CN108586612A

CN108586612A - A kind of preparation method for the monoclonal antibody being suitable for detecting human peripheral expression CD161 molecule subgroup lymphocytes

Info

Publication number: CN108586612A
Application number: CN201810304106.9A
Authority: CN
Inventors: 陈心春; 黄萍; 郭永超
Original assignee: SHENZHEN UNI-MEDICA TECHNOLOGY Co Ltd
Current assignee: SHENZHEN UNI-MEDICA TECHNOLOGY Co Ltd
Priority date: 2018-03-27
Filing date: 2018-04-08
Publication date: 2018-09-28

Abstract

A kind of preparation method being suitable for detect the monoclonal antibody that human peripheral expresses CD161 molecule subgroup lymphocytes, it belongs to a kind of hybridoma cell strain and monoclonal antibody technique field, specific steps include：It is prepared by the hybridoma cell strain of stably excreting CD161 monoclonal antibodies；The preparation purifying and verification of 161 protein monoclonal antibody of mouse-anti-human T lymphocyte CD of hybridoma secretion；Fluorescent molecular (such as APC is further made in mouse anti-human monoclonal's antibody of acquisition, FITC, PE) the straight labeling antibody of streaming, T lymphocytes for detecting expression CD161 molecules, streaming antibody, creative activity tuberculosis and tuberculosis latent infection flow cytometer detection kit are detected in conjunction with other lymphocytes.The anti-human CD161 antibody titers that the method for the present invention is obtained are high, specific very good, can directly mark a variety of tracers, by currently advanced detecting instrument, such as：Flow cytometer, chemiluminescence detector, magnetic separator etc. carry out the positive-selecting of CD161 expression cells, there is very positive meaning for prevention and control field lungy.

Description

A kind of list being suitable for detecting human peripheral expression CD161 molecule subgroup lymphocytes The preparation method of clonal antibody

Technical field：

The present invention relates to a kind of monoclonal antibodies for being suitable for detecting human peripheral and expressing CD161 molecule subgroup lymphocytes Preparation method, it belongs to a kind of hybridoma cell strain and monoclonal antibody technique field.

Background technology：

Although numerous scientific and technical personnel have been working hard point of the research various hypotype Expressions In Lymphocytes of human peripheral in recent years Son, these molecular studies have important relationship with lymphocyte quantity detection under various physiological status, and there are also correlative studys Report common Lymphocyte surface molecules, such as CD45, CD8, CD3, CD19, CD20.But also have some protein moleculars, Since current clinical detection meaning is less or uncertain, so its research is relatively fewer, the CD161 as this patent is related to divides Son belongs to c-type agglutinin superfamily, expresses in natural natural kill (NK) cell and T lymphocytic cell surfaces.CD161 is expressed It is thin in Memorability/functionality CD4+T cells, CD8+T cells, TCR γ T cells, part CD3+ thymocytes and Th17CD4+T Born of the same parents.Although the function of current CD161, there is no defining, some researches show that CD161 expression on Treg cells recently, CD161+Treg cells express the cytokine profiles such as IFN γ, IL-17, IL-2, and are expressed in inflammation part height.In recent years new Research also have article had studied compared with system and clearly CD161 expression T lymphocytes have in active tuberculosis patient it is bright Aobvious reduction, therefore CD161 albumen is very promising as Diagnosis of Tuberculosis marker.

Tuberculosis (Tuberculosis, TB) is chronic infectious disease caused by being infected by mycobacterium tuberculosis, tuberculosis Mycobacteria can not only cause pulmonary tuberculosis (85%), but also can cause the tuberculosis of a variety of organs outside lung.Although existing at present have The antituberculotic of effect, but tuberculosis is still the number one killer of current infectious diseases.Global annual about 2,000,000 people die of knot Core disease；The population of global about one third has infected mycobacterium tuberculosis, is referred to as tulase latent infection (LTBI) at these Crowd in, there is about 10% will finally progress to active tuberculosis.

Due to lacking effective tuberculosis vaccine at present, prevention and control lungy depend on early detection, treatment Or isolation active tuberculosis patient.However, diagnostic activities detection technique lungy cannot meet clinical and tuberculosis now The requirement of prevention and control, there are following wretched insufficiencies：(1) phlegm mycobacterium tuberculosis microorganism checking specificity is high, is currently to examine The goldstandard of disconnected active tuberculosis, but that there are sensibility is low (less than 40%), and time-consuming, and (tulase culture takes 1-2 Month), Laboratory biosafety requires high disadvantage.(2) M. tuberculosis genes detect, although realizing the mesh of quick diagnosis (1 day), but the genetic test directly carried out from sputum specimen does not significantly improve in terms of sensibility, and there are false negative or vacations Positive problem.(3) antibody test assert unsuitable diagnosis lungy by the World Health Organization in immunology detection；Cell is exempted from Epidemiology detection includes tuberculin skin test (TST) and tulase interferon release test (IGRA), cannot effectively distinguish activity Tubercular and tulase latent infection person, although the sensibility that the latter detects in active tuberculosis patient is significantly higher than other inspections It surveys.

In conclusion ratio of the CD161 protein expressing cells subgroups in lymphocyte, active tuberculosis patient can be apparent Less than tulase latent infection person and healthy population.Therefore CD161 protein moleculars can be used as the new target of diagnosis of tuberculosis Point molecule, for distinguishing active tuberculosis and tuberculosis latent infection, to more targetedly take tuberculosis treatment and every From measure.In addition, main attention has been placed on tuberculotherapy, blocked by pulmonary tuberculosis field, domestic and international scientific research institution, clinical hospitals Infection, the prevention of tubercular drugs hepatotoxicity wind agitation etc., so particularly important for filling up establishing for Precise Diagnosis method lungy.

Invention content：

The purpose of the present invention is to provide a kind of CD161 molecule subgroup lymphocytes are expressed suitable for detecting human peripheral The preparation method of monoclonal antibody is established with obtaining the CD161 monoclonal antibodies of high-titer, high specific using the monoclonal Antibody, the method by expressing CD161 molecular isoform cell proportions in Flow cytometry human peripheral lymphocyte, into And establish a kind of flow cytometer detection kit that can be clinically used for distinguishing active tuberculosis and tuberculosis latent infection.

The object of the present invention is achieved like this：

First：The preparation method (one) that human peripheral expresses the monoclonal antibody of CD161 molecule subgroup lymphocytes is stablized It is prepared by the hybridoma cell strain for secreting CD161 monoclonal antibodies：

(1) immunogene is prepared：

Utilize the MonoExpress Gold Antibody Service (mouse) of the bio tech ltd Jin Sirui Service prepares CD161 immunogen proteins using the method for DNA artificial sequence synthetic and prokaryotic expression protein；

Using the method for DNA artificial sequence synthetic prokaryotic expression protein by following concrete operation step in above-mentioned steps (1) Composition：

The DNA of a.CD161 extracellular domains is synthesized.

From the bio tech ltd Jin Sirui synthesis people CD161 (gi | 4504879 | ref | NP_002249.1 |) it is extracellular The encoding gene in domain (AA67-225) is inserted into the prokaryotic expression carrier E3 of the said firm, to realize its packet in Escherichia coli It is reached containing body surface.Albumen end is there are six histidine (His) label, ni-sepharose purification and the Western detection for albumen.

Synthetic gene sequence must pass through sequence verification, and totally 480 bases, particular sequence are as follows：

The sequence expression theoretical amino acid sequence, totally 160 amino acid add 6 histidines, particular sequence as follows：

The isoelectric point of albumen theory	The molecular weight of albumen theory
		7.81	19444.8Da

The Prokaryotic expression, purification of b.CD161 immunogen proteins and verification.

E3 plasmids with CD161 genes are converted into Escherichia coli, IPTG induced fusion protein expressions, ultrasonic method is crushed bacterium Body, CD161 immunogen protein of the nickel column method purifying with His labels.Utilize SDS-PAGE gel electrophoresises and Western Blot Detection, gel images processing system analyze the purity and size of protein.Purity of protein requires to reach 90% or more, it is believed that closes Lattice, the animal immune after can be used for.

