CN103675293A - Application of proteins of Rv3872, Rv0164 and/or Rv1926c in developing and/or designing product with functions of identification, diagnosis, auxiliary diagnosis, screening and/or auxiliary screening of active tuberculosis - Google Patents

Abstract

The invention provides application of proteins of Rv3872, Rv0164 and/or Rv1926c in developing and/or designing a product with functions of identification, diagnosis, auxiliary diagnosis, screening and/or auxiliary screening of the active tuberculosis. The invention further provides a protein chip prepared by the proteins of Rv3872, Rv0164 and/or Rv1926c. According to the invention, the protein chip can be used for detecting the levels of IgG antibodies corresponding to three protein antigens in the blood serum of a patient with active tuberculosis and those in the blood serum of a normal person, conjointly analyzing the testing results of antibodies corresponding to the three groups of proteins and judging whether people to be tested have active tuberculosis or not. Test results show that the specificity and the sensitivity of the optimal operating point of the protein chip for auxiliary diagnosis of the active tuberculosis are 91.1% and 82.4% respectively which are both above the diagnosis indexes of the active tuberculosis in prior art.

Description

Rv3872, Rv0164 and/or the Rv1926c albumen purposes in the phthisical product of preparation diagnostic activities

Technical field

The invention belongs to biomedicine field, be specifically related to the purposes of mycobacterium tuberculosis protein in the product of preparation diagnosis and/or auxiliary diagnosis disease.

Background technology

Since a plurality of centuries, tuberculosis continues to become in the whole world public health problem can not be ignored.At present the population in the whole world existing 1/3rd carries Much's bacillus, and 2010 1 year just newly-increased cases of tuberculosis 8,800,000 is only dead 1,450,000, on average less than 22 seconds, has a people to die from pulmonary tuberculosis, and tuberculosis is in first of Death of Infectious Diseases number.And China is one of the high burden of 22, whole world tuberculosis country, tuberculosis patient number is in global second, infected number surpasses 500,000,000, only within 2010 1 year, just there are new cases 90～1,200,000, occupy approximately 12% of global total newly-increased case, as adopted an effective measure not in time, at Future Ten, in year, may have 3,000 ten thousand people's morbidities, will cause serious public health problem and social concern, so must realize as early as possible phthisical effective control from national strategy aspect.

Science proof shows very easily propagated due to active tuberculosis, to the diagnosis of active tuberculosis and eliminate as early as possible the infection sources, controls that tuberculosis is popular to have very great significance.Yet phthisical radical cure can't be accomplished at present completely, show that according to the study active tuberculosis patient is after different SCCs is cured, there is the patient of 1%-9% to recur, and in some special populations, up to 20%(Hong Kong Chest Service/British Medical Research Council.Am Rev Respir Dis, 1991,143:700-706), and further the propagation with the not timely tubercular who diagnoses of recurrence of drug-resistant tuberculosis has very large relation.So cure patient's active tuberculosis diagnosis has a very big significance for the overall strategy of preventing and treating tuberculosis about tuberculosis.Activity judgement at present comprehensively clinical, x-ray shows and the decision of phlegm bacterium, and Main Basis is phlegm bacterium and x-ray.Sputum smear examination is simple and easy to do, and accuracy is higher, is the goldstandard that active tuberculosis is made a definite diagnosis, but some research in its recall rate even lower than 10%.Diagnostic method based on nucleic acid amplification, if the advantage of real-time quantitative PCR and DNA chip is highly sensitive and consuming time short, its problem is that specificity is difficult to ensure card, easily causes false positive results.And serodiagnosis based on antigen-antibody reaction, due to its simplicity, rapidity, becomes the clinical complementary diagnostic means of important active tuberculosis day by day, and can meet the requirement of distinguishing tuberculosis rehabilitation crowd and active tuberculosis.Therefore, in the long run in general, should be devoted to find all good tuberculosis marks of susceptibility and specificity.

