CN101974099A - Purification method of tubercle bacilipin Arab mannan and tubercle bacillus diagnostic reagent - Google Patents
Purification method of tubercle bacilipin Arab mannan and tubercle bacillus diagnostic reagent Download PDFInfo
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Abstract
The invention belongs to the technical field of medical treatment, and in particular relates to a purification method of tubercle bacilipin Arab mannan (LAM) and a tubercle bacillus diagnostic reagent. In the invention, by utilizing the structural differences that the LAM antigen contains three mannose while other impurities (LM, PIM, and the like) only contain one mannose, and the appetency of lectin (such as flat lentils lectin) to three manna branch structures is far higher than two manna and one manna, the LAM antigen purification is carried out by using the lectin to obtain the LAM antigen with the purity of above 95%. The high-purity antigen obtained by purification can form a reagent for LAM antigen detection and can also form a reagent for enriching urine LAM antigen, purifying LAM antibody and detecting the urine LAM antibody.
Description
Technical field
The invention belongs to field of medical technology, be specifically related to a kind of preparation method and tubercule bacillus diagnostic reagent of tubercule bacillus fat AM.
Background technology
LAM antigenTubercule bacillus survives in owner's body, breeds by unique cell wall structure.Its bacillus tubercle cell wall contains multiple composition (Fig. 1), as: porin (Porin), polysaccharide, lipid (Lipids), mycolic acid (mycolic acid), phosphatidylinositol mannosidase (phosphatidylinositol mannosides.PIM), fat mannosans (lipomannans, LM), and the fat AM (lipoarabinomannan, LAM).Wherein, LAM is the main component of mycobacterium cell walls, reaches 15 milligrams/g bacterium gross weight.The infected host's of LAM extensive influence immunity system.It is reported, can cause the T cell inhibitory effect from mycobacterium tuberculosis and the isolated LAM of leprosy bacillus, disturb macrophage activation with the Interferon, rabbit mediation, remove deleterious free oxygen free radical, arrestin kinase activity and plain (IL)-2 of genes encoding Jie and IL-5 and granulocyte synthesize/macrophage colony stimulating human T cytokine, induce the scavenger cell immediate early gene to express, promote the generation enhancing and the complement activation of monocyte tumour necrosis factor.Result of study shows as genus-specific antigen LAM, to have stronger immunogenicity and immunoloregulation function, has participated in many processes of mycobacterium tuberculosis infection, helps tuberculosis morbidity (pulmonary tuberculosis) and other mycobacterial infectionses.There is building-up process in the thalline from PI → PIMs → LM → AraLAM.Fig. 2 is fat AM (LAM), fat mannosans (LM) structure.
The antigenic acquisition of LAMLAM antigen generally extracts from the thalline of a large amount of cultivations and obtains, usually earlier with the cell walls fragmentation, at first extract, again by hydrophobic chromatography coupled ion displacement chromatography and gel-filtration through organic solvent (50-70% ethanol, phenol etc.), obtain pure antigen, preparation process is very complicated.Nonetheless, because fat AM (LAM) is very close with fat mannosans (LM), phosphatidylinositol mannosidase (PIM) structure etc., needs multistep process owing to proximate components such as LM, PIM, polysaccharide will be removed from the LAM antigenic solution.Cause batch preparations very difficult, often use Combination antigen.
The LAM antigen mycobacterium tuberculosis antibody that is used to circulate detectsBecause fat AM (LAM) can stimulate body to produce corresponding antibody, serum LAM antibody test can be used as effective aided diagnosis method of tuberculosis.
The purity of fat AM (LAM) has directly determined the quality (sensitivity and specific degree) of serum LAM antibody test quality product in the mycobacterium tuberculosis antibody diagnostic reagent.Some non-genus-specific antigens such as fat mannosans (LM), the serious interference detection results of meeting such as phosphatidylinositol mannosidase (PIM), the specificity of reduction reagent.
Therefore, high purity LAM antigen is most important for the quality product of the antibody diagnosis of tubercule bacillus.