(2) animal immune：

The CD161 immunogen proteins (90% or more purity) of purifying are mixed with Freund's adjuvant, animal selection and institute is immunized With the homologous BALB/c healthy mices three of myeloma cell, age of mouse was at 8-14 weeks, and abdominal part hypodermic, every immune every time 100 μ g albumen.Altogether it is immune three times, be spaced three weeks per between twice.Second of immune posterior orbit vein is used from 100 μ l of blood sampling Enzyme-linked immunosorbent assay (abbreviation ELSA) method measures antiserum, and antibody titer, which is equal to, is higher than 1:1000, it is believed that immune success rate, The highest mouse of titre is selected to be used for the preparation of hybridoma.

Described includes following operating procedure using ELSA methods measurement antiserum method：

A. it is coated with：It is dense that CD161 immunogen proteins in abovementioned steps (1) with phosphate buffer are configured to 1 μ g/ml Solution is spent, 100 μ l/well are coated with 96 hole polyethylene boards, and vacuum is drained, and 4 DEG C of sealing saves backup.

B. it closes：The 200 μ l of phosphate buffer that pH is 7.4 are added per hole to wash, include 1% lowlenthal serum.

C. it is loaded：Be added per hole third time it is immune after 3 days clear 50-100 μ l of mouse peripheral blood (1:1000-5000 is dilute Release), normal control, positive control and blank (phosphate buffer), 37 DEG C of incubation 1h, phosphate buffer washing 3 are set per plate It is secondary.

D. ELIAS secondary antibody is added per hole 100-200 μ l, washs.

E. it develops the color：Substrate 50-100 μ l are added per hole, are incubated at room temperature 10min, is terminated and is reacted using terminate liquid.

F. colorimetric：It is returned to zero with blank, is longer than at 450nm in wave through microplate reader and reads OD value (OD).

G. result judges：P/N=measures sample OD mean values/negative serum OD mean values, and P/N >=2.1 are the positive.

(3) preparation of feeder cells：

It takes a healthy ICR mouse, neck dislocation to put to death, after body surface disinfection and four limbs fixation, skin is cut off from thigh, Exposure peritonaeum, cotton ball soaked in alcohol sterilize peritonaeum.With 10ml syringes, 12^#Syringe needle, injection 5-10ml HAT culture mediums to abdominal cavity are right Hand fixes syringe, and left hand holds cotton ball soaked in alcohol gently abdomen massage, draws back intraperitoneal liquid, centrifuges, and sediment adds HAT culture solutions It is resuspended, it is spare as the feeder cells of fused cell culture later.

(4) splenocyte is prepared：

3 days after booster immunization mouse are taken, eyeball blood sampling is extractd and is put to death for detaching positive serum, neck dislocation, sterile opening Spleen is taken out in abdominal cavity, and splenocyte suspension is made, and is cultivated through standing, centrifugation, ice-water bath, centrifugation, dilution, counting and RPMI-1640 Liquid washs to obtain mouse boosting cell suspension, spare.

(5) myeloma cell pre-processes：

Under the Sp2/0 myeloma cell with greater activity is rinsed from culture vessel wall with RPMI-1640 culture solutions Come, is washed through collection, centrifugation, dilution, counting and RPMI-1640 culture solutions, it is spare.

(6) cell fusion and culture：

The myeloma cell of above-mentioned preparation and splenocyte are mixed in the centrifuge tube with cover of a 50ml, 1000rpm 10min is centrifuged, supernatant fully exhausts, in order to avoid influence the effect of Macrogol 4000 (PEG4000).Fusion pipe is placed in palm In, bottom is gently vibrated, two kinds of cells must be made to mix well.It is with 1ml suction pipes that the PEG4000 of preheating is slow in 45~60s Slowly it is added in above-mentioned centrifuge tube, side edged gently shakes up.The DMEM culture mediums of 37 DEG C of preheatings are added dropwise immediately, PEG4000 is made to dilute And it is ineffective, specific addition is the incomplete culture medium for adding 1ml preheatings in first minute with suction pipe, is added in second minute 2ml, third minute add 8ml (in accordance with first slow rear fast principle), 37 DEG C of standing 10min, 1000rpm centrifugation 10min to abandon supernatant. The HAT culture mediums of 5ml are added, gently suspend sedimentation cell, adds suitable above-mentioned (3) feeder cells, finally adds HAT extremely 50ml or so.96 porocyte culture plates are sub-packed in, culture plate are then set 37 DEG C, 5%CO₂Culture in incubator.

(7) screening and cloning positive hybridoma cell：

Cell culture to be fused observed the growing state of hybridoma to the 5th day, waited for that its cells and supernatant turns yellow Or clone distribution to hole floor space 1/10 or more when, appropriate cell conditioned medium is drawn, through limiting dilution 1:10 start, 3 times of gradients Dilution, totally 6 gradients, detect antibody content with ELSA methods, the antibody-secreting of high-titer are filtered out according to the secretion situation of antibody Hole carries out hybridoma cell strain and is subcloned, monoclonal antibody expression and conservation.

The ELSA methods measure culture supernatant antibody, and this method is made of following operating procedure：

A. it is coated with：It is molten that CD161 immunogen proteins in step (1) with phosphate buffer are configured to 1 μ g/ml concentration Liquid, 100 μ l/well are coated with 96 hole polyethylene boards, and vacuum is drained, and 4 DEG C of sealing saves backup.

C. it is loaded：Culture medium supernatant 50-100 μ l (1 are added per hole:10-2430 dilutes), set normal control, the positive per plate Control and blank (phosphate buffer), 37 DEG C of incubation 1h, phosphate buffer wash 3 times.

D. ELIAS secondary antibody is added per hole 100-200 μ l, washs.

G. result judges：P/N=measures sample OD mean values/feminine gender OD mean values, and P/N >=2.1 are the positive.

(2) preparing for 161 protein monoclonal antibody of mouse-anti-human T lymphocyte CD secreted by above-mentioned hybridoma is pure Change and verifies：

According to supernatant affinity testing result, 5 plants of hybridoma cell strains with high-affinity are selected to carry out conservation, and right It chooses one plant of cell strain and carries out a small amount of cultures and IgG purifying.It is as follows：

A. antibody producing：

Cell strain is expanded into culture extremely with DMEM culture mediums (containing 10%FBS, 1%Penicillin-Streptomycin) 20ml, harvests culture supernatant after 4 days, cell fragment is removed in centrifugation；

B. antibody purification：

Supernatant crosses proteinG purification columns, then 30 times of column volumes of phosphate buffer, then the glycine (PH with 0.1M =2.8) antibody of combination is eluted and collects, dialysis removes remaining glycine, and A280 methods detect antibody concentration.

C. antibody is verified：

The affinity of ELISA method detection IgG purification and CD161 antibody：It is coated with CD161 immunogenes respectively on elisa plate Albumen and His tag fusion proteins (1 μ g/ml, 100 μ l/well), after washing closing, the purifying CD161 that gradient dilution is added is anti- Body；The sheep anti-mouse igg that horseradish peroxidase-labeled is added is secondary antibody, TMB colour developings, microplate reader reading OD450 values.

Western Blot methods detect the affinity and specificity of IgG and CD161 antibody：SDS-PAGE electrophoresis CD161 exempts from Epidemic focus albumen and His tag fusion proteins with the CD161 antibody of 10 μ g/ml are primary antibody, mouse polyclonal sera after transferring film closing For positive primary antibody；Tag fusion protein is negative control.

(3) active tuberculosis is prepared by 161 protein monoclonal antibody of mouse-anti-human T lymphocyte CD of above-mentioned production, purifying With tuberculosis latent infection flow cytometer detection kit.