Desirable Diagnosis of Tuberculosis mark should meet following condition: (1) susceptibility is high; (2) specificity is high; (3) be present in body fluid, particularly, in blood, be easy to detect.But at present Mycobacterium tuberculosis serodiagnosis for antigen as antigen 5,38KD antigen, 30/31KD antigen and antigen 60 etc., all exist positive rate low, the problems such as cross-immunity with other mycobacterium, although in curative effect monitoring, prompting recurrence, judging prognosis and people at highest risk's generaI investigation, there is certain value, at present still can not making a definite diagnosis for active tuberculosis.In order to realize the high sensitivity to active tuberculosis, the diagnosis of high specific, is badly in need of searching out more responsive, more special active tuberculosis biomarker on molecular level.

Summary of the invention

An object of the present invention is to provide the product that detects anti-Rv3872 antibody, anti-Rv0164 antibody and/or anti-Rv1926c antibody horizontal in serum and there is the application in the product of discriminating, diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis purposes in preparation.

In described application, described Rv3872 be following a) or b) albumen:

A) protein that the amino acid sequence shown in the SEQ ID № .1 in sequence table forms;

B) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .1 have identical function by a) derivative protein;

Described Rv0164 is following c) or albumen d):

C) protein that the amino acid sequence shown in the SEQ ID № .2 in sequence table forms;

D) by the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .2 have identical function by c) derivative protein;

Described Rv1926c is following e) or albumen f):

E) protein that the amino acid sequence shown in the SEQ ID № .3 in sequence table forms;

F) by the amino acid residue sequence of the SEQ ID № .3 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .3 have identical function by e) derivative protein;

And/or, in the described product that has discriminating, diagnosis, auxiliary diagnosis, examination and/or an auxiliary examination active tuberculosis purposes, comprise the instructions that is described below content: compare with the people who does not normally suffer from any disease, active tuberculosis infects in people, has at least 2 kinds significantly to raise in anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody horizontal.

Described antibody is IgG.

Also object of the present invention be to provide a kind of for differentiating, the mark composition of diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis, by anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody, formed;

In described mark composition, the antibody antibody in vitro serum of behaving described in each;

Described antibody is IgG;

Described Rv3872 be following a) or b) albumen:

A) protein that the amino acid sequence shown in the SEQ ID № .1 in sequence table forms;

B) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .1 have identical function by a) derivative protein;

Described Rv0164 is following c) or albumen d):

C) protein that the amino acid sequence shown in the SEQ ID № .2 in sequence table forms;

D) by the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .2 have identical function by c) derivative protein;

Described Rv1926c is following e) or albumen f):

E) protein that the amino acid sequence shown in the SEQ ID № .3 in sequence table forms;

F) by the amino acid residue sequence of the SEQ ID № .3 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .3 have identical function by e) derivative protein.

A further object of the present invention be to provide anti-Rv3872 antibody in serum, anti-Rv0164 antibody and/or anti-Rv1926c antibody as a token of thing in exploitation, design and/or preparation, there is the application in the product of discriminating, diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis purposes;

Described Rv3872 be following a) or b) albumen:

A) protein that the amino acid sequence shown in the SEQ ID № .1 in sequence table forms;

B) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .1 have identical function by a) derivative protein;

Described Rv0164 is following c) or albumen d):

C) protein that the amino acid sequence shown in the SEQ ID № .2 in sequence table forms;

D) by the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .2 have identical function by c) derivative protein;

Described Rv1926c is following e) or albumen f):

E) protein that the amino acid sequence shown in the SEQ ID № .3 in sequence table forms;

F) by the amino acid residue sequence of the SEQ ID № .3 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .3 have identical function by e) derivative protein;

In the described product that has discriminating, diagnosis, auxiliary diagnosis, examination and/or an auxiliary examination active tuberculosis purposes, comprise the instructions that is described below content: compare with the people who does not normally suffer from any disease, active tuberculosis infects in people, has at least 2 kinds significantly to raise in anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody horizontal.

In described arbitrary application, described discriminating, diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis specifically refer to whether discriminating, diagnosis and/or auxiliary diagnosis people to be measured suffers from active tuberculosis; Or in examination and/or auxiliary examination crowd to be measured, suffers from the situation of active tuberculosis.

