CN105968197B

CN105968197B - A kind of anti-3 type Ureaplasma urealyticum MB protein antibodies and the immune chromatography reagent kit using the antibody

Info

Publication number: CN105968197B
Application number: CN201610318423.7A
Authority: CN
Inventors: 胡征; 董俊; 杨波
Original assignee: HUBEI HUALONG BIOLOGICAL PHARMACEUTICAL CO Ltd; Hubei University of Technology
Current assignee: HUBEI HUALONG BIOLOGICAL PHARMACEUTICAL CO Ltd; Hubei University of Technology
Priority date: 2016-05-13
Filing date: 2016-05-13
Publication date: 2019-08-16
Anticipated expiration: 2036-05-13
Also published as: CN105968197A

Abstract

The present invention relates to the immune chromatography reagent kits of anti-3 type Ureaplasma urealyticum MB protein antibodies and application the antibody test Ureaplasma urealyticum, anti- 3 type Ureaplasma urealyticum MB protein antibodies are the antibody for identifying linear epitope composed by 3 type Ureaplasma urealyticum MB protein 10 5-118 amino acids, and 3 type Ureaplasma urealyticum MB albumen are AAC41437.1 in GenBank sequence number；The amino acid sequence of 3 type Ureaplasma urealyticums MB protein 10 5-118 is KLPREPKPNEQLTI.3 type Ureaplasma urealyticum MB protein antibodies of rabbit-anti provided by the present invention have the characteristics that specificity is good, with high purity, potency is high, preparation cost is cheap.

Description

A kind of anti-3 type Ureaplasma urealyticum MB protein antibodies and the immunochromatography using the antibody Kit

Technical field

The invention belongs to field of biomedicine technology, it is related to the anti-3 type Ureaplasma urealyticum MB protein antibodies of one kind and application should The immune chromatography reagent kit of antibody test Ureaplasma urealyticum.

Background technique

Ureaplasma urealyticum (M.urealyticum) is to obtain unique one kind in Ureaplasma because growth needs urea Name.There are two biota, 14 serotypes to be normally present in the asymptomatic crowd of health, thus it is speculated that may be only for Ureaplasma urealyticum It is certain subgroups or Asian Development Bank with pathogenic.By dividing group the study found that in mankind's uropoiesis to different crowd urogenital tract It is first group that the major part of infection is lived away from home and caused to genital tract, most commonly seen with 3 types.After ureaplasma urealyticum infection, patient is most Non-evident sympton also easily causes doctor to fail to pinpoint a disease in diagnosis therefore, it is difficult to be perceived by patient.Ureaplasma urealyticum can invade urethra, uterine neck and preceding The big gland in front yard, causes urethritis, cervicitis and bartholinitis；When uplink infects, endometritis, pelvic inflammatory disease, defeated ovum can be caused Guan Yan, especially salpingitis are common.Female sex organ pathology caused by ureaplasma urealyticum infection sexually revises, and is infertile Major reason.Data at home and abroad prompt, ureaplasma urealyticum positive rate is up in the cervical mucus of Infertile Couples, sperm 50% or more, it can be seen that, ureaplasma urealyticum infection and infertility have correlativity.Ureaplasma urealyticum infection causes not Good reason is miscarriage, and someone checks that the positive rate of Ureaplasma urealyticum is up to 40% or more from the tissue of miscarriage.

The detection method of Ureaplasma urealyticum mainly has 3 classes at present: first is that isolated culture, is to confirm infection " gold mark It is quasi- ", but since the growth cycle of Ureaplasma urealyticum is extremely slow, cultivation cycle is long, and leading to the method clinically not can be carried out fastly Speed diagnosis；Second is that serological method, that is, use enzyme-linked immunization, colloidal gold immunization, micro-Immunofluorescence assay and indirect hemagglutination Test etc. detects ureaplasma urealyticum antibodies level in examinee's serum, can prompt the presence of ureaplasma urealyticum infection indirectly.So And serological test can only provide a kind of retrospective diagnosis, and sometimes for paired sera.In addition, antibody occur when Machine is not easy to grasp, and there are the differences of Ureaplasma urealyticum specific antibody again between children and adolescents and adult, also, solves urea branch Glycolipid antigen on somatoblast film and other microorganisms and body tissue are there are non-specific cross-reaction, therefore existing serology The detection quality of method is subject to certain restrictions；Third is that using the presence of Protocols in Molecular Biology detection Ureaplasma urealyticum DNA, wherein The most commonly used is polymerase chain reaction (PCR), and this method is quick, sensitive, special, are the important hands of current research Mp infection Section, but since PCR is more demanding to experimental facilities and operation, and easily there is false positive, it can't be as common in China Methods for clinical diagnosis.Therefore, it is very necessary to establish Ureaplasma urealyticum specific antigen diagnostic method.At present, it has been disclosed that report The method for detecting Ureaplasma urealyticum antigen is mainly double crush syndrome method, indirect immunofluorescence etc., but these methods are not Detection by bed can be carried out, needs to detect to specific occasion using specific instrument (such as microplate reader, luminoscope), not only not The enough convenient and efficient and time is longer, and clinical application is more inconvenient.

Therefore, the fast diagnosis method for establishing Ureaplasma urealyticum specific antigen is very necessary.Due to most of pathogenic Ureaplasma urealyticum is three type of serum, simultaneously because it is well-conserved in its outer membrane protein MB sequence, therefore obtain high special Property anti-3 type Ureaplasma urealyticum MB protein antibodies be exactly a highly important job.Currently, about anti-3 type Ureaplasma urealyticums MB protein antibodies report at most to be corresponding monoclonal antibody and polyclonal antibody.Monoclonal antibody the biggest advantage is to It is specific high, but preparation method is cumbersome, high production cost limits its application.Polyclonal antibody then has specific low, effect The defects such as valence is low.Therefore, the inexpensive anti-3 type Ureaplasma urealyticum MB protein antibodies for preparing high specific, high-titer, to realize Quick detection to 3 type Ureaplasma urealyticums, just seems particularly significant.

Summary of the invention

For these technical problems present in background technique, the purpose of the present invention is to provide 3 type solution urea branch of identification is former The immunochromatography reagent of the antibody and application of linear epitope composed by body MB protein 10 5-118 the amino acids antibody Box.

Anti- 3 type Ureaplasma urealyticum MB protein antibodies, it is characterised in that: described that 3 type Ureaplasma urealyticum MB protein antibodies is resisted to be Identify the antibody of linear epitope composed by 3 type Ureaplasma urealyticum MB protein 10 5-118 amino acids, the 3 type solution urea Mycoplasma MB albumen is AAC41437.1 in GenBank sequence number；The 14 of 3 type Ureaplasma urealyticum MB protein 10 5-118 A amino acid sequence is KLPREPKPNEQLTI；By the sequence of MB protein 10 5-118 14 amino acid of 3 type Ureaplasma urealyticum It is named as MB3Linearar；The 3 type MB protein antibodies of anti-Ureaplasma urealyticum are AbMB3Linearar.

One kind being formed by immune chromatography reagent kit based on anti-3 type Ureaplasma urealyticum MB protein antibodies as previously described, Be characterized in that: the kit is immune chromatography reagent kit based on quantum dot-labeled technology or based on colloidal gold-labeled method Immune chromatography reagent kit.