Fluorescent molecular (such as APC, FITC, PE) stream is further made in the mouse anti-human monoclonal's antibody obtained through above-mentioned steps The straight labeling antibody of formula, the T lymphocytes for detecting expression CD161 molecules detect streaming antibody in conjunction with other lymphocytes, can Realize that the clinical detection purpose of active tuberculosis and tuberculosis latent infection is distinguished in detection.

It is as follows：

(1) R-PE coupling methods prepare streaming fluorescence antibody：

The monoclonal antibody that this patent is purified, according to R-PHYCOERYTHRIN(RPE) The operating instruction of CONJUGATION KIT (Prozyme, PJ31K) carries out R-PE labels (Nanjing Jin Siruisheng to CD161 antibody Object Science and Technology Ltd. synthesizes), obtain CD161-PE streaming antibody.Remaining required streaming antibody can use a series of abilities Protein specific antibody known to domain, such as MACS, BD, Beckman, R＆D etc..Such as embodiments herein experiment is selected：BD companies CD45-APC/cy7 (article No.s：And CD3-FITC.Cy7 (article No.s 340943)：57851).

(2) sample collection:

With vacuum anticoagulant tube acquisition people's upper arm vein peripheral blood about 2ml, 4 DEG C stand preservation.Sample is divided into through clinical definite Three groups：Tuberculosis patient (TB), latent infection crowd (LTBI) and healthy population (HC).

(3) antibody incubation：

The 200 μ l of every Patients with Peripheral blood of above-mentioned TB, LTBI and HC made a definite diagnosis are drawn respectively in streaming pipe, in each streaming pipe In be separately added into (1) described three kinds of streaming antibody, each 10 μ l shake mixing, are protected from light are incubated 20min at room temperature.

(4) erythrocyte splitting：

According to volume ratio (hemolysin：ddH₂O=1:9) (hemolysin：BDIS article No.s：349202) hemolysin solution is prepared, It is continuously added in each streaming pipe, 2ml is added per streaming pipe, shakes mixing, is protected from light is incubated 5-10min at room temperature, wait in pipe Liquid become it is limpid it is transparent after, 1500rpm/min centrifuges 5min, and supernatant liquid is outwelled, and buckle on clean blotting paper and to do.

(5) cell is fixed：

3ml sterilizing 1M phosphate buffers (PBS) are respectively added in streaming pipe after each button is dry in above-mentioned (4), concussion is mixed Even, 1500rpm/min centrifuges 5min, abandons supernatant, and the 100 μ l incubations at room temperature 20min of paraformaldehyde solution that 4% (M/V) is added is solid Determine cell, abandon supernatant, 1ml 1M phosphate buffers are added and wash once, abandon clean supernatant.

(6) flow cytometer detection：

500 μ l 1M phosphate buffers are respectively added in the streaming pipe that above-mentioned (5) each abandon clean supernatant, cell are resuspended, It is detected with BD flow cytometers.

(7) testing result：

Utilize flow cytomery difference cell percentages：Lymphocyte accounts for ratio (L%), monokaryon in CD45+ cells Cell accounts for the ratio (M%) of CD45+ cells, CD3+ cells account for the ratio (CD45+CD3+%) of lymphocyte, CD161+ cells exist Positive rate (CD3+CD161+%) in CD3+ lymphocytes.

(8) result calculates：

It is specific as follows：

A. the ratio (L%/M%) of cent lymphocytes and monocyte percentage：It is thin using lymph in CD45+ cells The percentage (M%) of monocyte in the percentage (L%) divided by CD45+ cells of born of the same parents.

Expression quantity (L%/M% × CD3+CD161+%) of the b.CD161 positive cells in CD3+ cells：It is thin using lymph The ratio between born of the same parents' number and monocyte number (L%/M%) are multiplied by the percentage (CD3+CD161+%) of CD161+ in CD3+ cells, indicate Expression quantity of the CD161 positive cells in CD3+ cells.

C. different crowd (normal healthy controls-HC, latent infection-LTBI, tuberculosis are calculated separately according to above-mentioned computational methods People-TB) L%/M% × CD3+CD161+%, compares the expression of the above-mentioned cell of different crowd.

Analysis result finds that L%/M% × CD3+CD161+% is significantly lower than normal healthy controls and hides in tuberculosis patient The infected illustrates 161 monoclonal antibody of mouse-anti-human T lymphocyte CD prepared by this patent, is applied to flow cytometer detection, may be implemented The detection of active tuberculosis and tuberculosis latent infection is distinguished.

The anti-human CD161 antibody titers that the method for the present invention is obtained are high, specific very good, can directly mark A variety of tracers, by currently advanced detecting instrument, such as：Flow cytometer, chemiluminescence detector, magnetic separator etc. into The positive-selecting of row CD161 expression cells, has prevention and control field lungy very positive meaning.

Description of the drawings：

Fig. 1 is the gel images processing system analysis chart in the embodiment of the present invention 1.

Fig. 2 is the Westen Blot experimental analysis figures in the embodiment of the present invention 2.

Fig. 3 is the molecular weight of protein and purity expression figure in the embodiment of the present invention 3.

Fig. 4 is the molecular weight of Westen Blot experimental proteins and purity expression figure in the embodiment of the present invention 4.

Fig. 5 is that the HL60 cell non-specificity in the embodiment of the present invention 5 detects chart.

Fig. 6 is the CD161 positive cell specific detection charts in the embodiment of the present invention 6.

Fig. 7 is the flow cytomery result figure in the embodiment of the present invention 7.

Fig. 8 is the flow cytomery comparing result figure of different crowd in the embodiment of the present invention 7.

Specific implementation mode：

With reference to embodiment, the present invention will be further elaborated.

Embodiment 1.

E3 plasmids with CD161 genes are converted into Escherichia coli, IPTG induced fusion protein expressions, ultrasonic method is crushed bacterium Body, CD161 immunogen protein of the nickel column method purifying with His labels.Utilize SDS-PAGE gel electrophoresises and Western Blot Detection, gel images processing system analyze the purity and size of protein.Purity of protein requires to reach 90% or more, it is believed that closes Lattice, the animal immune after can be used for.The CD161 immunogen proteins (90% or more purity) of purifying and Freund's adjuvant is mixed It closes, animal selection and the homologous BALB/c healthy mices three of myeloma cell used is immunized, age of mouse was at 8-14 weeks, subcutaneous abdomen Injection, every is immunized 100 μ g albumen every time.Altogether it is immune three times, be spaced three weeks per between twice.Second of immune posterior orbit is quiet Arteries and veins measures antiserum from 100 μ l of blood sampling, using enzyme-linked immunosorbent assay (abbreviation ELSA) method, and antibody titer is equal to and is higher than 1:1000, it is believed that immune success rate selects the highest mouse of titre to be used for the preparation of hybridoma.

The expression CD161 immunogen proteins of other embodiments of the invention are also identical.

First, the molecular weight and purity of detection expression CD161 immunogen proteins.

The molecular weight and purity of SDS-PAGE experimental verification prokaryotic expression CD161 immunogen proteins.

E3 plasmids with CD161 genes are converted into Escherichia coli, IPTG induced fusion protein expressions, ultrasonic method is crushed bacterium Body tests detection and analysis protein using CD161 immunogen proteins of the nickel column method purifying with His labels using SDS-PAGE Purity and size.It is as follows：

(1) glue frame is assembled：

By clean, anhydrous slide alignment, cavity is formed, is inserted into the groove of frame of plastic, notices that arrow is upward, and ensure Lower end is concordant.It is put on gum-making rack and clamps, sealing strip is close in lower end.

(2) 10% separation gels configure：

According to preparing 10% separation gel 5ml as following formula：1.3ml distilled water, the 30%Acr-Bis (29 of 1.7ml:1), The 1.5M Tris-HCl (PH 8.8) of 1.5ml, 10% ammonium persulfate of the 10%SDS of 0.05ml, 0.05ml, 0.07ml's TEMED。

(3) encapsulating：

Gel solution is carefully injected into (glue height in the slit between long and short glass plate along glass rod with liquid-transfering gun after mixing Away from sample template broach lower edge about 1cm).