Another object of the present invention be to provide a kind of for differentiating, the kit of diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis, comprise detection chip, in described detection chip, be connected with at least 2 kinds of albumen in Rv3872, Rv0164 and Rv1926c albumen, every kind of albumen is set up separately a check point; Preferably, in described detection chip, be connected with Rv3872, Rv0164 and Rv1926c albumen;

Described Rv3872 be following a) or b) albumen:

A) protein that the amino acid sequence shown in the SEQ ID № .1 in sequence table forms;

B) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .1 have identical function by a) derivative protein;

Described Rv0164 is following c) or albumen d):

C) protein that the amino acid sequence shown in the SEQ ID № .2 in sequence table forms;

D) by the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .2 have identical function by c) derivative protein;

Described Rv1926c is following e) or albumen f):

E) protein that the amino acid sequence shown in the SEQ ID № .3 in sequence table forms;

F) by the amino acid residue sequence of the SEQ ID № .3 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .3 have identical function by e) derivative protein.

In described kit, described detection chip is that the solution point sample that contains respectively Rv3872, Rv0164 and/or Rv1926c albumen is made on carrier slide.

The described solution that contains respectively Rv3872, Rv0164 and/or Rv1926c albumen, by solute and solvent composition, described solute and the concentration in described solution thereof are:

Solvent is water.

In described solution, also comprise: the glycerine that percent by volume is 25%, the BSA of Tween20,0.05mg/ml and the NaN of 0.1g/L that percent by volume is 0.02% ₃.

Described point sample specifically adopts biochip point sample instrument point sample; The point sample volume of described point sample is 0.3-1nl; Preferred 0.5-1nl; 1nl most preferably.

What described carrier slide specifically adopted is three-dimensional H carrier slide.

After described point sample finishes, also need to stick the plastics fence in 12 holes, and after remaining on and spending the night in 4 ℃ of environment of 30%RH-40%RH humidity, slide is positioned over to-80 ℃ of low temperature of sealing in plastic casing and preserves; Preferably, humidity is 35%RH.

Described kit also comprises IgG positive control and/or the anti-human second antibody of Cy5 mark.

Described kit also comprises instructions, and described instructions is described below content:

Compare with the people who does not normally suffer from any disease, active tuberculosis infects in people, in anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody horizontal, has at least 2 kinds of antibody significantly to raise.

In described anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody horizontal, at least 2 kinds of remarkable risings of antibody judge as follows: in the testing result of testing sample, in anti-Rv3872 antibody, anti-Rv0164 antibody and/or anti-Rv1926c antibody, have at least 2 kinds of antibody to be positive, judge that this testing sample is that active tuberculosis is positive, otherwise be that active tuberculosis is negative;

In described kit, if the snr value that the concrete criterion of the described positive is each antibody between following value, thinks that this antibody is positive: the snr value of the antibody of Rv3872, between 8.98 and 10.30, comprises 8.98 and 10.30; The snr value of the antibody of Rv0164, between 8.95 and 10.43, comprises 18.95 and 10.43; The snr value of the antibody of Rv1926c, between 8.81 and 10.38, comprises 8.81 and 10.38.

Described anti-Rv3872 antibody, anti-Rv0164 antibody and/or anti-Rv1926c antibody specific are IgG.

The computing formula of described snr value is:

$\mathrm{SNR}=\frac{S-B}{\mathrm{\σ}}$

In formula, S and B represent respectively the signal value of the direct demonstration of scanner in described detection chip and the background value in non-sample spot region processed, and σ is the standard deviation that represents this original signal; The experiment number that described σ is corresponding is 3 times; Described S and B value obtain in the scanning of 635nm passage with scanner; Described scanner is GenePix.

Described kit also comprises the reagent that coordinates detection chip to use together, and described reagent comprises following 1)-4):

1) pH7.4PBS solution, it consists of:

2) the PBST solution of pH7.4, it consists of:

3) contain the pH7.4PBS solution of BSA:

4) fluorescently-labeled anti-human second antibody.

Described anti-human second antibody is specially anti-human IgG second antibody.

Last object of the present invention is to provide above-mentioned arbitrary described kit and has the application in the product of discriminating, diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis purposes in preparation.

Accompanying drawing explanation

Fig. 1 is that 3 prepared antigen protein Rv3872, Rv0164, the silver of Rv1926c dye quantitative result figure.