Preferably, when kit provided by the present invention is the immune chromatography reagent kit based on quantum dot-labeled technology, The preparation method of the kit is:

1) quantum dot-labeled antibody A bMB3Linearar:

0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide EDC are sequentially added into microcentrifugal tube, with MES buffer constant volume is 1ml, and mixed solution after 37 DEG C of reaction 5min, adds the antibody of 0.34mg being prepared AbMB3Linearar is protected from light 2h, and single-ended amino-polyethyleneglycols PEG2000-NH2 to final concentration of 1% (m/v) is added, envelope Unreacted activated carboxyl site is closed, continues to be protected from light 1h；Sample after reaction is centrifuged (molecular cut off with super filter tube 100k), 6500g is centrifuged 5min, until volume 200ul, sample after ultrafiltration is transferred in common EP pipe, centrifugation is obtained except reuniting Upper clear supernate and lower part are precipitated, and are centrifuged 3min under the conditions of 10000g；Upper clear supernate is added on splitter Superdex-200 Purifying, flows into cylinder naturally to upper clear supernate, is then rinsed with PBS, and the position of sample is observed with ultraviolet light cylinder, to Sample starts to collect since when lower part is flowed out, stops collecting after collecting 1ml；By the super filter tube (retention point of sample after purification Son amount 100k) to be transferred in common EP pipe centrifugation after 6500g centrifugal concentrating to 200ul except reuniting, to common EP pipe carry out from The condition of the heart is 10000g, 3min；Liquid is saved with phosphate after acquisition supernatant and dilutes 200 times, 4 DEG C save backup；So far it is made Solution containing quantum dot labelled antibody AbMB3Linearar；

Each component content is respectively in the MES buffer: 10.66g/L MES and 0.74g/L EDTA, the MES The pH 7.4 of buffer；

The phosphate save liquid preparation method be weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 1g bovine serum albumin(BSA) BSA and 0.1gNaN₃, it is dissolved in the deionized water of 90ml, with 1mol/L NaOH 100ml is settled to deionized water after tune pH to 7.3；

2) bonding pad is prepared

Polyester fiber film is immersed in the obtained solution containing quantum dot labelled antibody AbMB3Linearar of step 1) 1h takes out, and is cut into after rear specification is 4cm × 0.6cm/ item after 25 DEG C of drying, and 4 DEG C are sealed spare, so far obtained bonding pad；

3) sample pad is prepared

It takes glass fibre element film one to open, glass fibre element film is impregnated at least 3h in sample pad treatment fluid, then be placed in life In object safety cabinet after 37 DEG C of aeration-dryings, specification is cut into after 4cm × 2.5cm/ item, to obtain sample pad, 25 DEG C of sealings are protected It deposits；

The preparation method of the sample pad treatment fluid be weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 2g bovine serum albumin(BSA) BSA, 1ml Tween-20,2g sucrose and 0.5g polyvinylpyrrolidone PVP-10, it is molten Solution is settled to 100ml with deionized water in the deionized water of 90ml, with after 1mol/L NaOH tune pH to 7.3；

4) detection layers are prepared

4.1) preparation of anti-MB protein polyclone antibody:

4.1.1) the clone of related gene

To 3 type Ureaplasma urealyticum MB albumen, (the accession number in its NCBI Protein Data Bank is AAC41437.1 bioinformatic analysis) is carried out, epitope peptide the most abundant in the extracellular conserved domain of its N-terminal is obtained Section finds the corresponding DNA encoding sequence of the peptide fragment, while introducing restriction enzyme site NdeI, 3 ' ends at 5 ' ends of this DNA encoding sequence (Jin Siruisheng is transferred in complete sequence synthesis to chemical synthesis complete genome sequence respectively after introducing termination signal TAA and restriction enzyme site XhoI Object Science and Technology Ltd. completes, and when delivery, artificial synthesized genetic fragment was respectively connected on carrier pUC57), it is denoted as MB3, Gene complete sequence is as shown in sequence table.The protein sequence of MB3 gene coding is natural 3 type Ureaplasma urealyticum memebrane protein MB The 30-150aa of (accession number:AAC41437.1).By the carrier containing this section of artificial synthesized DNA fragmentation Target fragment is separately recovered in pUC57 according to a conventional method after carrying out double digestion with NdeI and XhoI respectively, spare.NdeI is used simultaneously And XhoI carries out double digestion to carrier pET-28a (+), and according to a conventional method respectively connects the MB3 gene obtained after double digestion Enter in pET-28a (+) carrier, and convert Escherichia coli TOP10, constructs pET-MB3 expression vector.It is demonstrate,proved through digestion and sequencing Real expression vector establishment is errorless.Carrier expression recombination MB3-His fusion protein.

4.1.2 the expression and purification of MB3-His fusion protein) is recombinated

Plasmid will be extracted after identifying correct positive colony bacterium culture, routinely technology is transferred to competence E.coli BL21 (DE3) in, bacterium solution is coated on the LB plate containing 50 μ g/mL kanamycins after the completion of conversion, according to a conventional method screening expression Bacterial strain.The single bacterium colony with exogenous protein expression ability and inoculation that picking pET-MB3 is converted enter in 100mL LB culture medium, In 37 DEG C of overnight incubations.After taking out bacterium solution, 100mL is inoculated in by 1:100 and is contained in the LB culture medium of 50 μ g/mL kanamycins, In 37 DEG C of cultures to OD₆₀₀When=0.6,1mol/L IPTG to final concentration of 1mmol/L is added, bacterium culture, induction are shaken in 37 DEG C Expressing fusion protein.10min collection thallus is centrifuged under 8000r/min after inducing 4h.By this thallus 20mL phosphate-buffered Liquid washs 3 times and with 10mL sample-loading buffer (20mM Na₃PO₄, 0.5M NaCl；30mM imidazoles, pH7.4) be resuspended after surpassed Sound is broken, operating condition are as follows: 50HZ, 200W, ultrasonic 3S, interval 5S work 100 times.After the completion of ultrasound, 12000g centrifugation 15min carries out electrophoresis detection after collecting precipitating and supernatant respectively.It was found that recombinant protein MB3-His is present in bacterium in a manner of soluble In body.

The purification step for recombinating MB3-His fusion protein is as follows:

His Trap affinity is used after the ultrasonication supernatant of above-mentioned acquisition is filtered with 0.45 μm of filter membrane Columns (GE healthcare Products), is purified with same method to specifications.The specific method is as follows:

1) it is filled distilled water with 5mL syringe, unscrews the plug of column, connected column with syringe with the connector of offer, Column is washed with 1mL/min flow velocity.

2) it is balanced with 10mL sample-loading buffer, 1mL/min flow velocity.

3) by fusion protein loading, 1mL/min flow velocity.

4) 10mL sample-loading buffer is used, column is washed with 1mL/min flow velocity.

5) 10mL elution buffer (20mM Na is used₃PO₄, 0.5M NaCl, 300mM imidazoles, pH7.4), it is flowed with 1mL/min Speed elution, is in charge of collection, and every pipe 1ml, 12%SDS-PAGE detection merge the sample containing destination protein in elution fraction.Through After bradford kit carries out determination of protein concentration, adjustment concentration is 0.2mg/mL.

4.1.3) the preparation of anti-MB protein polyclone antibody

With the recombination MB3-His fusion protein of above-mentioned purifying, according to 200 μ g, (200 μ g recombinate the total of MB3-His fusion protein Volume is 1mL) with 1mL Freund's complete adjuvant mix emulsification after be immunized Male New Zealand White Rabbit (by Hubei Province's disease prevention control Center processed provides), in dorsal sc multi-point injection, it is spaced after 7d and is immunized again once, with the recombination of above-mentioned purifying after 14d P1-His fusion protein is incomplete with 1mL Freund according to 200 μ g (it is 1mL that 200 μ g, which recombinate the total volume of MB3-His fusion protein) Adjuvant carries out booster immunization after mixing emulsification, and booster immunization is primary again in same way as described above again after booster immunization 7d.It is taken after 7d Haemanalysis antibody titer.If dissatisfied, one may be repeated to booster immunization twice, until antibody titer satisfaction (uses indirect ELISA Method measures antibody titer and is greater than 1 × 10⁵).The Culling heart blood if satisfied separates serum, with Protein G affinity column (GE Healthcare Products), in strict accordance with operational manual purified polyclonal antibodies IgG, with triumphant base Braford protein content Detection kit measurement antibody concentration is simultaneously adjusted to 1mg/mL with phosphate buffer, and -20 DEG C of preservations are spare, and anti-MB is so far made Protein polyclone antibody.