(4) fluid-tight：

Gently add one layer of water to completely cut off air along short glass plate edge in gel surface, keeps glue surface smooth.Stand about 30min Glue surface variation is observed, when seeing water and the glue surface of the solidification boundary that have refractive index different, shows that glue solidifies completely, outwells Layer water, is used in combination filter paper to blot remaining aqueous.

(5) 5% concentration glue configurations：

Glue 2ml is concentrated according to preparing 5% as following formula：1.4ml distilled water, the 30%Acr-Bis (29 of 0.33ml:1), The 1.5M Tris-HCl (PH 8.8) of 0.25ml, 10% ammonium persulfate of the 10%SDS of 0.02ml, 0.02ml, 0.02ml's TEMED。

(6) it is inserted into glue comb：

Gel solution is injected into the slit between long and short glass plate (above separation gel), gently with liquid-transfering gun after mixing Sample template comb is added, carefully avoids the appearance of bubble.About 30min, polymerization are complete.

(7) electrophoresis tank is installed：

The gel slab prepared is removed, comb is carefully pulled up.Two piece 10% of gel slab is inserted into U-shaped rubber frame respectively In the baltimore groove of both sides, can up lift makes gel slab be close to rubber.The rubber moulding frame for installing glass plate is lain in into the storage tank faced upward and put On frame, lower edge is aligned with storage tank frame lower edge, is put into electrophoresis tank.Pour into 1 × tris-gly electrophoretic buffers.

(8) sample treatment：

The 80 μ l of CD161 immunogen proteins sample for taking a concentration of 65 μ g/ml, add 20 5 × buffer of μ l (to add B- sulfydryls Ethyl alcohol), mixing.

(9) it is loaded：

10 μ l of processed sample solution (about 5 μ g of CD161 immunogen proteins content) are taken with liquid-transfering gun, are carefully added successively Enter into each gel spill sample cell, setting Marker is compareed and BSA (5 μ g, 60KD) controls.

(10) electrophoresis：

Electrophoresis tank is placed on electrophoresis apparatus, is powered on, positive and negative anodes are to good.Voltage is adjusted to about 150V and keeps constant pressure.Wait for bromine When phenol indigo plant label is moved to gel bottom, powered-down source is refunded electrophoretic buffer in bottle.

(11) glue is removed：

Electrophoresis tank is taken out, two boards are lifted down, and are tilted, then concentration glue is wiped off, are taken among length slide with scraping blade Under.

(12) it dyes：

The glue that upper step is taken out is put in the staining plate added with R250 dyeing liquors, dye liquor covers glue, is placed on shaking table, Rotating speed 45rmp/min, time 1h, after the completion dye liquor outwell and dye liquor be washed off with water.

(13) it decolourizes：

It takes out the glue dyed to be put in the dye vat added with destainer, destainer covers glue, is placed on shaking table, rotating speed 45rmp/min, time 1h, area color purify, and band is high-visible, outwells destainer after the completion.

(14) it takes pictures：

Glue after decoloration is placed in transparent file folder, the bubble above glue is driven out of and (can also be wiped with cotton ball soaked in alcohol with preceding Clean file), it is put on scanner, takes pictures.

(15) it analyzes：

The position of target stripe and net OD value are analyzed with gel images processing system, to analyze the molecular weight of protein And purity.Purity of protein requires to reach 90% or more, it is believed that qualified, the animal immune after can be used for.

The result is shown in Figure 1：The extracellular domain albumen size of people's CD161 immunogen proteins meets expection in 20KDa or so (19444.8Da), lipidated protein reaches 95% or more, illustrates that CD161 immunogen proteins are expressed successfully, can be used for mouse and exempt from Epidemic disease.

Embodiment 2.

The molecular weight and purity of Westen Blot experimental verification prokaryotic expression CD161 immunogen proteins.

E3 plasmids with CD161 genes are converted into Escherichia coli, IPTG induced fusion protein expressions, ultrasonic method is crushed bacterium Body utilizes Westen Blot experiment detection and analysis albumen using CD161 immunogen proteins of the nickel column method purifying with His labels The purity and size of matter.It is as follows：

(1) glue frame is assembled：

Glass plate is scrubbed clean, is washed down with distilled water, airing.With 10% ammonium persulfate (preferably now with the current).Solidifying Glass plate is installed on glue frame, with it is reeded inwards.A small amount of deionized water is added, see whether leak.Such as leakage, then pacify again Dress；It does not leak, then starts to match glue.

(2) 10% separation gels configure：

According to preparing 10% separation gel 5ml as following formula：1.3ml distilled water, the 30%Acr-Bis (29 of 1.7ml:1), The 1.5M Tris-HCl (PH 8.8) of 1.5ml, 10% ammonium persulfate of the 10%SDS of 0.05ml, 0.05ml, 0.07ml's TEMED.Gently oscillation solution makes mixing (excess gas, which is steeped oneself-meeting, influences glue polymerization).

(3) encapsulating：

(4) fluid-tight：

Gently add one layer of water to completely cut off air along short glass plate edge in gel surface, keeps glue surface smooth.About 30min is stood to see Glue surface variation is examined, when seeing water and the glue surface of the solidification boundary that have refractive index different, shows that glue solidifies completely, outwells upper layer Water is used in combination filter paper to blot remaining aqueous.

(5) 5% concentration glue configurations：

(6) it is inserted into glue comb：

Gel solution is injected into the slit between long and short glass plate (above separation gel), gently with liquid-transfering gun after mixing Sample template comb is added, carefully avoids the appearance of bubble.About 30min, polymerization are complete.After concentration gelling is solid.Glass plate is transferred to In electrophoresis tank, a little buffer solution is added to check whether leakage.Such as leakage, unloads and reinstall；It does not leak, slowly plus electrophoretic buffer is to filling it up with Inside groove.It is vertical to extract comb.

(7) electrophoresis tank is installed：

(8) sample treatment：

By the CD161 immunogen proteins of production, SDS sample-loading buffers are added, a concentration of 35ng/ μ l are adjusted, in boiling water bath In boil 5-7min and make albuminous degeneration, be put in cooled on ice at least 5min.10 000g centrifuge 10min and remove insoluble impurity later.

(9) it is loaded：

10 μ l of processed sample solution (CD161 immunogen proteins content about 35ng) are taken with liquid-transfering gun, carefully successively It is added in each gel spill sample cell, setting Marker is compareed.

(10) electrophoresis：

Voltage range is adjusted to 600V.Prior to electrophoresis 11min under 80V voltages or so, wait for that sample pressure is into a line, and in sight When will enter separation gel, 120V voltages are switched to.When bromophenol blue just runs out of separation gel, electric current is cut off.Electrophoresis takes around 80min。

(11) transferring film

First by transfer groove, scissors, tweezers etc. in ddH₂It impregnates, outwells in O.It pours electricity into again and turns buffer solution (at 4 DEG C Reason), while shearing filter paper and NC films according to the size of glue.Filter paper electricity is put into after shearing to turn to impregnate 10-15min in liquid.It removes Gel, the part that need to be dyed is cut spare, and another part, which is cut, not to be had to.Swimming lane and concentration glue, well, according to sample-adding Sequence cuts Yi little Jiao on the upper left side of glue, is also placed in electricity and turns to stand 10-15min in liquid.It has to remember Loading sequence with glue just Anti- relation of plane.According to the suitable of (red) anode-sponge-two layers of filter paper-film-glue-two layers of filter paper-sponge-the moon (black) pole after soaking Sequence is put into transfer groove.A bubble will be caught up with by often putting one layer well, and a bubble is caught up with again with pipe after to the last putting sponge well.So Transfer groove is put into electroporation (attention will ensure that transfer groove cathode is corresponding with the cathode of electroporation) afterwards, and is put into ice chest. Electrophoresis apparatus voltage is adjusted to 30V, 4 DEG C of transferring film 15h.

