kinds of tubercle bacillus differential advantage epitope antigens that utilize clonal expression, prepare the Preparation method and use of visual many target spots tuberculosis antibody detection protein chip.
Protein chip of the present invention adopts immune gold and silver detection system chromogenic chip.
The purpose that detects for the efficient parallel of realizing many target spots tuberculosis antibody; The present invention is through the further investigation to the protein chip field; A kind of tuberculosis antibody that is used to detect has been proposed; The many target spots visible protein chip technology that adopts detects multiple antibody in tubercular's serum simultaneously, thereby sets up visual many target spots tuberculosis antibody detection protein chip.
Visual many target spots tuberculosis antibody detection protein chip of the present invention; Tuberculosis antibody detection chip different from the past; Be many target point proteins of the tuberculosis antibody chip that is based upon on the seven species specific tubercle bacillus differential epitope antigen bases, and utilize " collaurum-Yin dyes " Color Appearance System to reach chip detection result visualization purpose.Protein chip provided by the invention only just can detect the response situation of serum and 7 a plurality of target spot antigens with 1 sample, realized the high flux, collimation and the high efficiency that detect.
For realizing above-mentioned purpose, the inventor has designed the matrix of being made up of target spot tuberculosis antigen more than 7, and point has 7 kinds of negre antigens on each matrix, and every kind of antigen is established 2 parallel points.Negative control point is antigen blank and pBVIL1 empty carrier, and positive control point is a human IgG.Adopt the some contact method that 7 kinds of tuberculosis antigens are fixed on the chip substrate; Interact with tuberculosis and patients serum; Adopt " collaurum-Yin dyes " to make the chip colour developing, judge the response situation of research tubercular's serum and each target spot antigen through the result; Tuberculosis antibody is reactive among the statistics patients serum, and analyzes its clinical meaning.
The present invention is achieved through following technical scheme: at first prepare the tuberculosis epitope antigen; Utilize molecular biology method to prepare; Carry out pre-service behind the tuberculosis antigen purifying with preparation; Adopt the some contact method to prepare many target spots of tuberculosis antibody detection chip, sealing is also dry, obtains protein chip of the present invention.Using this chip to carry out tuberculosis antibody when detecting, tuberculosis patient serum and chip are reacted, adopt the colloid gold label albumino reaction after the rinsing, with the liquor argenti nitratis ophthalmicus colour developing, can be visual or write down the result with the normal image scanner, analyze its clinical meaning.
The visual many target spots tuberculosis antibody detection protein chip of the present invention can detect 7 kinds of anti-mycobacterium tuberculosis antibody among the patients serum; The result shows; Compare with the susceptibility that phlegm smear and phlegm bacterium are cultivated detection method; Visual many target spots tuberculosis antibody detection protein chip susceptibility is respectively 98.5% and 96.6%, and specificity is 93.3%, is significantly higher than existing goldstandard detection method.Prepared visual many target spots tuberculosis antibody detection protein chip can be used for clinical assistant diagnosis lungy, improves phlegm smear and phlegm bacterium negative culture results patient's recall rate greatly.
Compared with prior art, the present invention has following characteristics:
1. the present invention is based upon on the visible protein chip detection technique basis
Tubercle bacillus differential advantage epitope antigen involved in the present invention has 7 kinds; Adopt protein chip just can reach the 7 kinds of antibody response degree in patient's serum that detect through an application of sample; Detection efficiency is far above existing enzyme joint inspection survey technology and gold marked reagent; Adopt " collaurum-Yin dyes " can realize the visual of protein chip testing result, need not to buy expensive chip scanner, be fit to clinical expansion.
2. the present invention has certain clinical meaning.
Many target spots antigen that the present invention adopted is the epi-position with tubercle bacillus differential, and it has concentrated present most of the detection to use antigen.7 kinds of antibody have clinical meaning separately, and are in close relations with clinical research.
Description of drawings
The SDS-PAGE of tubercle bacillus differential epitope antigen analyzes behind Fig. 1 purifying, wherein 1.ESAT-6; 2.CFP10; 3.38kD; 4.Mtb8; 5.Mtb8.4; 6.Mtb16.3; 7.MPT64; M. LMWP standard.