4.2) detection layers are prepared

Nitrocellulose filter is cut into 4cm × 4cm size；The anti-MB protein polyclone being prepared according to the above method is resisted It is respectively 2.0mg/mL and 1.0mg/mL that body and goat anti-rabbit igg, which are adjusted with phosphate buffer to final concentration,；It is anti-by what is diluted MB protein polyclone antibody is fitted into BIODOT and draws in film instrument spray head, and the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter, shape At detection line；The anti-rabbit IgG diluted is fitted into BIODOT to draw in film instrument spray head, the amount of 1.0 μ l/cm of setting is sprayed on cellulose nitrate Nature controlling line is used as on plain film, nature controlling line and detection line spacing are 0.7cm；37 DEG C of the nitrocellulose filter dry 2h that will have been sprayed, cut It is cut into the specification of 4cm × 4cm, 4 DEG C of hermetically dryings save；So far detection layers are made；

The preparation method of the phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate with And 0.2g sodium chloride, it is dissolved in the deionized water of 90ml, is settled to after 1mol/L NaOH tune pH to 7.3 with deionized water 100ml；

5) assembling detection card

5.1) bottom plate is cut into 4cm × 7.3cm size, it is spare；

5.2) absorbent filter is cut into 4cm × 3cm size, it is spare as water absorption pad；

5.3) adhered protection film on bottom plate that step 5.1) is prepared is taken off, is by detection layers described in step 4) Nitrocellulose filter with nature controlling line and detection line pastes on bottom plate, and smoothes out film surface；

5.4) the ready water absorption pad of step 5.2) is assembled on bottom plate, makes the left side of water absorption pad and the right end of detection layers There is the overlapping of 0.2cm, the right hand edge of water absorption pad is then aligned with the right hand edge of bottom plate and glues and smooth out；Again by knot described in step 2) It closes pad to be overlapped at the left edge of detection layers by 0.3cm, 0.3cm is sticked on bottom plate；

5.5) sample pad described in step 3) is then overlapped at the left edge of bonding pad by one side 0.3cm, another side with The left edge of bottom plate is aligned, and is sticked on bottom plate and is smoothed out；Assembled detection plate is cut into the detection of 4.0mm wide under cutting machine Card, 4 DEG C of hermetically dryings are kept in dark place.

Preferably, when kit provided by the present invention is the immune chromatography reagent kit based on colloidal gold-labeled method, The preparation method of the kit is:

1) colloidal gold labeled monoclonal antibody AbMB3Linearar:

1.1) 30nm colloidal gold solution is prepared:

99ml ultrapure water is added, by 1ml 1% (m/v) HAuCl in the 250ml triangular flask for taking a silication good₄Solution is added It is mixed in 250ml triangular flask and with ultrapure water, oil bath heating is simultaneously stirred to boiling；2ml is rapidly joined into 250ml triangular flask 1% (m/v) trisodium citrate aqueous solution, solution continue the 10min that boils, are changed into the solution in 250ml triangular flask by blue Stop heating when red, then the solution cooled to room temperature in 250ml triangular flask is added super into 250ml triangular flask Pure water polishing is to 100ml；

1.2) colloidal gold labeled monoclonal antibody AbMB3Linear:

1.2.1 colloidal gold solution prepared by 10ml step 1.1) is added in the 50ml triangular flask for) taking a silication good, to 240ul 0.2mol/L K is added in colloidal gold liquid₂CO₃Adjust pH to 8.5；

1.2.2) under magnetic stirrer, antibody A bMB3Linear is added in colloidal gold solution, until antibody is dense eventually Degree is 10ug/ml, is added dropwise when antibody is added, and continues to stir 45min~60min after adding；

1.2.3) reaction completes that 5% (m/v) bovine serum albumin(BSA) BSA to final concentration of 1% (m/v) is added, and stirring 15~ 30 minutes, 4 DEG C saved backup；

1.2.4 50ml centrifuge tube is packed into after) taking out the antibody A bMB3Linear marked, 2500g, 4 DEG C are centrifuged 5 points Clock obtains lower sediment and supernatant liquor, discards lower sediment, and supernatant liquor is transferred to another 50ml centrifuge tube, 12000g, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor, discard supernatant liquor, and lower sediment 10ml colloidal gold is buffered Precipitating is resuspended in liquid, then 12000g again, and 4 DEG C are centrifuged 30 minutes, obtains lower sediment and supernatant liquor again, most by lower sediment It is resuspended afterwards with 3ml colloidal gold buffer, 4 DEG C save backup, and obtain the solution containing colloidal gold labeled monoclonal antibody AbMB3Linear；

Each component content is respectively in the colloidal gold buffer: 10mM Tris, 1%m/vBSA, 1%v/v Tween- 20,5%m/v sucrose and 3 ‰ m/v polyvinylpyrrolidone PVP-10, the pH of the colloidal gold buffer are 10.5；

2) bonding pad is prepared

Polyester fiber film is immersed in the obtained solution containing colloidal gold labeled monoclonal antibody AbMB3Linear of step 1) 1h takes out, and is cut into after rear specification is 4cm × 0.6cm/ item after 25 DEG C of drying, and 4 DEG C are sealed spare, so far obtained bonding pad；

3) sample pad is prepared

It takes glass fibre element film one to open, glass fibre element film is impregnated at least 2h in sample pad treatment fluid, then be placed in life In object safety cabinet after 37 DEG C of aeration-dryings, specification is cut into after 4cm × 1.5cm/ item, to obtain sample pad, 25 DEG C of sealings are protected It deposits；

The preparation method of the sample pad treatment fluid be weigh 0.242g Tris, 1g bovine serum albumin(BSA) BSA, 1ml are spat Temperature -20,5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, are dissolved in the deionized water of 90ml, with 1mol/L NaOH 100ml is settled to deionized water after tune pH to 11；

4) detection layers are prepared

4.1) preparation of anti-MB protein polyclone antibody:

4.1.1) the clone of related gene

To 3 type Ureaplasma urealyticum MB albumen, (the accession number in its NCBI Protein Data Bank is AAC41437.1 bioinformatic analysis) is carried out, epitope peptide the most abundant in the extracellular conserved domain of its N-terminal is obtained Section finds the corresponding DNA encoding sequence of the peptide fragment, while introducing restriction enzyme site NdeI, 3 ' ends at 5 ' ends of this DNA encoding sequence (Jin Siruisheng is transferred in complete sequence synthesis to chemical synthesis complete genome sequence respectively after introducing termination signal TAA and restriction enzyme site XhoI Object Science and Technology Ltd. completes, and when delivery, artificial synthesized genetic fragment was respectively connected on carrier pUC57), it is denoted as MB3, Gene complete sequence is as shown in sequence table.The protein sequence of MB3 gene coding is natural 3 type Ureaplasma urealyticum memebrane protein MB The 30-150aa of (accession number:AAC41437.1).Its protein complete sequence is as shown in sequence table.The section will be contained Mesh is separately recovered after carrying out double digestion with NdeI and XhoI respectively in the carrier pUC57 of artificial synthesized DNA fragmentation according to a conventional method Segment, it is spare.Double digestion is carried out to carrier pET-28a (+) using NdeI and XhoI simultaneously, and according to a conventional method respectively will be through The MB3 gene obtained after double digestion is connected into pET-28a (+) carrier, and converts Escherichia coli TOP10, building pET-MB3 expression Carrier.Confirm that expression vector establishment is errorless through digestion and sequencing.Carrier expression recombination MB3-His fusion protein.

The purification step for recombinating MB3-His fusion protein is as follows:

2) it is balanced with 10mL sample-loading buffer, 1mL/min flow velocity.

3) by fusion protein loading, 1mL/min flow velocity.

4.1.3) the preparation of anti-MB protein polyclone antibody

4.2) detection layers are prepared

5) assembling detection card

5.1) bottom plate is cut into 4cm × 6cm size, it is spare；

5.2) absorbent filter is cut into 4cm × 2.5cm size, it is spare as water absorption pad；

5.4) the ready water absorption pad of step 5.2) is assembled on bottom plate, makes the left side of water absorption pad and the right end of detection layers There is the overlapping of 0.2cm, the right hand edge of water absorption pad is then aligned with the right hand edge of bottom plate and glues and smooth out；Again by knot described in step 2) It closes pad to be overlapped at the left edge of detection layers by 0.2cm, 0.4cm is sticked on bottom plate；

5.5) sample pad described in step 3) is then overlapped at the left edge of bonding pad by one side 0.2cm, another side with The left edge of bottom plate is aligned, and is sticked on bottom plate and is smoothed out；Assembled detection plate is cut into the detection of 4.0mm wide under cutting machine Card, 4 DEG C of hermetically dryings are kept in dark place.