Another clotting glue removed is put into culture dish, adds dyeing liquor that gel is completely covered, vibrates at room temperature 30min pours out dyeing liquor (reusable).New destainer is added, oscillation decoloration is high-visible to protein band, and sees Examine banding pattern.Period replaceable destainer.

(12) it closes：

Power supply is pulled up after the completion of transferring film, takes out transfer groove.Suitable PBST is added in a clean culture dish.With Tweezers take the film out and also cut a triangular cut at film same position corresponding with gel notch.Again by film towards the one of gel It is put into PBST downwards and washes 5min.It then takes out and is put into confining liquid (and downwards close to gel, similarly hereinafter), room temperature is shaken It swings, 2h.

(13) primary antibody reacts：

The film taking-up PBST oscillations closed are washed 3 times, each 5min.It is (commercially available to dilute primary antibody with 5% confining liquid CD161 antibody), a plastic foil (be careful not to hand contact plastic foil inside) more bigger than Buddhist nun's logical sequence film is cut, NC films are put into Wherein, it draws antibody liquid with syringe to be added in plastic foil, be sealed with sealing machine after catching up with most bubble.It is stored at room temperature 3h.

(14) secondary antibody reacts：

The film that primary antibody was incubated is taken out from refrigerator, is washed in PBST three times, each 5min.Come with 5% confining liquid Dilute secondary antibody (sheep anti-mouse igg).The plastic foil slightly larger than NC films is cut, is sealed after being put into Buddhist nun's logical sequence film.Two corresponding anti-solution is added, catches up with most gas Mouth is sealed after bubble again.It is stored at room temperature 2h.

(15) it develops the color：

Film is taken out, is washed in PBST 4 times, each 5min.DAB developing solutions are configured, the small effective masking foil packages of 4ml is taken, adds Enter mixing after 1ml HRP buffer, 50 μ l reagent As, 50 μ l reagents B, 50 μ l reagent Cs.It is protected from light dyeing.After there is clear signal, Incline and falls developing solution.Phosphate buffer embathes termination.

(16) it takes pictures：

The film of colour developing is placed in transparent file folder, the bubble above film is driven out of and (can also be wiped with cotton ball soaked in alcohol with preceding Clean file), it is put on scanner, takes pictures.

(17) it analyzes：

As a result see Fig. 2：The extracellular domain albumen size of people's CD161 immunogen proteins meets expection in 20KDa or so (19444.8Da), lipidated protein reaches 95% or more, illustrates the success of CD161 protein expressions, can be used for mouse immune.

Embodiment 3.

Hybridoma cell strain screening is carried out using ELISA method.

Cell culture to be fused observed the growing state of hybridoma to the 5th day, waited for that its cells and supernatant turns yellow Or clone distribution to hole floor space 1/10 or more when, appropriate cell conditioned medium is drawn, through limiting dilution (1:10 start, 3 times of ladders Degree dilutes, totally 6 gradients), antibody content is detected with ELISA, the antibody-secreting of high-titer is filtered out according to the secretion situation of antibody Hole carries out hybridoma cell strain and is subcloned, monoclonal antibody expression and conservation.

It is as follows：

(1) it is coated with：CD161 immunogen proteins are configured to 1 μ g/ml strength solutions with phosphate buffer, per 100 μ l of hole 96 hole polyethylene boards are coated with, vacuum is drained, and 4 DEG C of sealing saves backup.

(2) it closes：The 200 μ l of phosphate buffer that pH is 7.4 are added per hole to wash, include 1% lowlenthal serum.

(3) it is loaded：Medium supernatant 50-100 μ l (1 are added per hole:10-2430 dilutes), set normal control, sun per plate Property control and blank (phosphate buffer), 37 DEG C incubation 1h, phosphate buffer wash 3 times.

(4) ELIAS secondary antibody (sheep anti-mouse igg that horseradish peroxidase-labeled is added) is added per 200 μ l of hole, washs.

(5) it develops the color：100 μ l of substrate are added per hole, are incubated at room temperature 10min, is terminated and is reacted using terminate liquid.

(6) colorimetric：It is returned to zero with blank, is longer than at 450nm in wave through microplate reader and reads OD value (OD).

(7) result judges：P/N=measures sample OD mean values/negative serum OD mean values, and P/N >=2.1 are the positive.

It the results are shown in Table 1：

Table 1

In the 10 plants of hybridoma supematants selected, the antibody titer for having 7 plants is more than 1:2430, it can carry out hybridoma The subclone of strain and the big culture of expansion, for producing CD161 monoclonal antibodies.

According to above-mentioned to supernatant affinity testing result, 5 plants of hybridoma cell strains with high-affinity is selected to be protected Kind, and 10B3E6 cell strains are cultivated, (contain 10%FBS, 1%Penicillin- with DMEM culture mediums Streptomycin cell strain) is expanded into culture to 20ml, culture supernatant is harvested after 4 days, cell fragment supernatant mistake is removed in centrifugation ProteinG purification columns, then PBS washs 30 times of column volumes, then is eluted with the glycine (PH=2.8) of 0.1M and collect knot The antibody of conjunction, dialysis remove remaining glycine, and A280 methods detect antibody concentration.Antibody 2.0mg, antibody concentration are harvested altogether For 0.671mg/ml.

ELISA method detection purifies the affinity of CD161 antibody, is as follows：

(1) it is coated with：By CD161 immunogen proteins and His tag fusion proteins, 1 μ g/ml are configured to phosphate buffer Strength solution, 100 μ l/well are coated with 96 hole polyethylene boards, and vacuum is drained, and 4 DEG C of sealing saves backup.

(3) it is loaded：It is coated with 1 μ g/ml of CD161 immunogen proteins and His tag fusion proteins respectively on elisa plate, washes After washing closing, per hole be added gradient dilution 100 μ l of purifying CD161 antibody (10B3E6 clone hybridomas production, 1: 1000- 512000 dilutions), normal control, positive control and blank (phosphate buffer), 37 DEG C of incubation 1h are set per plate, phosphate delays Fliud flushing is washed 3 times.

(4) ELIAS secondary antibody (sheep anti-mouse igg of horseradish peroxidase-labeled) is added per 200 μ l of hole, washs.

As a result see Fig. 3 and table 2：The CD161 monoclonal antibodies of 10B3E6 clone's hybridomas production have very high sensitivity Degree, for titre up to 1: 512000, it is about 0.092 μ g/ml that software, which calculates its EC50,.

Table 2

Embodiment 4：

According to embodiment 3 to supernatant affinity testing result, 5 plants of hybridoma cell strains with high-affinity is selected to carry out Conservation, and 1083E6 cell strains are cultivated, (contain 10%FBS, 1%Penicillin- with DMEM culture mediums Streptomycin cell strain) is expanded into culture to 20ml, culture supernatant is harvested after 4 days, cell fragment, supernatant mistake are removed in centrifugation ProteinG purification columns, then PBS washs 30 times of column volumes, then is eluted with the glycine (PH=2.8) of 0.1M and collect knot The antibody of conjunction, dialysis remove remaining glycine, and A280 methods detect antibody concentration.Antibody 2.0mg, antibody concentration are harvested altogether For 0.671mg/ml, it is 7171460-1 antibody to be named as the antibody.

Western Blot methods detect the affinity and specificity of IgG and CD161 antibody：SDS-PAGE electrophoresis CD161 exempts from Epidemic focus albumen and His tag fusion proteins with the CD161 antibody of 10 μ g/ml are primary antibody, mouse polyclonal sera after transferring film closing For positive primary antibody；His tag fusion proteins are negative control；Phosphate buffer is blank control.

(1) glue frame is assembled：

(2) 10% separation gels configure：

According to preparing 10% separation gel 5ml as following formula:1.3ml distilled water, the 30%Acr-Bis (29 of 1.7ml:1), The 1.5M Tris-HCl (PH 8.8) of 1.5ml, 10% ammonium persulfate of the 10%SDS of 0.05ml, 0.05ml, 0.07ml's TEMED.Gently oscillation solution makes mixing (excess gas, which is steeped oneself-meeting, influences glue polymerization).