Fig. 2 is 3 prepared antigen protein Rv3872, Rv0164, the Western-Blotting qualification result figure of Rv1926c.

Fig. 3 is specificity and the susceptibility statistics figure of the method for application protein-chip auxiliary diagnosis active tuberculosis.

Embodiment

The experimental technique using in following embodiment if no special instructions, is conventional method.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.

The preparation of embodiment 1, protein-chip

(1) preparation of Mycobacterium tuberculosis antigen protein Rv3872, Rv0164 and Rv1926c

1, express

Saccharomyces cerevisiae by genetic engineering modified mistake utilizes galactose induction overexpression, and detailed process is:

First from mycobacterium Much's bacillus (Mycobacterium M.tuberculosis) H37Rv bacterial strain (Beijing Strain), by PCR, from the beginning clone and obtain albumen Rv3872, the gene code fragment of Rv0164 and Rv1926c, BP enzyme by Invitrogen company is connected to fragment on pDONR221 carrier (purchased from Invitrogen), be transformed in bacillus coli DH 5-Alpha and increase, extract carrier and by LR enzyme (Invitrogen), change again that (this carrier is at " Jian Zhu to the pEGH-A carrier that can express GST label through transforming, Heng Zhu, et al:J.Virol.May2009vol.83no.105219-5231 " in disclosed, the public can obtain from Ti Bikang bio tech ltd, Guangdong), again be transformed in bacillus coli DH 5-Alpha and increase, extraction plasmid is transformed into Pep4 Wine brewing yeast strain, and (this bacterial strain is at document " Heng Zhu, Michael Snyder, et al:Nature Genetics26, 283 – 289 (2000) doi:10.1038/81576 " in disclosed, the public can obtain from Ti Bikang bio tech ltd, Guangdong).In inducing culture, cultivate, when its OD600 is 0.6-0.8, adding final concentration is the galactose of 2g/L, induction 6h, the centrifugal collection of 4000rpm bacterium ,-80 ℃ of preservations.

The PCR primer sequence that PCR is used in from the beginning cloning is as follows:

The primer pair of the gene code fragment of amplification Rv3872 albumen:

Upstream primer: GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAAAAATGTCACATGATCCGATC

Downstream primer: GGGGACCACTTTGTACAAGAAAGCTGGGTACTATTCGGCGAAGACGCC

The primer pair of the gene code fragment of amplification Rv0164 albumen:

Upstream primer: GGGGACAAGTTTGTACAAAAAAGCAGGCTTAACGGCAATCTCGTGCTCACCGCG

Downstream primer: GGGGACCACTTTGTACAAGAAAGCTGGGTATTAGCTGGCCGCCAGCTGCTCG

The primer pair of the gene code fragment of amplification Rv1926c albumen:

Upstream primer: GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAAGCTCACCACAATGATCAA

Downstream primer: GGGGACCACTTTGTACAAGAAAGCTGGGTACTACGGCTCCCAAATCAG

The component containing in every 1L inducing culture (solvent is water) is as shown in table 1:

Table 1

2, purifying

1), prepare lysate:

In 50ml lysate, add 50 μ l mercaptoethanols, 125 μ lPMSF and two Roche protein inhibitors;

2), in-80 ℃ of refrigerators, take out the bacterium (cultivating the thalline of collecting from 120ml inducing culture) that above-mentioned steps 1 is collected, add 400 μ l zirconium oxide beads and 400 μ l lysates, in 4 ℃ of environment, shake 30s, postposition is 2min on ice, repeats four times;

3), take out after the centrifugal 2min of 11,000rpm, get supernatant in a new 15ml centrifuge tube;

4), repeat 2 and 3 step four times, supernatant is collected in same centrifuge tube;

5), add lysate to 12ml be 1/10 of original inducing culture volume, simultaneously with not having the lysate of inhibiting that glutathione beads is cleaned 3 times.In 12ml lysate, add the beads of 300 μ l;

6), add the lysate after beads to hatch 2h in 4 ℃;

7), 11, after the centrifugal 2min of 000rpm, get supernatant and be stored in 4 ℃.Beads respectively washes 3 times with cleaning fluid Ι and cleaning fluid II;

8), add 300 μ l eluents and hatch after 15min, centrifuging and taking supernatant is collected in a new centrifuge tube, repeats once.