The invention has the advantages that

The present invention provides anti-3 type Ureaplasma urealyticum MB protein antibodies, the anti-3 type Ureaplasma urealyticum MB protein antibodies identifications Linear epitope composed by 14 amino acid of 3 type Ureaplasma urealyticums MB protein 10 5-118,3 type Ureaplasma urealyticum MB Albumen is AAC41437.1 in GenBank sequence number；The ammonia of 14 amino acid of 3 type Ureaplasma urealyticums MB protein 10 5-118 Base acid sequence is KLPREPKPNEQLTI；By the sequence designations of MB protein 10 5-118 14 amino acid of 3 type Ureaplasma urealyticum For MB3Linear；Anti- 3 type Ureaplasma urealyticum MB protein antibodies are AbMB3Linear.Based on Ureaplasma urealyticum single linear antigen Above-mentioned 3 type Ureaplasma urealyticum MB protein antibodies of rabbit-anti prepared by epitope are good, with high purity with specificity, potency is high, is prepared into This cheap feature can be used for producing the highly sensitive detection Ureaplasma urealyticum of the various principles based on antigen-antibody reaction Detection kit.Meanwhile resisting 3 type Ureaplasma urealyticum MB protein antibodies that two different immunochromatographies have also been prepared based on this Kit.Two different immune chromatography reagent kits can quickly, accurately detect the Ureaplasma urealyticum in biological sample, include Antibody of the present invention；Two kinds of immune chromatography reagent kits are used equally for the auxiliary diagnosis of ureaplasma urealyticum infection, have higher Sensitivity and specificity, combined it is simple, quickly, the advantages such as stable and manufacturing cost is low, be suitable for clinical samples Inspection be also suitable for epidemiological survey and since large batch of quick inspection can be carried out.Therefore, of the present invention 3 type Ureaplasma urealyticum MB protein antibodies of rabbit-anti, two kinds of immune chromatography reagent kits all have broad application prospect and practical valence Value.

Specific embodiment

The present invention is further understood in order to facilitate those skilled in the art, it is detailed that preferred embodiment is cited below particularly Illustrate the present invention.

" m/v " in present specification in concentration unit, when m unit is g, v unit is mL, belongs to the normal of this field Know.

The source for a variety of materials that the present invention is used or used and the preparation of related reagent

1, sample pad treatment fluid: weighing 0.242g Tris, 1g bovine serum albumin(BSA) (BSA), 1ml Tween-20,5g sucrose, 0.3g polyvinylpyrrolidone (PVP-10), is dissolved in the deionized water of 90ml, with spending after 1mol/L NaOH tune pH to 11 Ionized water is settled to 100ml.

2, phosphate saves liquid: weighing 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 1g ox Seralbumin BSA and 0.1gNaN₃, it is dissolved in the deionized water of 90ml, with being spent after 1mol/L NaOH tune pH to 7.3 Ionized water is settled to 100ml；

3, phosphate buffer (PBS): weighing 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, It is dissolved in the deionized water of 90ml, is settled to 100ml with deionized water with after 1mol/L NaOH tune pH to 7.3.

4,0.0121g trishydroxymethylaminomethane, 0.17g sodium chloride, the dissolution of 0.025g lysozyme sample treatment liquid: are weighed In 90ml deionized water, 100ml is settled to deionized water with after hydrochloric acid tune pH to 8.0.

5, it antibody A bMB3Linear: for present invention self-control, is diluted, is shaken up with PBS, make antibody concentration 3mg/ in solution ml。

6, it anti-MB protein polyclone antibody: for present invention self-control, is diluted, is shaken up with PBS, make antibody concentration in solution 3mg/ml。

7, it goat anti-rabbit igg: for Wuhan Boster Biological Technology Co., Ltd.'s product, is diluted, is shaken up with PBS, made in solution Anti-TNF-α bulk concentration is 1mg/ml.

8, quantum dot: quantum dot used is water-soluble CdSe/ZnS quantum of carboxylated amphipathic polymer modification in the present invention Point, launch wavelength 565nm, from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., name of product is that carboxyl is water-soluble for purchase Property quantum dot -565.

9, glass fibre element film: with a thickness of 0.4mm, water absorption 42mg/cm², glass fiber diameter is 0.6-3 μm, tool There is good hydrophily, buys in Shanghai Jinbiao Bio-Tech Co., Ltd. (model BT40).

10, polyester fiber film: with a thickness of 0.48mm, absorption speed 18s/4cm, there is fabulous hydrophily, for tying The preparation of pad is closed, is bought in Shanghai Jinbiao Bio-Tech Co., Ltd. (model DL42).

11, nitrocellulose filter: model Millipore Corp SHF135 has liner plate, and purchase is in Millipore public affairs Department.

12, absorbent filter: with a thickness of 0.95mm, absorption speed 60s/4cm, water absorption 700mg/cm², have good Water imbibition, as production water absorption pad material.It buys in Shanghai Jinbiao Bio-Tech Co., Ltd. (model CH37K).

13, bottom plate: for high whiteness PVC material, surface is coated with single layer high polymer pressure sensitive adhesive SM31-40, buys in Shanghai Jin Biao Biotechnology Co., Ltd.

14, it Ureaplasma urealyticum: is purchased from American type culture collection (ATCC), number ATCC27815.

15, the microbiological specimens used in the present invention are purchased from American type culture collection (ATCC).

Technical solution provided by the present invention is described in detail below with reference to embodiment:

The preparation of 1 rabbit-anti of embodiment, 3 type Ureaplasma urealyticum MB albumen single-epitope antibody

3 type Ureaplasma urealyticum MB albumen single-epitope antibody of rabbit-anti the preparation method is as follows:

1) after structure biology analysis and Related Experimental Study, 3 type Ureaplasma urealyticum MB albumen (GenBank sequences are selected Row number AAC41437.1) 105-118 14 amino acid as preparation 3 type Ureaplasma urealyticum MB protein antibodies of rabbit-anti it is linear Epitope, this section of amino acid sequence are KLPREPKPNEQLTI, are MB3Linear by this sequence designations；

2) automatic with polypeptide after the C-terminal of amino acid sequence MB3Linear described in step 1) being added a cysteine Synthesizer synthesis polypeptide simultaneously purifies, polypeptide and carrier protein KLH after purification, forms MB3Linear-KLH compound protein；

3) compound protein synthesized by step 2) is emulsified, in rabbit subcutaneous abdomen multi-point injection after emulsification, successively injects three It is secondary, every minor tick 7-10 days；

4) after third time injection 10-12 days, collection isolated contains 3 type Ureaplasma urealyticum MB albumen list epitope of rabbit-anti The serum of antibody, ELISA detect the potency of 3 type Ureaplasma urealyticum MB albumen single-epitope antibody of rabbit-anti in serum, the antibody Potency is in 1:60000 or more；

5) step 2) is synthesized and the polypeptide purified and the Sepharose 4B of cyanogen bromide-activated is coupled, it is affine to form polypeptide Chromatographic column；

6) serum containing 3 type Ureaplasma urealyticum MB albumen single-epitope antibody of rabbit-anti that step 4) obtains is added to step 5) in the affinity column prepared, and after 4 DEG C are incubated overnight, antibody elution obtains 3 type Ureaplasma urealyticum MB albumen list of rabbit-anti Epitope antibodies；Identify that the antibody of this purifying 97% or more, is named as AbMB3Linear by its purity through SDS-PAGE.

Step 2) -6 in the present embodiment) it is all existing mature technology, Duo Jia biotechnology company can provide sequencing Technological service.In the present embodiment in above-mentioned steps related experiment link specific implementation, entrust Nanjing Jin Sirui biology section Skill Co., Ltd completes.

" antibody " of the present invention should be construed to cover any specificity of the binding structural domain with required specificity Binding factor.Thus, this term covers the function of antibody fragment homologous therewith, derivative, humanized antibody and antibody It can coordinate and homologue.The example of antibody is that immunoglobulin hypotype (such as IgG, IgE, IgM, IgD and IgA) and its hypotype are sub- Class；It is also possible to segment such as Fab, scFv, Fv, dAb, Fd and double-chain antibody comprising antigen-binding domains.

The preparation of the anti-MB protein polyclone antibody of embodiment 2:

1) clone of related gene

2) expression and purification of MB3-His fusion protein is recombinated

The purification step for recombinating MB3-His fusion protein is as follows:

A) it is filled distilled water with 5mL syringe, unscrews the plug of column, connected column with syringe with the connector of offer, Column is washed with 1mL/min flow velocity.