LAM antigen is used for negre antigen and detectsDetection of antigen not only can be used for the discovery and the diagnosis of infectious disease pathogens, can also carry out real-time assessment to the patient in the treatment, is the method that the transmissible disease diagnosis is praised highly.In human body, animal body, infected tubercule bacillus or tubercule bacillus breeds in vivo, can find fat AM (LAM) in the urine, because this detects the restriction of not infected lesions position (lung, stomach, kidney, bone, spinal cord, brain, bladder etc.).Therefore, detect the breakthrough means that AM (LAM) in the urine may become diagnosis of tuberculosis.
But fat AM (LAM) concentration lower (0.01-1ng/mL) in the tuberculosis patient urine, have only higher sensitivity immunological method or through other concentrate, enriching method could measure after handling, and the prerequisite that improves immunological method sensitivity is high purity, high-affinity antibody, and the preparation of this antibody needs high purity fat AM (LAM).
Summary of the invention
An object of the present invention is to provide a kind of method of easy acquisition high purity tubercule bacillus fat AM (LAM), it is the mycobacterium tuberculosis antibody detection reagent and the LAM antigen detecting agent of basis preparation with the high purity LAM antigen that obtains that another purpose provides a kind of.
The present invention utilizes LAM antigen to contain the structural difference (Fig. 1, Fig. 2) that 3 other impurity of seminose (LM, PIM etc.) only contain 1 seminose, utilize lectin (as flat LcA) to the avidity of 3 seminose branched structures avidity characteristic again far above 2 seminoses and 1 seminose, use lectin to carry out the LAM antigen purification, obtain purity and reach the above LAM antigen of 90 %, more excellent obtained purity reaches the above LAM antigen of 95 %.The high purity antigen that above-mentioned purifying obtains can be formed reagent and be used for the LAM antibody test; The high purity antigen that above-mentioned in addition purifying obtains also can be formed reagent and be used for the antigenic enrichment of urine LAM, LAM antibody purification and urine LAM Detection of antigen.
1, the method for acquisition high purity tubercule bacillus fat AM provided by the invention (LAM) is the method that adopts existing affinity purification, and its basic step comprises: the dress post, go up sample, wash, dissociate, electrophoretic separation and analysis.Wherein, adopt lectin (as flat LcA) as affinity purification reagent.
In the inventive method, the indication lectin comprises: and ConA or concanavalin A (Concanavalin ensifomis agglutinin, COA); Lens culinaris agglutinin (Lens cuinaris agglutinin, LCA); Pisum sativum agglutinin (Pisum satiwum agglutinin); DBL (Dolichos bifows agglutinin, DBA); Sophora japonica lectin (Sophora japonica agglutinin, SjA); Peanut agglutinin (Peanut agglutinin; PNA); The wheat germ element (Wheat germ agglutinin, WGA); Soybean agglutinin (Soybean agglutinin, SBA) etc.
In the inventive method, the indication lectin refer in particular to can with the lectin of seminose, glucose specific combination, comprising: ConA or concanavalin A (Concanavalin ensifomis agglutinin, COA); Flat LcA (Lens cuinaris agglutinin, LCA); Pisum sativum agglutinin (Pisum satiwum agglutinin, PSA) etc.
In the inventive method, and the preferred flat LcA of indication lectin (Lens cuinaris agglutinin, LCA).
Among the present invention, the antigenic purifying of described LAM, the enrichment of urine LAM antigen are meant by general ordinary method such as affinity chromatography or other solid phase (magnetic bead etc.) method to be undertaken;
Among the present invention, described LAM antibody test is meant that the high purity LAM antigen that utilizes the lectin affinity purification carries out TPPA in the serum; Can improve the specificity of detection reagent.
Among the present invention, described LAM Detection of antigen is meant that high purity LAM antigen cross-linking with the lectin affinity purification is to the chromatography column material, obtain high purity antibody by large scale purification, and then set up the highly sensitive immunological method and be used for urine fat AM Detection of antigen.
Among the present invention, described LAM Detection of antigen can also be to utilize lectin to replace antibody and get up to detect with antibodies, can select to use amplification systems such as enzyme, gold sol, chemoluminescence, fluorescence, time resolved fluorescence to detect.