The eluent obtaining is dissolved with this albumen.

In above-mentioned purge process, the composition of damping fluid used (solvent is water) is in Table 2-table 5.

Table 2 lysate (1L)

Table 3 cleaning fluid I (1L)

Table 4 cleaning fluid II (1L)

Table 5 eluent (1L)

3, identify

In following experimentation, described solvent or the percentage of liquid are percent by volume.

1), material preparation

Configure the SDS-PAGE glue of two 12%, 1.0mm, 15 holes.A silver dyes, a Western Blotting.Get each 20 μ l of albumen that above-mentioned purifying is good, add 4 μ l6X Loading buffer, the BSA sample of simultaneously preparing specification concentration gradient dyes quantitative contrast as silver, boils sample 5min.

2), run glue

Every hole adds the above-mentioned sample preparing of 12 μ l, BSA gradient sample, 2.5 μ Marker(Takara successively) record order.80V30min，140V1h。

3) silver dyes operation steps:

Fixing: 30min or longer time 40% ethanol 10% glacial acetic acid add water to 250ml

Sensitization: 30min75ml ethanol 30% ethanol 17g sodium acetate 28.2g sodium acetate trihydrate 0.5g sodium thiosulfate (dodium thiosulfate) adds water to final volume 250ml

Washing: 3x10min

Silver dyes: 20min0.625g AgNO3100 μ l37% formaldehyde (adding before use) adds water to final volume 250ml

Washing: 2x1min

Colour developing: the time 6.25g Na that depends on the circumstances ₂cO ₃50 μ l37% formaldehyde (adding before use) add water to final volume 250ml

Stop: 10min1g glycocoll adds water to final volume 250ml

Preserve: 1% glacial acetic acid, 4 ℃

4) Western-Blotting step:

Transferring film: 15V40min(is half-dried to be turned, Bio-Rad).Transferring film damping fluid: glycocoll 2.9g; Tris5.8g; SDS0.37g; Methyl alcohol 200ml; Add ddH ₂o is settled to 1000ml

Sealing: 5% skim milk (Bio-Rad) 1h.

First antibody is hatched: Anti-GST mouse-anti (NovaGen) final concentration 1 μ g/ml1h

Second antibody is hatched: sheep mouse-anti fluorescence 800 passages (Odyssey) final concentration 1 μ g/ml1h

Odyssey scanner scanning, preserves picture.

Carry out silver and dye result quantitative and that Western-Blotting identifies respectively as depicted in figs. 1 and 2.Fig. 1 result shows that the amount of prepared Rv3872, Rv0164 and Rv1926c albumen is 50 μ g/ml; Fig. 2 result proves, prepared Rv3872, Rv0164 and Rv1926c albumen are correct.

Through order-checking, prepared Rv3872, Rv0164 and Rv1926c albumen have respectively the amino acid sequence shown in SEQ ID № .1-3 in sequence table successively.

(2) put in advance the preparation of the protein-chip of antigen

In the eluate solution of the Rv3872 antigen protein containing 50 μ g/ml of above-mentioned preparation, adding final concentration is 25%(percent by volume) glycerine, 0.02%(percent by volume) the BSA of Tween20,0.05mg/ml and the NaN of 0.1g/L ₃.Adopt biochip point sample instrument that above-mentioned mixed liquor is put in carrier slide (carrier slide is commercial three-dimensional H carrier slide, purchased from CapitalBio Corporation) upper, every about 1nL of point sample, 2 parallel points.Adopt biochip point sample instrument (purchased from CapitalBio Corporation) some system.

Using above-mentioned Rv3872 antigen protein replace with Rv0164 antigen protein (being 50 μ g/ml), Rv1926c antigen protein (being 50 μ g/ml), as IgG (1mg/ml) standard items or the anti-human second antibody of Cy5 mark (20 μ g/ml) of positive control, in mixed liquor, other component and final concentration are constant, prepare the mixed liquor that contains respectively Rv0164 antigen protein, Rv1926c antigen protein, IgG standard items or the anti-human second antibody of Cy5 mark.