(3) encapsulating：

(4) fluid-tight：

(5) 5% concentration glue configurations：

Glue 2ml is concentrated according to preparing 5% as following formula:1.4ml distilled water, the 30%Acr-Bis (29 of 0.33ml:1), The 1.5M Tris-HCl (PH 8.8) of 0.25ml, 10% ammonium persulfate of the 10%SDS of 0.02ml, 0.02ml, 0.02ml's TEMED。

(6) it is inserted into glue comb：

After concentration gelling is solid.Glass plate is transferred in electrophoresis tank, and a little buffer solution is added to check whether leakage.Such as leakage, weight is unloaded New installation；It does not leak, slowly plus electrophoretic buffer is to filling it up with inside groove.It is vertical to extract comb.

(7) electrophoresis tank is installed：

(8) sample treatment：

By the CD161 immunogen proteins of production, SDS sample-loading buffers are added, a concentration of 50ng/ μ l are adjusted, in boiling water bath In boil 5-7min and make albuminous degeneration, be put in cooled on ice at least 5min.10 000g centrifuge 10min and remove insoluble impurity later.

(9) it is loaded：

Processed 10 μ l (content 500ng) of CD161 immunogen proteins solution are taken with liquid-transfering gun, are carefully added sequentially to In each gel spill sample cell, setting Marker is compareed.

(10) electrophoresis：

Voltage range is adjusted to 600V.Prior to electrophoresis 11min under 80V voltages or so, wait for that sample pressure is into a line, will When into separation gel, 120V voltages are switched to.When bromophenol blue just runs out of separation gel, electric current is cut off.Electrophoresis takes around 80min。

(11) transferring film

First by transfer groove, scissors, tweezers etc. in ddH₂It impregnates, outwells in O.It pours electricity into again and turns buffer solution (at 4 DEG C Reason), while shearing filter paper and NC films according to the size of glue.Filter paper electricity is put into after shearing to turn to impregnate 10-15min in liquid.It removes Gel, the part that need to be dyed is cut spare, and another part, which is cut, not to be had to, swimming lane and concentration glue, well, according to sample-adding Sequence cuts Yi little Jiao on the upper left side of glue, is also placed in electricity and turns to stand 10-15min in liquid.It has to remember Loading sequence with glue just Anti- relation of plane.According to (red) anode-sponge-two layers of filter paper-film-glue-two layers of filter paper-sponge-the moon (black) pole after soaking Sequence is put into transfer groove.A bubble will be caught up with by often putting one layer well, and a bubble is caught up with again with pipe after to the last putting sponge well. Then transfer groove is put into electroporation (attention will ensure that transfer groove cathode is corresponding with the cathode of electroporation), and is put into ice Box.Electrophoresis apparatus voltage is adjusted to 30V, 4 DEG C of transferring film 15h.

(12) it closes：

Power supply is pulled up after the completion of transferring film, takes out transfer groove.Suitable PBST bufferings are added in a clean culture dish Liquid.It is taken the film out with tweezers and also cuts a triangular cut at film same position corresponding with gel notch.Again by film towards solidifying 5min is washed in being lowered into PBST on one side for glue.It then takes out and is put into confining liquid (and downwards close to gel, similarly hereinafter), Shaken at room temperature 2h.

(13) primary antibody reacts：

The film taking-up PBST oscillations closed are washed 3 times, each 5min.Primary antibody is diluted with 5% confining liquid：Adjustment 7171460-1 antibody concentrations are 10ng/ μ l and 5ng/ μ l；Adjust a concentration of 10ng/ μ l of His fusion proteins；Mouse Polyclonal blood It is thin to release 100 times.A plastic foil (be careful not to hand contact plastic foil inside) more bigger than Buddhist nun's logical sequence film is cut, NC films are put into Wherein, it draws antibody liquid with syringe to be added in plastic foil, be sealed with sealing machine after catching up with most bubble.It is stored at room temperature 3h.

(14) secondary antibody reacts：

(15) it develops the color：

Film is taken out, is washed in PBST 4 times, each 5min.DAB developing solutions are configured, the small effective masking foil packages of 4ml is taken, adds Enter mixing after 1ml HRP buffer, 50 μ l reagent As, 50 μ l reagents B, 50 μ l reagent Cs.It is protected from light dyeing.After there is clear signal, Incline and falls developing solution.PBS embathes termination.

(16) it takes pictures：

(17) it analyzes：

The position of target stripe and net OD value are analyzed with gel images processing system, to analyze the molecule of protein Amount, purity and specificity.

As a result see Fig. 4：The 7171460-1 monoclonal antibodies (clone 10B3E6) of production, to CD161 immunogen proteins Ability with specific recognition is CD161 protein monoclonal antibodies.

Embodiment 5.

Leukemia tumor cells system (HL60), does not express CD161 albumen, so theoretically will not be with CD161 antibody knots It closes, it is straight to can be used for verifying fluorescence (such as APC, FITC, PE) made of the anti-human CD161 monoclonal antibodies in mouse source of this patent production Mark the specificity of streaming antibody.

With the CD161 streaming antibody products of common similar PE labels in the market, BD antibody (article No. 556081；Clone number DX12)；MACS antibody (article No. 130-109-637；Clone REA631)；Beckman antibody (article No. IM3450；Clone number 191B8) carry out contrast experiment.

It is as follows：

(1) a concentration of 10e6cell/ml for adjusting HL60 cell culture medium cell suspensions, draws 500 μ l in streaming respectively Pipe, 1500rpm/min centrifuge 5min, supernatant liquid are outwelled, and buckle and do on clean blotting paper；

(2) 1ml sterilizing 1M phosphate buffers are respectively added in the streaming pipe after above-mentioned each button is dry, shake mixing, 1500rpm/min centrifuges 5min, and supernatant liquid is outwelled, and buckles and do on clean blotting paper；

(3) the 100 μ l of paraformaldehyde solution that 4% (M/V) is respectively added in the streaming pipe after above-mentioned each button is dry are fixed, Supernatant is abandoned, 1ml 1M phosphate buffers are added and shake mixing, 1500rpm/min centrifuges 5min, supernatant liquid is outwelled, and It is buckled on clean blotting paper dry；

(4) 90 μ l sterilizing 1M phosphate buffers are respectively added in the streaming pipe after above-mentioned each button is dry, in each streaming The CD161-PE streamings antibody and the homemade CD161-PE streamings antibody of this patent of above-mentioned 3 kinds of commercially available brands are separately added into pipe 10 μ l shake mixing, are protected from light are incubated 20min at room temperature, and 1500rpm/min centrifuges 5min, supernatant liquid is outwelled, and dry It is buckled on net blotting paper dry；

(5) 1ml sterilizing 1M phosphate buffers are respectively added in the streaming pipe after above-mentioned each button is dry, shake mixing, 1500rpm/min centrifuges 5min, and supernatant liquid is outwelled, and buckles and do on clean blotting paper；

(6) 200 μ l sterilizing 1M phosphate buffers are respectively added in the streaming pipe after above-mentioned each button is dry, are flowed with BD C6 Formula cell instrument is detected.

Testing result：It is illustrated in fig. 5 shown below, self-control stream is anti-, the background average fluorescent strength difference of BD, MACS and Beckman For：3195.36,5997.98,6170.12,5348.33.So three streaming antibody brands with mainstream currently on the market (BD, MACS, Beckman) is compared, and the homemade CD161-PE streamings monoclonal antibody of the present invention has lower background value, says The specific higher of bright antibody identification CD161 molecules, non-specific binding are low.

Embodiment 6.