The visual many target spots tuberculosis antibody detection protein chip of Fig. 2 reaction matrix design drawing, wherein 1a and 1b are ESAT-6 antigen; 2a and 2b are Mtb8 antigen; 3a and 3b are Mtb8.4 antigen; 4a and 4b are CFP10 antigen; 5a and 5b are Mtb16.3 antigen; 6a and 6b are 38kD antigen; 1c and 1d are MPT64 antigen; 2c, 2d, 3c and 3d are the IL1 negative control; 4c, 4d, 5c and 5d are pBVIL1 empty carrier negative control; 6c and 6d are the IgG positive control.
Fig. 3 visual many target spots tuberculosis antibody detection protein chip testing result and judgement, wherein A is No. 85729 TB patients serum samples, is judged to be the positive; B is No. 85518 TB patients serum samples, is judged to be suspicious; C is No. 1 Donor serum sample, is judged to be feminine gender.
Embodiment
One, material
Prokaryotic expression carrier pBVIL1 is made up by this chamber.Tubercle bacillus 7 species specific antigen gene plasmids are made up and are preserved by this chamber.Testing required Pyrobest archaeal dna polymerase and Taq archaeal dna polymerase is the TaKaRa Company products, DNA restriction enzyme Sal I, Xho I and Xba I, and the T4DNA ligase is all available from Promega company.Anion-exchange column Q-Sepharose FF and solvent resistant column Sephardex G-50 are available from Pharmacia Biotech company; Urea, sucrose are available from the chemical reagents corporation in Beijing; Lysozyme, BSA are available from Promega company, and the glass substrate of modification is provided by Shenzhen Yi Shengtang company.
Two, methods and results
1. the preparation of 7 kinds of specific epitopes antigens of tubercle bacillus
According to the synthetic upstream and downstream primer of nucleotide sequence design of specific epitopes antigenic peptides section, employed restriction enzyme is respectively BamHI and XhoI.
The primer sequence that is used for amplifying specific epitope antigen peptide section 38kD is following:
38kD-F：5’-GGGATCCGCGGCGGGCTGTGGCTCGA-3’
38kD-R：5’-GCTCGAGGCTGGAAATCGTCGCGA-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section ESAT6 is following:
ESAT6-F：5’-GGGATCCCATTCCCTCCTTGACGA-3’
ESAT6-R：5’-GCTCGAGGTTCAGCTCGGTAGCCGT-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section CFP10 is following:
CFP 10-F：5’-CGGATCCGAAGCAGCCAATAAGCAG-3’
CFP 10-R：5’-GCTCGAGCGAGGACAGCGCCTGCTG-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section MPT64 is following:
MPT64-F：5’-GGGATCCCCCAAGACCTACTGCGA-3’
MPT64-R：5’-GCTCGAGCGGCGCTATCGATACCT-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section Mtb8 is following:
Mtb8-F：5’-GGGATCCACCAGCCCCACATCCT-3’
Mtb8-R：5’-GCTCGAGAATGACCCGAGCGACGCGGA-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section Mtb8.4 is following:
Mtb8.4-F：5’-GGGATCCCCGGGGTCGCCTCCGCA-3’
Mtb8.4-R：5’-GCTCGAGTTGCGCGGCCATGGCA-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section Mtb16.3 is following:
Mtb16.3-F：5’-GGGATCCCGGACAAGACGACACAGA-3’
Mtb16.3-R：5’-GCTCGAGGCCCTCGACTCGTTTCTT-3’
With tubercle bacillus 7 species specific antigen gene plasmids is template, the genetic fragment of 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and the Mtb16.3 specific epitopes antigenic peptides section of having increased respectively.The pcr amplification condition is following: in advance 95 ℃ of sex change are 2 minutes, 94 ℃ of sex change 30 seconds; 58 ℃ of renaturation 30 seconds; Extend 72 ℃ of 30-90 second (different and different) with mrna length, 32 circulations of increasing, 72 ℃ were extended 7 minutes again.The fragment of pcr amplification gained is identified through electrophoresis, is shown the genetic fragment that all obtains corresponding size.With digestion with restriction enzyme and connect into the carrier through the pBVIL1 of same restrictions property endonuclease digestion, order-checking is accomplished by the sincere Bioisystech Co., Ltd of Li Jiafu with the PCR product.