With above-mentioned identical point sample method, to containing the mixed liquor of Rv0164 antigen protein, Rv1926c antigen protein, IgG standard items or the anti-human second antibody of Cy5 mark, put on above-mentioned identical carrier slide respectively, form microarray, every slide can be put 12 and repeat parallel seal interval.After point sample finishes, stick the plastics fence in 12 holes, and remain in 4 ℃ of environment of 35%RH humidity after 16h, slide is positioned over to-80 ℃ of low temperature of sealing in plastic casing and preserves.

Embodiment 2, application protein-chip auxiliary diagnosis active tuberculosis

(1) preparation of test serum sample

Whole blood sample in room temperature place within 2 hours or 4 ℃, spend the night after in 1000g centrifugal about 20 minutes, get supernatant and can detect immediately; Or carry out packing, and sample is put in to-20 ℃ or-80 ℃ of preservations, but should avoid multigelation.4 ℃ of samples after thawing should be again centrifugal, then detects.

(2) related various damping fluids and the preparation of reagent in diagnostic procedure

(1) sample diluting liquid (in Table 6): pH7.4PBS solution

Table 6

(2) the PBST solution of cleansing solution (in Table 7): pH7.4

Table 7

(3) chip confining liquid (in Table 8): containing the pH7.4PBS solution of BSA

Table 8

(4) concentrate of the anti-human second antibody of Cy5 mark: use commercially available anti-human IgG-Cy5 fluorescence labeling two anti-, being diluted to concentration is 1mg/ml, is sub-packed in lucifuge tubule.

(3) method of application protein-chip auxiliary diagnosis active tuberculosis

Use the protein-chip of embodiment 1 preparation can detect in tested person's blood sample 3 proteantigen Rv3872, Rv0164 and the Rv1926c level of corresponding IgG antibody separately, the testing result of the corresponding antibodies of these 3 protein of Conjoint Analysis, can judge whether tested person is active tuberculosis.

1, concrete operation step

1) chip that 1 of embodiment is made to sealing is from-80 ℃ of taking-ups, room temperature rewarming 10 minutes.

2) sealing: chip is put into sink, add about 50ml chip confining liquid (table 8), shaking table 50rpm, room temperature 1h.

3) get rid of fast liquid unnecessary on chip, be placed in wet box.

4) dilution of testing sample and application of sample: by sample diluting liquid (table 6) dilution of above-mentioned preparation for 1:100 by volume of test serum sample, the solution containing test serum of getting after 30 μ L dilutions joins in the fence-enclosing space of sealing.Reaction 1h, room temperature.Detected sample preparation in 15 minutes before use.

5) chip is taken out from wet box, be placed in sink, add the cleansing solution of the above-mentioned preparation of about 50ml, shaking table 50rpm, room temperature 5min, repeats 3 times.

6) get rid of fast liquid unnecessary on chip, be placed in wet box.

7) the Cy5 fluorescence anti-human two that each enclosure space adds 30 μ L to be diluted to final concentration 1 μ g/ml resists on chip, lucifuge reaction 1h.

8) chip is taken out from wet box, be placed in sink, add the cleansing solution of the above-mentioned preparation of about 50ml, shaking table 50rpm, room temperature 5min, repeats 3 times.Use again the ultrapure washing of about 50ml once, 5min.

9) centrifugal drying, with Genepix scanner reading out data under 635nm passage.

2, the testing sample judgement to 3 kinds of antigen protein resistances respectively

By the result that Genepix scanner obtains in the scanning of 635nm passage, be to weigh by the signal to noise ratio (snr) of point of sample, the signal to noise ratio (snr) computing formula of point of sample is:

$\mathrm{SNR}=\frac{S-B}{\mathrm{\σ}}$

In formula, S and B represent respectively signal value original in chip (being the numerical value that scanner directly shows) and background value (signal value in non-sample spot region processed), and σ is the standard deviation (experiment number that σ is corresponding is 3 times) that represents this original signal.