Lymphocyte, cell detection report inspection are expressed in Sai Ersi biotechnologys (Shanghai) Co., Ltd. of Australia purchase CD161 It surveys, cell purity is 95% or more, 95% or more live cell fraction, no mycoplasma and Chlamydia pollution.With the commercialization cell System can be used for verifying fluorescent molecular (such as APC, FITC, PE) made of the mouse source anti-human monoclonal antibodies of this patent production and directly mark The accuracy and stability of streaming antibody.

It is as follows：

(1) a concentration of 10e6cell/ml for adjusting CD161 cell culture medium cell suspensions, draws 500 μ l in stream respectively Formula pipe, 1500rpm/min centrifuge 5min, supernatant liquid are outwelled, and buckle and do on clean blotting paper；

It is illustrated in fig. 6 shown below, interpretation of result is as follows：

Self-control stream is anti-, and BD, MACS and the positive ratio of Beckman detections are 95% or more, respectively：98.8%, 97.1%, 94.1% and 94.7%, unanimously with product positive criteria (95% or more), illustrate the homemade CD161-PE streams of this patent Formula monoclonal antibody testing result qualification is credible.

Self-control stream is anti-, and the detection average fluorescent strength of BD, MACS and tetra- kinds of antibody of Beckman is respectively： 1044766.25,907610.55,989598.86 and 883510.82.So three streaming antibody with mainstream currently on the market Brand (BD, MACS, Beckman) is compared, and the homemade CD161-PE streamings monoclonal antibody average fluorescent strength of this patent is most strong, Illustrate that it, with higher specificity, can generate stronger signal.

Self-control stream is anti-, and the fluorescence intensity CV values of BD, MACS and tetra- kinds of antibody of Beckman are respectively：27.01%, 32.32%, 30.74% and 34.01%.So with three streaming antibody brands of mainstream currently on the market (BD, MACS, Beckman it) compares, the CV values of the homemade CD161-PE streamings monoclonal antibody fluorescence intensity of this patent are minimum, illustrate that it has Higher stability.

Embodiment 7：

Present embodiments provide fluorescent molecular made of the mouse anti-human monoclonal's antibody prepared using the present invention (such as APC, FITC, PE) streaming antibody is directly marked, the T lymphocytes of CD161 molecules are expressed for detection in human peripheral sample, in conjunction with Other lymphocyte detection streaming antibody are, it can be achieved that the clinical detection mesh of active tuberculosis and tuberculosis latent infection is distinguished in detection 's.

Crowd is divided into three groups：Tuberculosis patient (TB, 27), latent infection crowd (LTBI, 49) and healthy population (HC, 60), by the percentage (L%) and monocyte (M%) of every Whole blood lymphocyte of FCM analysis, and The percentage of CD3+CD161+ cells in lymphocyte.Then L%/M% × CD3+CD161+% is calculated.As a result, it has been found that its L%/M% × CD3+CD161+% is significantly lower than normal healthy controls and latent infection person (as shown in Figure 8) in tuberculosis patient. Concrete operations are as follows：

(1) the 200 μ l of every Patients with Peripheral blood of above-mentioned TB, LTBI and HC made a definite diagnosis are drawn respectively in streaming pipe, in each streaming 10 μ L of following fluorescent labeled antibody are separately added into pipe：Homemade CD161-PE monoclonals streaming antibody, the CD45- of BD companies APC/cy7 (article No.s：And CD3-FITC.Cy7 (article No.s 340943)：57851) mixing, is shaken, is protected from light is incubated 20min at room temperature；

(2) according to volume ratio (hemolysin：ddH₂O=1:9) (hemolysin：BDIS article No.s：349202) it is molten to prepare hemolysin Liquid is continuously added in each streaming pipe, and 2ml is added per streaming pipe, shakes mixing, is protected from light is incubated 5-10min at room temperature；

(3) after liquid in pipe become it is limpid it is transparent after, 1500rpm/min centrifuge 5min, supernatant liquid is outwelled, and clean Blotting paper on buckle it is dry；

(4) 3ml sterilizing 1M phosphate buffers are respectively then added in the streaming pipe after above-mentioned each button is dry, concussion is mixed Even, 1500rpm/min centrifuges 5min, abandons supernatant, and the 100 μ l of paraformaldehyde solution that 4% (M/V) is added are fixed, and are abandoned supernatant, are added Enter 1ml 1M phosphate buffers to wash once；

(5) it is detected with BD FACSCanto II flow cytometers.

Testing result：

As shown in fig. 7, utilizing flow cytomery difference cell percentages：Lymphocyte accounts for ratio in CD45+ cells (L%), monocyte accounts for the ratio (M%) of CD45+ cells, CD3+CD161+ cells account for the ratio (CD3+CD4 of lymphocyte + %).

As a result it calculates：

After the percentage for obtaining above-mentioned cell by flow cytomery, CD161+ cells in different cells are analyzed Ratio, it is specific as follows：

(1) ratio (L%/M%) of cent lymphocytes and monocyte percentage：Utilize lymph in CD45+ cells The percentage (M%) of monocyte in the percentage (L%) divided by CD45+ cells of cell.

(2) expression quantity (L%/M% × CD3+CD161+%) of the CD161 positive cells in CD3+ cells：Utilize lymph The ratio between cell number and monocyte number (L%/M%) are multiplied by the percentage (CD3+CD161+%) of CD161+ in CD3+ cells, table Show expression quantity of the CD161 positive cells in CD3+ cells.

(5) according to above-mentioned computational methods calculate separately different crowd (normal healthy controls, latent infection, tuberculosis patient) L%/ M% × CD3+CD161+% compares the expression of the above-mentioned cell of different crowd, as shown in Figure 8.Wherein ordinate represents lymphocyte The ratio (L%/M%) of number and monocyte number is multiplied by the percentage of CD161 positive cells, and abscissa indicates different crowd.System Meter the result shows that, L%/M% × CD3+CD161+% in tuberculosis patient (TB).

(6) it is analyzed in software GraphPad Prism5 using above-mentioned data, can must determine the threshold of reaction：Work as L%/M% When × CD3+CD161+% is less than 88.47%, differentiates tuberculosis patient and tuberculosis latent infection person, normal healthy controls, sensibility exist Between 81.48%-88.89%, specificity is between 71.43%-88.33%.

161 protein monoclonal antibody of mouse-anti-human T lymphocyte CD prepares active tuberculosis in the present embodiment and tuberculosis is latent For distinguishing tuberculosis latent infection and active tuberculosis, the kit further includes infection flow cytometer detection kit：CD45- APC/cy7, CD3-FITC.Cy7 and CD161-PE；(hemolysin and ddH2O volume ratios are 1 to hemolysin solution:9)；1M phosphate Buffer solution；The paraformaldehyde of 4% (M/V).

The application method of mentioned reagent box, includes the following steps：

(1) 200 μ l of peripheral blood to be checked are drawn in streaming pipe；Following fluorescent labeled antibody is added in streaming pipe, and [BD is public CD45-APC/cy7 (the article No.s of department：340943), CD3-FITC.Cy7 (article No.s：And homemade CD161-PE (article No.s 57851)： PNIM3450) each 20 μ l], mixing is shaken, is protected from light is incubated 20min at room temperature.

(2) according to volume ratio (hemolysin：ddH₂O=1:9) (hemolysin：BDIS article No.s：349202) it is molten to prepare hemolysin Liquid is continuously added with the amount of 2ml in streaming pipe, shakes mixing, is protected from light is incubated 5-10min at room temperature.

(3) after liquid in pipe become it is limpid it is transparent after, 1500rpm/min centrifuge 5min, supernatant liquid is outwelled, and clean Blotting paper on buckle it is dry.

(4) 3ml sterilizing 1M phosphate buffers are then added in streaming pipe, shake mixing, 1500 rpm/min centrifugations 5min abandons supernatant, and the 100 μ l of paraformaldehyde solution that 4% (M/V) is added are fixed, and abandons supernatant, and 1ml 1M phosphate buffers are added It washes primary.