B) it is balanced with 10mL sample-loading buffer, 1mL/min flow velocity.

C) by fusion protein loading, 1mL/min flow velocity.

D) 10mL sample-loading buffer is used, column is washed with 1mL/min flow velocity.

E) 10mL elution buffer (20mM Na is used₃PO₄, 0.5M NaCl, 300mM imidazoles, pH7.4), it is flowed with 1mL/min Speed elution, is in charge of collection, and every pipe 1ml, 12%SDS-PAGE detection merge the sample containing destination protein in elution fraction.Through After bradford kit carries out determination of protein concentration, adjustment concentration is 0.2mg/mL.

3) preparation of anti-MB protein polyclone antibody

The preparation and application of immune chromatography reagent kit of the embodiment 3 based on quantum dot-labeled technology

1. quantum dot-labeled antibody A bMB3Linear

0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide (EDC) are sequentially added into microcentrifugal tube, With MES buffer (10.66g/L MES, 0.74g/L EDTA pH 7.4) constant volume for 1ml, ceaselessly mixed solution, 37 DEG C anti- After answering 5min, antibody A bMB3Linear prepared by the embodiment 1 of 0.34mg is added, 2h is protected from light, single-ended amino is added Polyethylene glycol (PEG2000-NH2) closes unreacted activated carboxyl site, continues to be protected from light to final concentration of 1% (m/v) 1h.Sample after reaction is centrifuged (molecular cut off 100k) with super filter tube, and 6500g is centrifuged 5min, until volume 200ul, by ultrafiltration Sample is transferred in common EP pipe afterwards, and centrifugation is except reunion (10000g, 3min).Upper clear supernate is added to splitter (Superdex- 200) purify on, flowed into cylinder naturally to it, then with PBS rinse (liquid flows down naturally), with ultraviolet light cylinder with When observe sample position, when sample start since lower part flow out when collect, collect 1ml after stop collect.It will after purification Sample is transferred to centrifugation in common EP pipe with super filter tube (molecular cut off 100k) after 6500g centrifugal concentrating to 200ul (10000g, 3min) is except reunion.Liquid is saved with phosphate after acquisition supernatant and dilutes 200 times, 4 DEG C save backup.So far it is made and contains There is the solution of quantum dot-labeled antibody A bMB3Linear.

2. the preparation of bonding pad

Polyester fiber film is immersed into 1h in the obtained solution containing quantum dot labelled antibody AbMB3Linear of step 1, It takes out, is cut into after 25 DEG C of drying after rear specification is 4cm × 0.6cm/ item, 4 DEG C are sealed spare, so far obtained bonding pad.

3. the preparation of sample pad

It takes glass fibre element film one to open, it is impregnated at least 3h in sample pad treatment fluid, then be placed in Biohazard Safety Equipment After 37 DEG C of aeration-dryings, specification is cut into obtain sample pad, 25 DEG C are sealed after 4cm × 2.5cm/ item.

4. the preparation of detection layers

Nitrocellulose filter is cut into 4cm × 4cm size；The anti-MB protein polyclone antibody that embodiment 2 is prepared Being adjusted with phosphate buffer to final concentration with goat anti-rabbit igg is respectively 2.0mg/mL and 1.0mg/mL；The rabbit-anti that will have been diluted Recombination P1-His fusion protein polyclonal antibody IgG is fitted into BIODOT and draws in film instrument spray head, and the amount of 1.0 μ l/cm of setting is sprayed on nitre On acid cellulose film, detection line is formed；The anti-rabbit IgG diluted is fitted into BIODOT to draw in film instrument spray head, 1.0 μ l/cm are set Amount be sprayed on nitrocellulose filter as nature controlling line, nature controlling line and detection line spacing are 0.7cm；The nitrocellulose that will have been sprayed 37 DEG C of film dry 2h, are cut into the specification of 4cm × 4cm, and 4 DEG C of hermetically dryings save；So far detection layers are made.

5. detecting the assembling of card

Bottom plate is cut into 4cm × 7.3cm size, it is spare.

Absorbent filter is cut into 4cm × 3cm size, it is spare as water absorption pad.

Assembly working first takes the adhered protection film on bottom plate off in operating in Biohazard Safety Equipment, will be described in step 4 Detection layers have nature controlling line and the nitrocellulose filter of detection line pastes on bottom plate, and carefully smooth out film surface.Secondly, by thing The water absorption pad first cut out is assembled on bottom plate, and having its left side and detection layers right end, 0.2cm's is overlapping, right hand edge then with bottom The right hand edge alignment of plate is glued and is carefully smoothed out.Bonding pad described in step 2 is overlapped in the left edge of detection layers by 0.3cm again Place, 0.3cm are sticked on bottom plate 7.Sample pad described in step 3 is finally then overlapped in by one side 0.3cm to the left edge of bonding pad Place, another side is aligned with the left edge of bottom plate, sticks on bottom plate and carefully smoothes out.Assembled detection plate is cut out under cutting machine At the detection card of 4.0mm wide, 4 DEG C of hermetically dryings are kept in dark place.

6. the composition of the immune chromatography reagent kit based on quantum dot-labeled technology

Immune chromatography reagent kit based on quantum dot-labeled technology detection card as described in step 5 and sample treatment liquid institute group At.

7. the application method of the immune chromatography reagent kit based on quantum dot-labeled technology

The 500 μ l of urine sample for obtaining person to be checked according to a conventional method, adds it to the modeling equipped with 500 μ l sample treatment liquids In expects pipe, be placed at room temperature for take out after ten minutes 120 μ L drops in detection card sample pad on, after 15 minutes under uv analyzer (model WD-9403A, Liuyi Instruments Plant, Beijing's production, burst of ultraviolel wavelength 365nm) observation testing result.If urine sample In contain Ureaplasma urealyticum antigen, then with the quantum dot-labeled antibody A bMB3Linear in bonding pad ining conjunction with, pass through chromatography work With elder generation in conjunction with the anti-MB protein polyclone antibody on nitrocellulose filter after will form at detection line under ultraviolet light excitation A macroscopic fluorescence detection line, the quantum dot-labeled antibody being not associated with continue chromatography in conjunction with goat anti-rabbit igg after Macroscopic Article 2 fluorescence nature controlling line is formed under ultraviolet light excitation；If only going out in urine sample to be checked without related antigen An existing fluorescence nature controlling line.If fluorescence nature controlling line does not occur, detection card failure.

8. the application effect of the immune chromatography reagent kit based on quantum dot-labeled technology is illustrated

The application method of the signified immune chromatography reagent kit based on quantum dot-labeled technology is referring to step 7 in the present embodiment The operating procedure.

1) specific test

With pathogenic pathogen such as people's mycoplasma pneumoniae (ATCC15531), legionella pneumophilia (ATCC 33152), people III Type parainfluenza virus viral (ATCC VR-93), stream of people's haemophilus influenza (ATCC 53781), chlamydia pneumoniae (AR-39 plants, ATCC number 53592), 3 type of adenovirus hominis (GB plants, ATCC number VR-3), 7 type of adenovirus hominis (Gomen plants, ATCC number VR- 7), influenza virus A hominis (H1N1, ATCC number VR-1743), people's influenza B virus (ATCC number VR-790), people's breathing Road syncytial virus (ATCC number VR26), streptococcus pneumonia (ATCC number 700670) etc. are detected instead of Ureaplasma urealyticum, Phosphate buffer dilution of the kit detection containing these microorganisms is all negative.

2) clinical trial example

Using Ureaplasma urealyticum detection " goldstandard "-cultivation as reference, the urine of 100 urological department urinary tract infection persons is taken Kit described in sample step 6 is detected, and cultivation positive rate is 26% (26/100), this kit is 25% (25/ 100), the coincidence rate of 2 kinds of methods is % (95/100).Concrete outcome is as shown in table 1.

The testing result of 1 clinical samples of table

The preparation and application of immune chromatography reagent kit of the embodiment 3 based on colloidal gold-labeled method

1. colloidal gold labeled monoclonal antibody AbMB3Linear

A.30nm the preparation of colloidal gold

The 250ml triangular flask that a silication is good is taken, 99ml ultrapure water is added, it is added in 1ml 1% (m/v) HAuCl4 solution Middle mixing, oil bath heating are simultaneously stirred to boiling.Rapidly join 2ml 1% (m/v) trisodium citrate aqueous solution thereto, solution after Continuous boiling 10min (solution is changed into red by blue during this).Stop heating, allow solution cooled to room temperature, so Ultrapure water polishing is added thereto afterwards to 100ml.