The signal to noise ratio (S/N ratio) interval (described signal to noise ratio (S/N ratio) interval comprises the endpoints thereof in given signal to noise ratio (S/N ratio) interval) of the snr value of point of sample on protein-chip and following table 9-table 11 is contrasted, to carry out the judgement of testing sample to the positive of corresponding antigens resistance, negative findings:

Anti-Rv3872(is in Table 9):

Table 9

Anti-Rv0164(is in Table 10):

Table 10

Anti-Rv1926c(is in Table 11):

Table 11

3, the judgement of testing sample assay

Criterion:

Compare with the people who does not normally suffer from any disease, active tuberculosis infects in people, in anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody horizontal, has at least 2 kinds of antibody significantly to raise, and is embodied in:

If in the testing result of testing sample, in described anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody, (when 2 parallel point of samples that judge same antigen on the protein-chip of the standard whether antibody is positive: embodiment 1 preparation are all judged to be the positive, this antibody is positive to have at least 2 kinds of antibody to be positive.), judge that this testing sample is that active tuberculosis is positive, otherwise be that active tuberculosis is negative.

(4), specificity and the susceptibility of the method for application protein-chip auxiliary diagnosis active tuberculosis

Adopt serum (100 parts of the patients that clinical diagnosis is active tuberculosis, 100 parts of the pulmonary tuberculosis rehabilitation patients of 200 parts of active tuberculosis associated patient; Serum sample is provided by Guangdong Province tuberculosis control center, serum sample obtain the informed consent of all passing through patient and examinee.) protein-chip of embodiment 1 has been carried out to specificity and sensitivity Detection, with above-mentioned criterion, judge that whether person to be measured is positive as active tuberculosis.

Testing result as shown in Figure 3.Horizontal ordinate false positive rate representative (1-specificity) in Fig. 3, ordinate True Positive Rate represents susceptibility.The specificity that is the best operating point of susceptibility/(1-specificity) the protein-chip auxiliary diagnosis active tuberculosis that calculates embodiment 1 according to positive likelihood ratio is 91.1%, susceptibility is 82.4%, is all much higher than the index of active tuberculosis diagnosis in prior art.

In 100 parts of active tuberculosis patients, there are 91 parts to be detected the positive; In 100 parts of pulmonary tuberculosis rehabilitation patients, there are 17 parts to be detected the positive.

Claims (10)

1. the product that detects anti-Rv3872 antibody, anti-Rv0164 antibody and/or anti-Rv1926c antibody horizontal in serum has the application in the product of discriminating, diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis purposes in preparation.

2. application according to claim 1, is characterized in that:

Described Rv3872 be following a) or b) albumen:

A) protein that the amino acid sequence shown in the SEQ ID № .1 in sequence table forms;

B) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .1 have identical function by a) derivative protein;

Described Rv0164 is following c) or albumen d):

C) protein that the amino acid sequence shown in the SEQ ID № .2 in sequence table forms;

D) by the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .2 have identical function by c) derivative protein;

Described Rv1926c is following e) or albumen f):

E) protein that the amino acid sequence shown in the SEQ ID № .3 in sequence table forms;

F) by the amino acid residue sequence of the SEQ ID № .3 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .3 have identical function by e) derivative protein;

And/or, in the described product that has discriminating, diagnosis, auxiliary diagnosis, examination and/or an auxiliary examination active tuberculosis purposes, comprise the instructions that is described below content: compare with the people who does not normally suffer from any disease, active tuberculosis infects in people, has at least 2 kinds significantly to raise in anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody horizontal.

For differentiating, the mark composition of diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis, by anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody, formed;

Described Rv3872 be following a) or b) albumen:

A) protein that the amino acid sequence shown in the SEQ ID № .1 in sequence table forms;

B) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .1 have identical function by a) derivative protein;

Described Rv0164 is following c) or albumen d):

C) protein that the amino acid sequence shown in the SEQ ID № .2 in sequence table forms;

D) by the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .2 have identical function by c) derivative protein;

Described Rv1926c is following e) or albumen f):

E) protein that the amino acid sequence shown in the SEQ ID № .3 in sequence table forms;

F) by the amino acid residue sequence of the SEQ ID № .3 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .3 have identical function by e) derivative protein.