With resulting recombinant plasmid transformed coli strain DH5 α, thermal induction is expressed, and collects thalline; To precipitate and claim weight in wet base; To precipitate with the 20mmol/L pH8.0TE damping fluid of 10 times of volumes and to hang, and add lysozyme (1mg/ml suspension), at room temperature magnetic agitation is 10 minutes.Ultrasonic disruption bacterium in ice bath surpassed for 30 seconds at every turn, at interval 30 seconds, surpassed altogether 10 times.8 ℃, 1,2000rpm, centrifugal 20 minutes, abandon supernatant, deposition is washed once with 1mol/LNaCl (preparing with TE), washes 2 times collecting precipitation again with TE.Deposition adds 1% beta-mercaptoethanol with 8M urea (preparing with PH8.0TE) dissolving.In 20 ℃ 1, centrifugal 10 minutes of 2000rpm goes deposition to get supernatant again.The inclusion body solution of above-mentioned dissolving is crossed Q-Sepharose FF anion-exchange column; After equilibrium liquid (pH8.0,20mmol/L TE contains the 6mol/L urea, 0.1% beta-mercaptoethanol) cleaning; NaCl (preparing) wash-out with variable concentrations with equilibrium liquid; Collect each eluting peak, identify, collect 0.05mol/L NaCl eluting peak through SDS-PAGE.After Sephardex G-50 solvent resistant column, collect first eluting peak.The antigen of purifying carries out SDS-PAGE to be analyzed, and the result shows and obtained the higher specific epitopes antigenic peptides section (Fig. 1) of purity.
2. the design of visual many target spots tuberculosis antibody detection protein chip reaction matrix
Designed the reaction matrix of being made up of tubercle bacillus differential epitope antigen in 7, point has 7 kinds of negre antigens, IL1 negative control, pBVIL1 empty carrier negative control and IgG positive control on each matrix, and every kind of antigen is established 2 parallel points.Fig. 2 is visual many target spots tuberculosis antibody detection protein chip reaction matrix design drawing mode chart, and wherein 1a and 1b are ESAT-6 antigen; 2a and 2b are Mtb8 antigen; 3a and 3b are Mtb8.4 antigen; 4a and 4b are CFP10 antigen; 5a and 5b are Mtb16.3 antigen; 6a and 6b are 38kD antigen; 1c and 1d are MPT64 antigen; 2c, 2d, 3c and 3d are the IL1 negative control; 4c, 4d, 5c and 5d are pBVIL1 empty carrier negative control; 6c and 6d are the IgG positive control.
3. the preparation of visual many target spots tuberculosis antibody detection protein chip
The operating point contact method is carried out point sample, and every some point sample amount is 1nL, and antigen concentration is 1mg/mL, 0.01mol/LPBS (pH=7.4) dissolved dilution antigen.Behind the point sample chip room temperature is placed 4h, (1%BSA, pH=7.4) sealing is 2 hours, with the unconjugated blank site of sealing substrate surface for confining liquid 0.1mol/L PBS.With filter paper the liquid around the microarray is blotted the back drying, 4 ℃ of preservations are subsequent use.
One, material
Colloid gold label albumin A, p-dihydroxy-benzene sodium citrate, citric acid, silver nitrate are available from Beijing chemical reagents corporation.
Two, methods and results
1. Preparation of Colloidal Gold
Adopt trisodium citrate reduction method to be prepared into 15nm particle diameter colloidal gold solution; Getting 1% 4 chlorauride 1ml adds the 99ml deionized water to be diluted to concentration is 0.01%; Get 100ml 0.01% tetrachloro Jinsui River solution and be heated to boiling; Add rapidly 1% trisodium citrate 1.0ml, boil and occur after about 5 minutes stopping heating when transparent orange is red, add deionized water after cooling and be supplemented to original volume.The colloidal gold solution for preparing should be the transparent orange redness, does not have deposition.Observe under the Electronic Speculum not have and assemble, be uniformly dispersed, measuring its mean diameter is 15nm.4 ℃ of preservations of qualified colloidal gold solution are subsequent use.