(5) it is detected with BD FACSCanto II flow cytometers, calculates lymphocyte and account for ratio in CD45+ cells (L%), monocyte accounts for the ratio (M%) of CD45+ cells, CD3+ cells account for the ratio (CD3+%) of lymphocyte；CD161+ Positive rate (CD3+CD161+%) of the cell in lymphocyte.

(6) interpretation of result and judgement：The data of L%/M% are first calculated, then calculate L%/M% × CD3+CD161+%, The judgment threshold of middle L%/M% × CD3+CD161+% is 65.40%.

It is judged as the positive when the numerical value of L%/M% × CD3+CD161+% is less than or equal to respective judgment threshold, it can Drawing a conclusion, it is active tuberculosis patient；It is judged as feminine gender when more than respective judgment threshold；Then it could be assumed that it is non- Active tuberculosis, as tuberculosis latent infection person or healthy individuals.

Claims

1. a kind of preparation method for the monoclonal antibody being suitable for detecting human peripheral expression CD161 molecule subgroup lymphocytes, It includes following step：

A：It is prepared by the hybridoma cell strain of CD161 monoclonal antibodies：

A-a：Utilize the MonoExpress Gold Antibody Service of the bio tech ltd Jin Sirui (mouse)) it services, CD161 immunogen proteins is prepared using the method for DNA artificial sequence synthetic and prokaryotic expression protein；Specifically The CD161 immunogen protein purity reaches 90% or more, spare；

A-b：The CD161 immunogen proteins of above-mentioned purifying are mixed with Freund's adjuvant, it is thin with myeloma used that animal selection is immunized The homologous BALB/c healthy mices three of born of the same parents, age of mouse was at 8-14 weeks, and abdominal part hypodermic, every is immunized 100 μ g albumen every time；Altogether It is immune three times, be spaced three weeks per between twice；Second of immune posterior orbit vein is from 100 μ l of blood sampling, using Enzyme-linked Immunosorbent Assay Test method detects antiserum, and antibody titer, which is equal to, is higher than 1:1000, the highest mouse of titre is selected, it is spare；

A-c：It takes a healthy ICR mouse, neck dislocation to put to death, after body surface disinfection and four limbs fixation, skin is cut off from thigh Skin, exposure peritonaeum, cotton ball soaked in alcohol sterilize peritonaeum.With 10ml syringes, 12^#Syringe needle, injection 5-10ml HAT culture mediums to abdominal cavity, The right hand fixes syringe, and left hand holds cotton ball soaked in alcohol gently abdomen massage, draws back intraperitoneal liquid, centrifuges, and sediment adds HAT to cultivate Liquid is resuspended, spare as the feeder cells of fused cell culture later；

A-d：3 days after booster immunization BALB/c mouses are taken, eyeball blood sampling is extractd and is put to death for detaching positive serum, neck dislocation, nothing Bacterium opens abdominal cavity and takes out spleen, splenocyte suspension is made, through standing, centrifugation, ice-water bath, centrifugation, dilution, counting and RPMI- 1640 culture medium washs to obtain mouse boosting cell suspension, spare；

A-e：Under the Sp2/0 myeloma cell with greater activity is rinsed from culture vessel wall with RPMI-1640 culture solutions Come, is washed through collection, centrifugation, dilution, counting and RPMI-1640 culture solutions, it is spare；

A-f：The myeloma cell prepared through above-mentioned steps and splenocyte are mixed in the centrifuge tube with cover of a 50ml, 1000rpm centrifuges 10min, and supernatant fully exhausts, and mixing；With 1ml suction pipes by the PEG4000 of preheating in 45-60s slowly plus Into above-mentioned centrifuge tube, shake up, immediately be added dropwise 37 DEG C preheating DMEM culture mediums, make PEG4000 dilute and it is ineffective；

A-g：Cell culture to be fused observed the growing state of hybridoma to the 5th day, waited for that its cells and supernatant becomes When yellow or clone is distributed to 1/10 or more of hole floor space, appropriate cell conditioned medium is drawn, through limiting dilution 1:10 start, 3 times of ladders Degree dilution, totally 6 gradients, antibody content is detected with ELSA methods, and the antibody point of high-titer is filtered out according to the secretion situation of antibody It secretes hole and carries out hybridoma cell strain subclone, monoclonal antibody expression and conservation；

B：To above-mentioned steps obtain hybridoma secretion 161 protein monoclonal antibody of mouse-anti-human T lymphocyte CD it is pure Change and verifies：

According to supernatant affinity testing result, 5 plants of hybridoma cell strains with high-affinity are selected to carry out conservation, and choose one Strain cell strain carries out a small amount of cultures and IgG purifying, is as follows：

B-a. antibody producing：With containing 10%FBS, 1%Penicillin-Streptomycin DMEM culture mediums, by cell strain Expand culture to 20ml, culture medium supernatant is harvested after 4 days, cell fragment is removed in centrifugation；

B-b. antibody purification：Supernatant crosses proteinG purification columns, then 30 times of column volumes of phosphate buffer, then with 0.1M's Glycine (PH=2.8) elutes and collects the antibody of combination, and dialysis removes remaining glycine, and it is dense that A280 methods detect antibody Degree；

B-c. antibody is verified：The affinity of ELISA method detection IgG purification and CD161 antibody, is coated with respectively on elisa plate Gradient dilution is added after washing closing in CD161 immunogen proteins and His tag fusion proteins (1 μ g/ml, 100 μ l/well) Purify CD161 antibody；The sheep anti-mouse igg that horseradish peroxidase-labeled is added is secondary antibody, TMB colour developings, microplate reader reading OD450 Value；The affinity and specificity of IgG and CD161 antibody are detected with Western Blot methods.

2. the monoclonal antibody as claimed in claim 1 for being suitable for detecting human peripheral expression CD161 molecule subgroup lymphocytes Preparation method, it is characterised in that in the hybridoma cell strain preparation process of the CD161 monoclonal antibodies use ELSA It is as follows that method measures sero-fast concrete operations：

CD161 immunogen proteins in step A-a are configured to 1 μ g/ml strength solutions, 100 μ l/well with phosphate buffer 96 hole polyethylene boards are coated with, vacuum is drained, and 4 DEG C of sealing saves backup；The 200 μ l of phosphate buffer that pH is 7.4 are added per hole It washs, include 1% lowlenthal serum；Be added per hole third time it is immune after 3 days clear 50-100 μ l of mouse peripheral blood (1:1000- 5000 dilutions), normal control, positive control and blank (phosphate buffer), 37 DEG C of incubation 1h, phosphate buffer are set per plate Washing 3 times；ELIAS secondary antibody is added per hole 100-200 μ l, washs；Substrate 50-100 μ l are added per hole, is incubated at room temperature 10min, makes It is terminated and is reacted with terminate liquid；It is returned to zero with blank, is longer than at 450nm in wave through microplate reader and reads OD value OD；As a result judge：P/ N=measures sample OD mean values/negative serum OD mean values, and P/N >=2.1 are the positive.

3. a kind of 161 protein monoclonal antibody of mouse-anti-human T lymphocyte CD with described in claim 1 prepares active tuberculosis With tuberculosis latent infection flow cytometer detection kit, it is characterised in that it includes CD161-PE streaming antibody, remaining required streaming is anti- Body uses protein specific antibody known in the art：MACS or BD or Beckman or R＆D.

4. the active tuberculosis described in a kind of claim 3 and tuberculosis latent infection flow cytometer detection kit, it is characterised in that Active tuberculosis and tuberculosis latent infection flow cytometer detection kit prepare streaming fluorescence antibody using R-PE coupling methods：Mouse is anti-human 161 protein monoclonal antibody of T lymphocyte CDs according toR-PHYCOERYTHRIN(RPE)CONJUGATION The operating instruction of KIT (Prozyme, PJ31K) carries out R-PE labels to CD161 antibody, obtains CD161-PE streaming antibody；Remaining Required streaming antibody uses protein specific antibody known in the art：MACS or BD or Beckman or R＆D.