In serum anti-Rv3872 antibody, anti-Rv0164 antibody and/or anti-Rv1926c antibody as a token of thing in exploitation, design and/or preparation, there is the application in the product of discriminating, diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis purposes;

Described Rv3872 be following a) or b) albumen:

A) protein that the amino acid sequence shown in the SEQ ID № .1 in sequence table forms;

B) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .1 have identical function by a) derivative protein;

Described Rv0164 is following c) or albumen d):

C) protein that the amino acid sequence shown in the SEQ ID № .2 in sequence table forms;

D) by the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .2 have identical function by c) derivative protein;

Described Rv1926c is following e) or albumen f):

E) protein that the amino acid sequence shown in the SEQ ID № .3 in sequence table forms;

F) by the amino acid residue sequence of the SEQ ID № .3 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .3 have identical function by e) derivative protein;

In the described product that has discriminating, diagnosis, auxiliary diagnosis, examination and/or an auxiliary examination active tuberculosis purposes, comprise the instructions that is described below content: compare with the people who does not normally suffer from any disease, active tuberculosis infects in people, has at least 2 kinds significantly to raise in anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody horizontal.

One kind for differentiating, the kit of diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis, comprise detection chip, in described detection chip, be connected with at least 2 kinds of albumen in Rv3872, Rv0164 and Rv1926c albumen, every kind of albumen is set up separately a check point; Preferably, in described detection chip, be connected with Rv3872, Rv0164 and Rv1926c albumen;

Described Rv3872 be following a) or b) albumen:

A) protein that the amino acid sequence shown in the SEQ ID № .1 in sequence table forms;

B) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .1 have identical function by a) derivative protein;

Described Rv0164 is following c) or albumen d):

C) protein that the amino acid sequence shown in the SEQ ID № .2 in sequence table forms;

D) by the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .2 have identical function by c) derivative protein;

Described Rv1926c is following e) or albumen f):

E) protein that the amino acid sequence shown in the SEQ ID № .3 in sequence table forms;

F) by the amino acid residue sequence of the SEQ ID № .3 in sequence table through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with albumen shown in SEQ ID № .3 have identical function by e) derivative protein.

6. kit according to claim 5, is characterized in that: described detection chip is that the solution point sample that contains respectively Rv3872, Rv0164 and/or Rv1926c albumen is made on carrier slide.

7. according to the kit described in claim 5 or 6, it is characterized in that: described kit also comprises instructions, described instructions is described below content:

Compare with the people who does not normally suffer from any disease, active tuberculosis infects in people, in anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody horizontal, has at least 2 kinds of antibody significantly to raise.

8. according to the arbitrary described kit of claim 5-7, it is characterized in that:

In described anti-Rv3872 antibody, anti-Rv0164 antibody and anti-Rv1926c antibody horizontal, at least 2 kinds of remarkable risings of antibody judge as follows: in the testing result of testing sample, in anti-Rv3872 antibody, anti-Rv0164 antibody and/or anti-Rv1926c antibody, have at least 2 kinds of antibody to be positive, judge that this testing sample is that active tuberculosis is positive, otherwise be that active tuberculosis is negative;

If the snr value that the concrete criterion of the described positive is each antibody between following value, thinks that this antibody is positive: the snr value of the antibody of Rv3872, between 8.98 and 10.30, comprises 8.98 and 10.30; The snr value of the antibody of Rv0164, between 8.95 and 10.43, comprises 18.95 and 10.43; The snr value of the antibody of Rv1926c, between 8.81 and 10.38, comprises 8.81 and 10.38.

9. according to the arbitrary described kit of claim 5-8, it is characterized in that: described kit also comprises the reagent that coordinates detection chip to use together, and described reagent comprises following 1)-4):

1) pH7.4PBS solution, it consists of:

2) the PBST solution of pH7.4, it consists of:

3) contain the pH7.4PBS solution of BSA:

4) fluorescently-labeled anti-human second antibody.

10. the arbitrary described kit of claim 5-9 has the application in the product of discriminating, diagnosis, auxiliary diagnosis, examination and/or auxiliary examination active tuberculosis purposes in preparation.

Images