2. colloid gold label A albumen probe preparation
Polyglycol (molecular weight 20,000) with deionized water preparation 0.1M sal tartari and 3%.At first regulate colloidal gold solution pH to 6.0, press the optimal proportion (generally pressing 1ml colloidal gold solution mark 10 μ g A albumen) of colloidal gold solution and A albumen with 0.1M sal tartari.After the stirring at room 15 minutes, it is 0.05% that the PEG of adding 3% makes its final concentration, continues abundant stirring and evenly mixing 15 minutes; Room temperature left standstill 30 minutes, centrifugal 45 minutes of 4 ℃ of 14000rpm, and deposition is with containing 0.01M PBS (1%BSA, 0.2% Sodium azide; PH7.2-7.4) dissolving; 4 ℃ of 14000rpm centrifugal 45 minutes once more, and deposition adds 0.01M PBS (1%BSA, 0.2% Sodium azide, pH7.2-7.4) the damping fluid dissolving of initial volume 1/10; 0.22 after the aseptic filtration of μ m nitrocellulose filter, 4 ℃ of preservations are subsequent use.
3. tuberculosis patient and donors with normal blood sample detection of antibodies and signals collecting
With tuberculosis patient and the dilution in 1: 10 of donors with normal blood sample, every matrix 30 μ L are with chip incubated at room 1h respectively.With PBST washing 5 times, add colloid gold label A albumen probe, every matrix 30 μ L incubated at room 30 minutes, wash 5 times after, wash 2 times with distilled water again.Add freshly prepared silver-colored development A liquid 20 μ L, B liquid 20 μ L, vibration gently, lucifuge effect 10 minutes.With distilled water washing back airing.Visual or use Canon LiDE20 scanner is analyzed the colored intensity of each point.Judgment principle as a result: in 7 kinds of antigens any more than 2 kinds or 2 kinds antigen positive all be judged to be the positive, only a kind of antigen positive be judged to be suspicious, 7 kinds of all negative feminine genders (Fig. 3) that are judged to be of antigen.
One, material
Visual many target spots tuberculosis antibody detection protein chip that embodiment 2 is developed.Clinical tuberculosis patient positive blood sample and donors with normal blood sample.
Two, methods and results
Use visual many target spots tuberculosis antibody detection protein chip of being developed to detect tuberculosis patient and blood donor's blood sample, according to decision principle judgement feminine gender or positive findings as a result, and compare with phlegm smear and phlegm bacterium cultivation results, the result sees table 1.
In 48 routine clinical definite tuberculosis patients, positive 17 examples of phlegm smear, negative 31 examples, susceptibility are 35.4%.And visual antibody detection protein chip detects positive 47 examples, and suspicious 1 example, susceptibility are 98.5%.Phlegm smear 17 routine positive wherein, visual antibody detection protein chip is also all positive; And in phlegm smear 31 routine negative samples, visual antibody detection protein chip detects 29 parts of positives, and 2 parts suspicious, sees table 2.
In 29 routine clinical definite tuberculosis patients, the phlegm bacterium is cultivated positive 14 examples, negative 15 examples, susceptibility 48.3%.And visual antibody detection protein chip detects positive 28 examples, and 1 example is suspicious, susceptibility 96.6%.Wherein the phlegm bacterium is cultivated 14 routine positive, and visual antibody detection protein chip is also all positive; And cultivate in the 15 routine negative samples the phlegm bacterium, it is positive that visual antibody detection protein chip detects 14 examples, and 1 example is suspicious, sees table 3.
It is positive that visual antibody detection protein chip detects 1 example in 30 parts of blood donor's samples, and 1 example is suspicious.Specificity is 93.3%.