B. colloidal gold labeled monoclonal antibody AbMB3Linear

1) the 50ml triangular flask that a silication is good is taken, colloidal gold liquid prepared by 10ml step a is added, is added into golden liquid 240ul 0.2mol/L K₂CO₃Adjust pH to 8.5；

2) under magnetic stirrer, antibody A bMB3Linear is added in colloidal gold solution, until antibody is final concentration of 10ug/ml should be added dropwise when antibody is added, and continue to stir 45min~60min after adding；

3) reaction completes that 5% (m/v) bovine serum albumin(BSA) (BSA) to final concentration of 1% (m/v), stirring 15~30 is added Minute, 4 DEG C save backup.

4) 50ml centrifuge tube is packed into after taking out the antibody A bMB3Linear marked, 2500g, 4 DEG C are centrifuged 5 minutes, obtain To lower sediment and supernatant liquor, discard lower sediment, supernatant liquor is transferred to another 50ml centrifuge tube, 12000g, 4 DEG C from The heart 30 minutes, lower sediment and supernatant liquor are obtained, supernatant liquor is discarded, lower sediment 10ml colloidal gold buffer is resuspended Precipitating, then 12000g again, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor again, lower sediment is finally used 3ml colloidal gold buffer is resuspended, and 4 DEG C save backup, and obtain the solution containing colloidal gold labeled monoclonal antibody AbMB3Linear；

Each component content is respectively in above-mentioned colloidal gold buffer: 10mM Tris, 1% (m/v) BSA, 1% (v/v) Tween-20,5% (m/v) sucrose and 3 ‰ (m/v) polyvinylpyrrolidone PVP-10, the pH of the colloidal gold buffer are 10.5。

2. the preparation of bonding pad

Polyester fiber film is immersed into 1h in the obtained solution containing colloidal gold labeled monoclonal antibody AbMB3Linear of step 1, It takes out, is cut into after 25 DEG C of drying after rear specification is 4cm × 0.6cm/ item, 4 DEG C are sealed spare, so far obtained bonding pad.

3. the preparation of sample pad

It takes glass fibre element film one to open, it is impregnated at least 2h in sample pad treatment fluid, then be placed in Biohazard Safety Equipment After 37 DEG C of aeration-dryings, specification is cut into obtain sample pad, 25 DEG C are sealed after 4cm × 2.5cm/ item.

4. the preparation of detection layers

5. detecting the assembling of card

Bottom plate is cut into 4cm × 6cm size, it is spare.

Absorbent filter is cut into 4cm × 2.5cm size, it is spare as water absorption pad.

Assembly working first takes the adhered protection film on bottom plate off in operating in Biohazard Safety Equipment, will be described in step 4 Detection layers have nature controlling line and the nitrocellulose filter of detection line pastes on bottom plate, and carefully smooth out film surface.Secondly, by thing The water absorption pad first cut out is assembled on bottom plate, and having its left side and detection layers right end, 0.2cm's is overlapping, right hand edge then with bottom The right hand edge alignment of plate is glued and is carefully smoothed out.Bonding pad described in step 2 is overlapped in the left side of detection layers 3 by 0.2cm again At edge, 0.4cm is sticked on bottom plate.Sample pad described in step 3 is finally then overlapped in by one side 0.2cm to the left edge of bonding pad Place, another side is aligned with the left edge of bottom plate, sticks on bottom plate and carefully smoothes out.Assembled detection plate is cut out under cutting machine At the detection card of 4.0mm wide, 4 DEG C of hermetically dryings are kept in dark place.

6. the composition of the immune chromatography reagent kit based on colloidal gold-labeled method

Based on the immune chromatography reagent kit of colloidal gold-labeled method detection card as described in step 5 and sample treatment liquid institute group At.

7. the application method of the immune chromatography reagent kit based on colloidal gold-labeled method

The 500 μ l of urine specimen for obtaining person to be checked according to a conventional method, adds it to the modeling equipped with 500 μ l sample treatment liquids In expects pipe, after 50 DEG C of water-bath 20min, rear 120 μ L drops of taking out visually observe after 15 minutes in the sample pad of detection card and detect knot Fruit.If containing Ureaplasma urealyticum antigen in urine specimen, tied with the antibody A bMB3Linear of the colloid gold label in bonding pad It closes, will form at detection line after acting on elder generation in conjunction with the anti-MB protein polyclone antibody on nitrocellulose filter by chromatography Macroscopic one red detection line, the colloidal gold labeled monoclonal antibody being not associated with continue chromatography rear shape in conjunction with goat anti-rabbit igg At macroscopic Article 2 red nature controlling line；If only there is a red Quality Control without related antigen in urine specimen to be checked Line.If red nature controlling line does not occur, detection card failure.

8. the application effect of the immune chromatography reagent kit based on colloidal gold-labeled method is illustrated

The application method of the signified immune chromatography reagent kit based on colloidal gold-labeled method is referring to step 7 in the present embodiment The operating procedure.

1) specific test

With common causative such as legionella pneumophilia (ATCC 33152), I type human parainfluenza viruses (ATCC VR-94), II type Human parainfluenza viruses (ATCC VR-92), type III human parainfluenza viruses' virus (ATCC VR-93), stream of people's haemophilus influenza (ATCC 53781), chlamydia pneumoniae (AR-39 plants, ATCC number 53592), 3 type of adenovirus hominis (GB plants, ATCC number VR- 3), 7 type of adenovirus hominis (Gomen plants, ATCC number VR-7), influenza virus A hominis (H1N1, ATCC number VR-1743), people (ATCC is compiled for influenza B virus (ATCC number VR-790), human respiratory syncytial virus (ATCC number VR26), streptococcus pneumonia Etc. number 700670) detected instead of Ureaplasma urealyticum, kit detects the phosphate buffer dilution containing these microorganisms It is all feminine gender.

2) clinical trial example

Using Ureaplasma urealyticum detection " goldstandard "-cultivation as reference, the urine of 100 urological department urinary tract infection persons is taken Kit described in sample step 6 is detected, and cultivation positive rate is 26% (26/100), this kit is 24% (24/ 100), the coincidence rate of 2 kinds of methods is 96% (96/100).Concrete outcome is as shown in table 1.

The testing result of 1 clinical samples of table

It should be pointed out that the foregoing is merely illustrative of the preferred embodiments of the present invention, it is not intended to limit the invention, it is all Any modification, equivalent replacement for being made within spirit of that invention and principle etc. should be included in protection scope of the present invention it It is interior.

Claims

1. one kind is formed by immune chromatography reagent kit based on anti-3 type Ureaplasma urealyticum MB protein antibodies, it is characterised in that: described Kit is the immune chromatography reagent kit based on quantum dot-labeled technology or the immunochromatography reagent based on colloidal gold-labeled method Box；

Anti- 3 type Ureaplasma urealyticum MB protein antibodies are AbMB3Linearar, and the preparation method of AbMB3Linearar is:

1) after structure biology analysis and Related Experimental Study, 14 of selected 3 type Ureaplasma urealyticum MB protein 10 5-118 Linear epitope of the amino acid as preparation 3 type Ureaplasma urealyticum MB protein antibodies of rabbit-anti, this section of amino acid sequence are This sequence designations is MB3Linear by KLPREPKPNEQLTI；

2) it after the C-terminal of amino acid sequence MB3Linear described in step 1) being added a cysteine, is automatically synthesized with polypeptide Instrument synthesis polypeptide simultaneously purifies, polypeptide and carrier protein KLH after purification, forms MB3Linear-KLH compound protein；

3) compound protein synthesized by step 2) is emulsified, in rabbit subcutaneous abdomen multi-point injection after emulsification, is successively injected three times, often Minor tick 7-10 days；

4) after third time injection 10-12 days, collection isolated contains 3 type Ureaplasma urealyticum MB albumen single-epitope antibody of rabbit-anti Serum, ELISA detect serum in 3 type Ureaplasma urealyticum MB albumen single-epitope antibody of rabbit-anti potency, the potency of the antibody In 1:60000 or more；