The visual many target spots tuberculosis antibody of table 1 protein chip detects the comparison of tuberculosis patient and blood donor's blood sample and phlegm smear and phlegm bacterium cultivation results
| No | Status | ESAT-6 | Mtb8 | Mtb8.4 | CFP10 | Mtb16.3 | 38kD | MPT64 | Chip results | The phlegm smear | The phlegm bacterium is cultivated |
| 85701 | TB | - | + | - | + | - | + | - | + | - | - |
| 64040 | TB | + | - | - | ++ | - | - | + | + | - | ND |
| 85759 | TB | + | - | - | +++ | - | ++ | - | + | + | + |
| 85713 | TB | + | - | - | ++ | - | ++ | - | + | - | ND |
| 85689 | TB | - | - | - | + | - | + | - | + | + | + |
| 85749 | TB | - | +++ | - | + | - | ++ | - | + | + | ND |
| 85518 | TB | - | - | - | + | - | - | - | ± | - | ND |
| 85707 | TB | ± | - | - | ++ | - | + | - | + | - | ND |
| 85714 | TB | ++ | - | + | ++ | - | +++ | ± | + | - | - |
| 85652 | TB | + | - | + | ++ | - | +++ | + | + | + | + |
| 85900 | TB | + | + | +++ | - | + | - | + | + | - | |
| 85463 | TB | + | - | - | ++ | ± | + | - | + | - | ND |
| 85694 | TB | - | - | ++ | + | - | +++ | - | + | + | + |
| 85470 | TB | + | - | - | ++ | - | + | - | + | + | + |
| 85487 | TB | ± | - | - | + | - | + | - | + | - | ND |
| 85473 | TB | + | + | + | ++ | - | +++ | ± | + | + | + |
| 85657 | TB | - | - | - | ++ | - | + | - | + | - | ND |
| 85661 | TB | ± | - | - | ++ | - | + | - | + | - | + |
| 85643 | TB | - | - | - | + | - | + | - | + | - | ND |
| 85680 | TB | - | - | - | + | - | - | - | ± | - | - |
| 85721 | TB | + | - | - | + | + | + | ± | + | - | - |
| 85728 | TB | ± | - | - | ++ | + | + | - | + | + | + |
| 85700 | TB | + | - | - | ++ | - | + | - | + | - | ND |
| 85705 | TB | ± | - | - | + | - | + | - | + | - | - |
| 85695 | TB | + | + | ± | ++ | - | +++ | - | + | - | + |
| 85489 | TB | ± | - | - | ++ | - | + | - | + | - | - |
| 85704 | TB | ± | + | - | ± | ± | ++ | ± | + | - | - |
| 56377 | TB | + | - | - | ++ | - | - | - | + | - | - |
| 85486 | TB | + | - | - | ++ | - | + | - | + | - | - |
| 85627 | TB | + | - | ± | + | - | - | - | + | - | ND |
| 85450 | TB | + | - | ± | ++ | + | ++ | - | + | - | - |
| 85479 | TB | + | + | + | + | + | ++ | + | + | + | - |
| 85662 | TB | - | - | - | ++ | - | - | - | + | + | ND |
| 85484 | TB | + | - | ± | + | - | + | - | + | - | ND |
| 85655 | TB | ++ | - | ++ | + | ± | + | - | + | - | - |
| 56741 | TB | + | - | - | + | - | ++ | + | + | - | - |
| 85729 | TB | - | - | - | ++ | - | ++ | ++ | + | + | + |
| 71656 | TB | + | + | - | ++ | - | +++ | ± | + | + | + |
| 85536 | TB | ± | - | + | ++ | - | - | - | + | - | ND |
| 85650 | TB | ± | - | - | ++ | - | ± | - | + | - | ND |
| 85622 | TB | - | - | - | ++ | - | - | - | + | + | + |
| 85614 | TB | + | - | ± | + | - | + | - | + | - | ND |
| 85517 | TB | + | - | - | - | - | ++ | +++ | + | + | + |
| 85496 | TB | - | - | - | ++ | + | ± | - | + | + | + |
| 85495 | TB | ± | +++ | - | + | - | +++ | + | + | + | ND |
| 85651 | TB | ± | + | ± | ++ | - | ± | - | + | - | ND |