5) step 2) is synthesized and the polypeptide purified and the Sepharose4B of cyanogen bromide-activated is coupled, form polypeptide affinity chromatography Column；

6) serum containing 3 type Ureaplasma urealyticum MB albumen single-epitope antibody of rabbit-anti that step 4) obtains is added to step 5) system In standby affinity column, and after 4 DEG C are incubated overnight, antibody elution obtains 3 type Ureaplasma urealyticum MB albumen list epitope of rabbit-anti Antibody；Identify that the antibody of this purifying 97% or more, is named as AbMB3Linear by its purity through SDS-PAGE；

When the kit is the immune chromatography reagent kit based on quantum dot-labeled technology, the preparation method of the kit is:

1) quantum dot-labeled antibody A bMB3Linearar:

0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide EDC are sequentially added into microcentrifugal tube, it is slow with MES Fliud flushing constant volume is 1ml, and mixed solution after 37 DEG C of reaction 5min, adds the antibody of 0.34mg being prepared AbMB3Linearar is protected from light 2h, and single-ended amino-polyethyleneglycols PEG2000-NH2 to final concentration of 1%m/v is added, closing Unreacted activated carboxyl site, continues to be protected from light 1h；Sample after reaction is centrifuged with super filter tube, and 6500g is centrifuged 5min, until Sample after ultrafiltration is transferred in common EP pipe by volume 200ul, and centrifugation obtains upper clear supernate and lower part precipitating except reuniting, 3min is centrifuged under the conditions of 10000g；Upper clear supernate is added on splitter Superdex-200 and is purified, is flowed naturally to upper clear supernate Enter in cylinder, then rinsed with PBS, the position of sample is observed with ultraviolet light cylinder, when sample starts to flow out from lower part Start to collect, stops collecting after collecting 1ml；By sample after purification with super filter tube to be shifted after 6500g centrifugal concentrating to 200ul Interior centrifugation is managed except reuniting to common EP, and the condition being centrifuged to common EP pipe is 10000g, 3min；With phosphoric acid after acquisition supernatant Salt saves liquid and dilutes 200 times, and 4 DEG C save backup；So far the solution containing quantum dot labelled antibody AbMB3Linearar is made；

Each component content is respectively in the MES buffer: 10.66g/L MES and 0.74g/L EDTA, the MES buffering The pH7.4 of liquid；

The preparation method that the phosphate saves liquid is to weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g chlorine Change sodium, 1g bovine serum albumin(BSA) BSA and 0.1gNaN₃, it is dissolved in the deionized water of 90ml, extremely with 1mol/L NaOH tune pH 100ml is settled to deionized water after 7.3；

2) bonding pad is prepared

Polyester fiber film is immersed into 1h in the obtained solution containing quantum dot labelled antibody AbMB3Linearar of step 1), It takes out, is cut into after 25 DEG C of drying after rear specification is 4cm × 0.6cm/ item, 4 DEG C are sealed spare, so far obtained bonding pad；

3) sample pad is prepared

It takes glass fibre element film one to open, glass fibre element film is impregnated at least 3h in sample pad treatment fluid, then be placed in biological peace In full cabinet after 37 DEG C of aeration-dryings, specification is cut into obtain sample pad, 25 DEG C are sealed after 4cm × 2.5cm/ item；

The preparation method of the sample pad treatment fluid is to weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g chlorine Change sodium, 2g bovine serum albumin(BSA) BSA, 1ml Tween-20,2g sucrose and 0.5g polyvinylpyrrolidone PVP-10, is dissolved in In the deionized water of 90ml, 100ml is settled to deionized water with after 1mol/L NaOH tune pH to 7.3；

4) detection layers are prepared

4.1) preparation of anti-MB protein polyclone antibody:

4.1.1) the clone of related gene

Bioinformatic analysis is carried out to 3 type Ureaplasma urealyticum MB albumen, obtains the 3 extracellular guarantors of type Ureaplasma urealyticum MB albumen n end The peptide fragment the most abundant of epitope in structural domain is kept, finds the corresponding DNA encoding sequence of the peptide fragment, while in this DNA encoding Chemical synthesis is complete respectively after 5 ' end introducing restriction enzyme site NdeI, 3 ' the end introducing termination signal TAA and restriction enzyme site XhoI of sequence Gene order is denoted as MB3；The 30- that the protein sequence of MB3 gene coding is natural 3 type Ureaplasma urealyticum memebrane protein MB 150aa；By normal after carrier pUC57 containing this section of artificial synthesized DNA fragmentation is carried out double digestion with NdeI and XhoI respectively Target fragment is separately recovered in rule method, spare；Double digestion is carried out to carrier pET-28a (+) using NdeI and XhoI simultaneously, and is pressed The MB3 gene obtained after double digestion is connected into pET-28a (+) carrier by conventional method respectively, and converts Escherichia coli TOP10 constructs pET-MB3 expression vector；Confirm that expression vector establishment is errorless through digestion and sequencing；Carrier expression recombination MB3-His fusion protein；

Plasmid will be extracted after identifying correct positive colony bacterium culture, routinely technology is transferred to competence E.coli BL21 (DE3) In, bacterium solution is coated on the LB plate containing 50 μ g/mL kanamycins after the completion of conversion, according to a conventional method screening expression bacterial strain； The single bacterium colony with exogenous protein expression ability and inoculation that picking pET-MB3 is converted enter in 100mL LB culture medium, in 37 DEG C overnight incubation；After taking out bacterium solution, 100mL is inoculated in by 1:100 and is contained in the LB culture medium of 50 μ g/mL kanamycins, in 37 DEG C culture is to OD₆₀₀When=0.6,1mol/L IPTG to final concentration of 1mmol/L is added, bacterium culture, induced fusion are shaken in 37 DEG C Protein expression；10min collection thallus is centrifuged under 8000r/min after inducing 4h；This thallus is washed with 20mL phosphate buffer It washs 3 times and carries out ultrasonication, operating condition after being resuspended with 10mL sample-loading buffer are as follows: 50HZ, 200W, ultrasonic 3S, interval 5S, Work 100 times；After the completion of ultrasound, 12000g centrifugation 15min carries out electrophoresis detection after collecting precipitating and supernatant respectively；It was found that recombination Albumen MB3-His is present in thallus in a manner of soluble；

Each component content is respectively in the sample-loading buffer: 20mM Na₃PO₄, 0.5M NaCl and 30mM imidazoles；On described The pH7.4 of sample buffer；

The purification step for recombinating MB3-His fusion protein is as follows:

His Trap affinity is used after the ultrasonication supernatant of above-mentioned acquisition is filtered with 0.45 μm of filter membrane Columns is purified with same method to specifications；The specific method is as follows:

A) it is filled distilled water with 5mL syringe, unscrews the plug of column, connected column with syringe with the connector of offer, with 1mL/min flow velocity washes column；

B) it is balanced with 10mL sample-loading buffer, 1mL/min flow velocity；

C) by fusion protein loading, 1mL/min flow velocity；

D) 10mL sample-loading buffer is used, column is washed with 1mL/min flow velocity；

E) 10mL elution buffer is used, with the elution of 1mL/min flow velocity, is in charge of collection, every pipe 1ml, 12%SDS-PAGE detection are closed And contain the sample of destination protein in elution fraction；After bradford kit carries out determination of protein concentration, concentration is adjusted For 0.2mg/mL；

Each component content is respectively in the elution buffer: 20mM Na₃PO₄, 0.5M NaCl and 300mM imidazoles is described to wash The pH7.4 of de- buffer；

4.1.3) the preparation of anti-MB protein polyclone antibody

Hero is immunized after mixing emulsification according to 200 μ g and 1mL Freund's complete adjuvant with the recombination MB3-His fusion protein of above-mentioned purifying Property new zealand white rabbit, in dorsal sc multi-point injection, be spaced after 7d be immunized again it is primary, with the weight of above-mentioned purifying after 14d Group P1-His fusion protein carries out booster immunization, booster immunization 7d after mixing emulsification according to 200 μ g and 1mL incomplete Freund's adjuvant Booster immunization is primary again in same way as described above again afterwards；Haemanalysis antibody titer is taken after 7d；Until antibody titer indirect ELISA Method measures antibody titer and is greater than 1 × 10⁵Culling heart blood afterwards separates serum, with Protein G affinity column, in strict accordance with behaviour Book purified polyclonal antibodies IgG is explained, measure antibody concentration with triumphant base Braford protein content detection kit and uses phosphoric acid Salt buffer is adjusted to 1mg/mL, and -20 DEG C of preservations are spare, and anti-MB protein polyclone antibody is so far made；