| 85502 | TB | ± | - | - | ++ | - | ± | - | + | - | ND |
| 85644 | TB | + | - | + | ++ | - | + | - | + | - | - |
| 1 | Donor | - | - | - | - | - | - | - | - | ||
| 2 | Donor | - | - | - | - | - | - | - | - | ||
| 3 | Donor | - | - | - | - | - | - | - | - | ||
| 4 | Donor | - | - | - | - | - | - | - | - | ||
| 5 | Donor | - | - | - | - | - | - | - | - | ||
| 6 | Donor | - | - | - | - | - | - | - | - | ||
| 7 | Donor | - | - | - | + | - | - | - | ± | ||
| 8 | Donor | - | - | - | - | - | - | - | - | ||
| 9 | Donor | - | - | - | + | - | + | - | + | ||
| 10 | Donor | - | - | - | - | - | - | - | - | ||
| 11 | Donor | - | - | - | - | - | - | - | - | ||
| 12 | Donor | - | - | - | - | - | - | - | - | ||
| 13 | Donor | - | - | - | - | - | - | - | - | ||
| 14 | Donor | - | - | - | - | - | - | - | - | ||
| 15 | Donor | - | - | - | - | - | - | - | - | ||
| 16 | Donor | - | - | - | - | - | - | - | - | ||
| 17 | Donor | - | - | - | - | - | - | - | - | ||
| 18 | Donor | - | - | - | - | - | - | - | - | ||
| 19 | Donor | - | - | - | - | - | - | - | - | ||
| 20 | Donor | - | - | - | - | - | - | - | - | ||
| 21 | Donor | - | - | - | - | - | - | - | - | ||
| 22 | Donor | - | - | - | - | - | - | - | - | ||
| 23 | Donor | - | - | - | - | - | - | - | - | ||
| 24 | Donor | - | - | - | - | - | - | - | - | ||
| 25 | Donor | - | - | - | - | - | - | - | - | ||
| 26 | Donor | - | - | - | - | - | - | - | - | ||
| 27 | Donor | - | - | - | - | - | - | - | - | ||
| 28 | Donor | - | - | - | - | - | - | - | - | ||
| 29 | Donor | - | - | - | - | - | - | - | - | ||
| 30 | Donor | - | - | - | - | - | - | - | - |
ND：no detection
Visual antibody detection protein chip of table 2 tubercle bacillus and phlegm smear testing result are relatively
Visual antibody detection protein chip of table 3 tubercle bacillus and phlegm bacterium are cultivated testing result relatively
Sequence table .txt
Sequence table
< 110>Institute of Basic Medical Sciences, Academy of Military Medical Sciences, PLA
< 120>a kind of visualized tuberculosis antibody detection protein chip, Preparation method and use
<160>7
<170>PatentIn version 3.3
<210>1
<211>354
<212>PRT
< 213>tubercle bacillus (Mycobacterium tuberculosis)
<400>2
Ala Ala Gly Cys Gly Ser Lys Pro Pro Ser Gly Ser Pro Glu Thr Gly
1 5 10 15
Ala Gly Ala Gly Thr Val Ala Thr Thr Pro Ala Ser Ser Pro Val Thr
20 25 30
Leu Ala Glu Thr Gly Ser Thr Leu Leu Tyr Pro Leu Phe Asn Leu Trp
35 40 45
Gly Pro Ala Phe His Glu Arg Tyr Pro Asn Val Thr Ile Thr Ala Gln
50 55 60
Gly Thr Gly Ser Gly Ala Gly Ile Ala Gln Ala Ala Ala Gly Thr Val
65 70 75 80
Asn Ile Gly Ala Ser Asp Ala Tyr Leu Ser Glu Gly Asp Met Ala Ala
85 90 95
His Lys Gly Leu Met Asn Ile Ala Leu Ala Ile Ser Ala Gln Gln Val
100 105 110
Asn Tyr Asn Leu Pro Gly Val Ser Glu His Leu Lys Leu Asn Gly Lys
115 120 125
Val Leu Ala Ala Met Tyr Gln Gly Thr Ile Lys Thr Trp Asp Asp Pro
130 135 140
Gln Ile Ala Ala Leu Asn Pro Gly Val Asn Leu Pro Gly Thr Ala Val
145 150 155 160
Val Pro Leu His Arg Ser Asp Gly Ser Gly Asp Thr Phe Leu Phe Thr
165 170 175
Gln Tyr Leu Ser Lys Gln Asp Pro Glu Gly Trp Gly Lys Ser Pro Gly
180 185 190
Phe Gly Thr Thr Val Asp Phe Pro Ala Val Pro Gly Ala Leu Gly Glu
195 200 205
Asn Gly Asn Gly Gly Met Val Thr Gly Cys Ala Glu Thr Pro Gly Cys
210 215 220
Val Ala Tyr Ile Gly Ile Ser Phe Leu Asp Gln Ala Ser Gln Arg Gly
225 230 235 240
Leu Gly Glu Ala Gln Leu Gly Asn Ser Ser Gly Asn Phe Leu Leu Pro
245 250 255
Asp Ala Gln Ser Ile Gln Ala Ala Ala Ala Gly Phe Ala Ser Lys Thr
260 265 270
Pro Ala Asn Gln Ala Ile Ser Met Ile Asp Gly Pro Ala Pro Asp Gly
275 280 285
Tyr Pro Ile Ile Asn Tyr Glu Tyr Ala Ile Val Asn Asn Arg Gln Lys
290 295 300
Asp Ala Ala Thr Ala Gln Thr Leu Gln Ala Phe Leu His Trp Ala Ile
305 310 315 320
Thr Asp Gly Asn Lys Ala Ser Phe Leu Asp Gln Val His Phe Gln Pro
325 330 335
Leu Pro Pro Ala Val Val Lys Leu Ser Asp Ala Leu Ile Ala Thr Ile
340 345 350
Ser Ser
<210>2
<211>41
<212>PRT
< 213>tubercle bacillus
<400>4
His Ser Leu Leu Asp Glu Gly Lys Gln Ser Leu Thr Lys Leu Ala Ala
1 5 10 15
Ala Trp Gly Gly Ser Gly Ser Glu Ala Tyr Gln Gly Val Gln Gln Lys
20 25 30
Trp Asp Ala Thr Ala Thr Glu Leu Asn
35 40
<210>3
<211>37
<212>PRT
< 213>tubercle bacillus
<400>6
Glu Ala Ala Asn Lys Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn
1 5 10 15
Ile Arg Gln Ala Gly Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln
20 25 30
Gln Ala Leu Ser Ser
35
<210>4
<211>154
<212>PRT
< 213>tubercle bacillus
<400>8
Pro Lys Thr Tyr Cys Glu Glu Leu Lys Gly Thr Asp Thr Gly Gln Ala
1 5 10 15
Cys Leu Ile Gln Met Ser Asp Pro Ala Tyr Asn Thr Asn Ile Ser Leu
20 25 30
Pro Ser Tyr Tyr Pro Asp Gln Lys Ser Leu Glu Asn Tyr Ile Ala Gln
35 40 45
Thr Arg Asp Lys Phe Leu Ser Ala Ala Thr Ser Ser Thr Pro Arg Glu
50 55 60
Ala Pro Tyr Glu Leu Asn Ile Thr Ser Ala Thr Tyr Gln Ser Ala Ile
65 70 75 80
Pro Pro Arg Gly Thr Gln Ala Val Val Leu Lys Val Tyr Gln Asn Ala
85 90 95
Gly Gly Thr His Pro Thr Thr Thr Tyr Lys Ala Phe Asp Trp Asp Gln
100 105 110
Ala Tyr Arg Lys Pro Ile Thr Tyr Asp Thr Leu Trp Gln Ala Asp Thr
115 120 125
Asp Pro Leu Pro Val Val Phe Pro Ile Val Gln Gly Glu Leu Ser Lys
130 135 140
Gln Thr Gly Gln Gln Val Ser Ile Ala Pro
145 150
<210>5
<211>30
<212>PRT
< 213>tubercle bacillus
<400>10
Thr Ser Pro Thr Ser Trp Glu Gln Ala Ala Ala Glu Ala Val Gln Arg
1 5 10 15
Ala Arg Asp Ser Val Asp Asp Ile Arg Val Ala Arg Val Ile
20 25 30
<210>6
<211>64
<212>PRT
< 213>tubercle bacillus
<400>12
Ala Gly Val Ala Ser Ala Asp Pro Val Asp Ala Val Ile Asn Thr Thr
1 5 10 15
Cys Asn Tyr Gly Gln Val Val Ala Ala Leu Asn Ala Thr Asp Pro Gly
20 25 30
Ala Ala Ala Gln Phe Asn Ala Ser Pro Val Ala Gln Ser Tyr Leu Arg
35 40 45
Asn Phe Leu Ala Ala Pro Pro Pro Gln Arg Ala Ala Met Ala Ala Gln
50 55 60
<210>7
<211>143
<212>PRT
< 213>tubercle bacillus
<400>14
Ala Asp Lys Thr Thr Gln Thr Ile Tyr Ile Asp Ala Asp Pro Gly Glu
1 5 10 15
Val Met Lys Ala Ile Ala Asp Ile Glu Ala Tyr Pro Gln Trp Ile Ser
20 25 30
Glu Tyr Lys Glu Val Glu Ile Leu Glu Ala Asp Asp Glu Gly Tyr Pro
35 40 45