4.2) detection layers are prepared

Nitrocellulose filter is cut into 4cm × 4cm size；The anti-MB protein polyclone antibody that step 4.1) is prepared and sheep It is respectively 2.0mg/mL and 1.0mg/mL that anti-rabbit IgG, which is adjusted with phosphate buffer to final concentration,；The anti-MB albumen that will have been diluted Polyclonal antibody is fitted into BIODOT and draws in film instrument spray head, and the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter, forms detection Line；The anti-rabbit IgG diluted is fitted into BIODOT to draw in film instrument spray head, the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter As nature controlling line, nature controlling line and detection line spacing are 0.7cm；37 DEG C of the nitrocellulose filter dry 2h that will have been sprayed, are cut into 4cm The specification of × 4cm, 4 DEG C of hermetically dryings save；So far detection layers are made；

The preparation method of the phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate and 0.2g sodium chloride, is dissolved in the deionized water of 90ml, is settled to after 1mol/L NaOH tune pH to 7.3 with deionized water 100ml；

5) assembling detection card

5.1) bottom plate is cut into 4cm × 7.3cm size, it is spare；

5.3) adhered protection film on bottom plate that step 5.1) is prepared is taken off, detection layers described in step 4) is had Nature controlling line and the nitrocellulose filter of detection line paste on bottom plate, and smooth out film surface；

5.4) the ready water absorption pad of step 5.2) is assembled on bottom plate, there is the left side of water absorption pad with the right end of detection layers The overlapping of 0.2cm, the right hand edge of water absorption pad are then aligned with the right hand edge of bottom plate and glue and smooth out；Again by combination described in step 2) Pad is overlapped at the left edge of detection layers by 0.3cm, and 0.3cm is sticked on bottom plate；

5.5) sample pad described in step 3) is then overlapped at the left edge of bonding pad by one side 0.3cm, another side and bottom plate Left edge alignment, stick on bottom plate and smooth out；Assembled detection plate is cut into the detection card of 4.0mm wide under cutting machine, 4 DEG C hermetically drying is kept in dark place；

When the kit is the immune chromatography reagent kit based on colloidal gold-labeled method, the preparation method of the kit is:

1) colloidal gold labeled monoclonal antibody AbMB3Linearar:

1.1) 30nm colloidal gold solution is prepared:

99ml ultrapure water is added, by 1ml1%m/v HAuCl in the 250ml triangular flask for taking a silication good₄250ml tri- is added in solution It is mixed in the bottle of angle and with ultrapure water, oil bath heating is simultaneously stirred to boiling；2ml1%m/v lemon is rapidly joined into 250ml triangular flask Lemon three sodium water solutions of acid, solution continue the 10min that boils, the stopping when solution in 250ml triangular flask is changed into red by blue Then ultrapure water polishing is added extremely into 250ml triangular flask in solution cooled to room temperature in 250ml triangular flask by heating 100ml；

1.2) colloidal gold labeled monoclonal antibody AbMB3Linear:

1.2.1 colloidal gold solution prepared by 10ml step 1.1) is added, to colloid in the 50ml triangular flask for) taking a silication good 240ul0.2mol/L K is added in golden liquid₂CO₃Adjust pH to 8.5；

1.2.2) under magnetic stirrer, antibody A bMB3Linear is added in colloidal gold solution, until antibody is final concentration of 10ug/ml is added dropwise when antibody is added, and continues to stir 45min~60min after adding；

1.2.3) reaction completion addition 5%m/v bovine serum albumin(BSA) BSA to final concentration of 1%m/v, stirring 15~30 minutes, 4 It DEG C saves backup；

1.2.4 50ml centrifuge tube is packed into after) taking out the antibody A bMB3Linear marked, 2500g, 4 DEG C are centrifuged 5 minutes, obtain To lower sediment and supernatant liquor, discard lower sediment, supernatant liquor is transferred to another 50ml centrifuge tube, 12000g, 4 DEG C from The heart 30 minutes, lower sediment and supernatant liquor are obtained, supernatant liquor is discarded, lower sediment 10ml colloidal gold buffer is resuspended Precipitating, then 12000g again, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor again, lower sediment is finally used 3ml colloidal gold buffer is resuspended, and 4 DEG C save backup, and obtain the solution containing colloidal gold labeled monoclonal antibody AbMB3Linear；

Each component content is respectively in the colloidal gold buffer: 10mM Tris, 1%m/vBSA, 1%v/v Tween-20, 5%m/v sucrose and 3 ‰ m/v polyvinylpyrrolidone PVP-10, the pH of the colloidal gold buffer are 10.5；

2) bonding pad is prepared

Polyester fiber film is immersed into 1h in the obtained solution containing colloidal gold labeled monoclonal antibody AbMB3Linear of step 1), is taken Out, be cut into after 25 DEG C of dryings rear specification be 4cm × 0.6cm/ item after, 4 DEG C be sealed it is spare, so far be made bonding pad；

3) sample pad is prepared

It takes glass fibre element film one to open, glass fibre element film is impregnated at least 2h in sample pad treatment fluid, then be placed in biological peace In full cabinet after 37 DEG C of aeration-dryings, specification is cut into obtain sample pad, 25 DEG C are sealed after 4cm × 1.5cm/ item；

The preparation method of the sample pad treatment fluid be weigh 0.242g Tris, 1g bovine serum albumin(BSA) BSA, 1ml Tween-20, 5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, are dissolved in the deionized water of 90ml, extremely with 1mol/L NaOH tune pH 100ml is settled to deionized water after 11；

4) detection layers are prepared

4.1) preparation of anti-MB protein polyclone antibody:

4.1.1) the clone of related gene

Bioinformatic analysis is carried out to 3 type Ureaplasma urealyticum MB albumen, obtains the 3 extracellular guarantors of type Ureaplasma urealyticum MB albumen n end The peptide fragment the most abundant of epitope in structural domain is kept, finds the corresponding DNA encoding sequence of the peptide fragment, while in this DNA encoding Chemical synthesis is complete respectively after 5 ' end introducing restriction enzyme site NdeI, 3 ' the end introducing termination signal TAA and restriction enzyme site XhoI of sequence Gene order is denoted as MB3；The 30- that the protein sequence of MB3 gene coding is natural 3 type Ureaplasma urealyticum memebrane protein MB 150aa；Its protein complete sequence is as shown in sequence table；Carrier pUC57 containing this section of artificial synthesized DNA fragmentation is used respectively Target fragment is separately recovered after carrying out double digestion in NdeI and XhoI according to a conventional method, spare；Simultaneously using NdeI and XhoI to load Body pET-28a (+) carries out double digestion, and the MB3 gene obtained after double digestion is connected into pET-28a respectively according to a conventional method In (+) carrier, and Escherichia coli TOP10 is converted, constructs pET-MB3 expression vector；Confirm that expression carries through digestion and sequencing Body building is errorless；Carrier expression recombination MB3-His fusion protein；

The purification step for recombinating MB3-His fusion protein is as follows:

B) it is balanced with 10mL sample-loading buffer, 1mL/min flow velocity；

C) by fusion protein loading, 1mL/min flow velocity；

4.1.3) the preparation of anti-MB protein polyclone antibody

4.2) detection layers are prepared

5) assembling detection card

5.1) bottom plate is cut into 4cm × 6cm size, it is spare；

5.4) the ready water absorption pad of step 5.2) is assembled on bottom plate, there is the left side of water absorption pad with the right end of detection layers The overlapping of 0.2cm, the right hand edge of water absorption pad are then aligned with the right hand edge of bottom plate and glue and smooth out；Again by combination described in step 2) Pad is overlapped at the left edge of detection layers by 0.2cm, and 0.4cm is sticked on bottom plate；

5.5) sample pad described in step 3) is then overlapped at the left edge of bonding pad by one side 0.2cm, another side and bottom plate Left edge alignment, stick on bottom plate and smooth out；Assembled detection plate is cut into the detection card of 4.0mm wide under cutting machine, 4 DEG C hermetically drying is kept in dark place.