Lys Arg Ala Arg Met Leu Met Asp Ala Ala Ile Phe Lys Asp Thr Leu
50 55 60
Ile Met Ser Tyr Glu Trp Pro Glu Asp Arg Gln Ser Leu Ser Trp Thr
65 70 75 80
Leu Glu Ser Ser Ser Leu Leu Lys Ser Leu Glu Gly Thr Tyr Arg Leu
85 90 95
Ala Pro Lys Gly Ser Gly Thr Glu Val Thr Tyr Glu Leu Ala Val Asp
100 105 110
Leu Ala Val Pro Met Ile Gly Met Leu Lys Arg Lys Ala Glu Arg Arg
115 120 125
Leu Ile Asp Gly Ala Leu Lys Asp Leu Lys Lys Arg Val Glu Gly
130 135 140
Claims (5)
1. visual many target spots tuberculosis antibody detection protein chip is characterized in that: be fixed with 7 kinds of tubercle bacillus differential advantage epitope antigens shown in the SEQ ID NO:1-7 on the protein chip.
2. prepare the method for the said protein chip of claim 1, may further comprise the steps:
(1) 7 kinds of tubercle bacillus differential advantage epitope antigens shown in the preparation SEQ ID NO:1-7;
(2) antigen with preparation is fixed in chip substrate;
(3) seal with confining liquid, dry then.
3. according to the said method of claim 2, wherein fixing above-mentioned antigen adopts the some contact method in chip substrate.
4. according to the said method of claim 2, wherein said chip substrate is the slide of amino silaneization.
5. the said protein chip of claim 1 detects the purposes in the composition of anti-mycobacterium tuberculosis antibody in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101348079A CN101639476B (en) | 2008-07-31 | 2008-07-31 | Visualized tuberculosis antibody detection protein chip, preparation method and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101348079A CN101639476B (en) | 2008-07-31 | 2008-07-31 | Visualized tuberculosis antibody detection protein chip, preparation method and application |
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| Publication Number | Publication Date |
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| CN101639476B true CN101639476B (en) | 2012-08-08 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006026404A2 (en) * | 2004-08-26 | 2006-03-09 | Sequella, Inc. | Assay for detecting tuberculosis in nonhuman primates |
| US20070054335A1 (en) * | 2004-08-26 | 2007-03-08 | Javanbakhsh Esfandiari | Universal rapid test and method for detection of tuberculosis in multiple host species |
-
2008
- 2008-07-31 CN CN2008101348079A patent/CN101639476B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006026404A2 (en) * | 2004-08-26 | 2006-03-09 | Sequella, Inc. | Assay for detecting tuberculosis in nonhuman primates |
| US20070054335A1 (en) * | 2004-08-26 | 2007-03-08 | Javanbakhsh Esfandiari | Universal rapid test and method for detection of tuberculosis in multiple host species |
Non-Patent Citations (1)
| Title |
|---|
| 陈坤 等.结核分支杆菌Mtb8、Mtb8.4和Mtb16.3抗原的基因克隆、表达及在血清诊断中的应用.《中国医学检验杂志》.2006,(第6期),388-390. * |
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| Publication number | Publication date |
|---|---|
| CN101639476A (en) | 2010-02-03 |
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