2, the high purity LAM antigen that obtains with aforesaid method also is provided is the mycobacterium tuberculosis antibody detection reagent of basis preparation in the present invention.
The essentially consist of this mycobacterium tuberculosis antibody detection reagent: confining liquid, washings, gold sol marker (goat anti-human igg's gold sol marker), diafiltration Sptting plate (containing NC film, thieving paper, LAM antigen, human IgG).
The principal character of this reagent is to use in the diafiltration Sptting plate LAM of 95% above purity antigen coated, and LAM antigen consumption can be adjusted according to the gold sol marker concentrations, is advisable with 0.01-2mg/mL concentration bag, and is preferably good for 0.05-1 mg/mL.
Phosphoric acid or TRIS damping fluid in confining liquid, the washings, its PH value scope is advisable with 6.5-8.5, between the best 7.0-8.0, best 7.2.
Contain an amount of tensio-active agent tween 20 in confining liquid, the washings and reach the sealing purpose, the content of tween 20 is the 0.01-0.1% of confining liquid or washings by weight, best 0.05%.
Also containing an amount of polyoxyethylene glycol (PEG) in confining liquid, the washings increases the permeability of film, improve antigen antibody reaction speed, the PEG molecular weight between 2000-40000, best 10000-20000, the PEG consumption is the 0.1-2% of confining liquid or washings by weight, best 1%.
3, the high purity LAM antigen that obtains with aforesaid method also is provided is the LAM antigen detecting agent of basis preparation in the present invention.
The essentially consist of this reagent: LCA enriching column or LCA enrichment pipe and sandwich assay immunity detection reagent.
LCA enriching column (LCA-Sepharose 4B) or LCA enrichment pipe (immune magnetic particle of LCA bag quilt) are used for the pre-treatment of sample, and the LAM that contains in the urine is carried out enrichment or concentrated, reach and improve the sensitivity that detects.
Among LCA-Sepharose 4B on the Sepharose 4B crosslinked LCA amount be not less than 1mg/mL, more than best 2 mg/mL; Use immune magnetic particle to contain the LCA amount and be not less than 0.01mg/mg, be preferably in more than 0.03 mg/mg.
The sandwich assay immunity detection reagent is made up of enzymic-labelled antibody, reaction substrate, washings and microdetermination Sptting plate.
The antibody (antibody of enzymic-labelled antibody, microdetermination Sptting plate bag quilt) that the sandwich assay immunity detection reagent uses must obtain by affinitive layer purification, promptly utilize affinitive layer purification antibody, the antibody that obtains through affinity chromatography has higher specificity and avidity to LAM antigen, can reach the sensitivity that LAM sandwich assay immunoreagent detects.
LAM is coupled to solid phase carrier and obtains affinity column, the principal character that is the affinity chromatography preparation is that the high purity LAM that embodiment 1 obtains is coupled to solid phase carrier obtains affinity column (LAM-beads), crosslinkedly select to use methods such as cyanogen bromide-activated, sodium periodate oxidation, carbodiimide coupling to connect, the best lower concentration sodium periodate method for oxidation that adopts.
Described lower concentration sodium periodate oxidation style refers to that used sodium periodate concentration is lower than conventional amount used, and oxidation LAM antigen gets sodium periodate concentration and is advisable with 1mM-10mM, preferably 5mM.
Tubercule bacillus LAM antigen detecting agent preparation process as shown in Figure 6.
Description of drawings
Fig. 1 is the bacillus tubercle cell wall structure.
Fig. 2 is fat AM (LAM), fat mannosans (LM) structure.
Fig. 3 is that ConA-(COA-Sepharose 4B, 2-5) (LCA-Sepharose 4B 7-10) is used for fat AM (LAM) purifying, the electrophorogram of 12%SDS-PAGE after silver dyes colour developing behind the purifying with flat LcA.
1:Crude is a sample solution, and mycobacterial cell wall reference method is extracted the purifying after product.Wherein:
2,7:Filtration was a post liquid, promptly went up the sample effluent liquid,
3,4,8,9:Elute is a washings,
5,10:Wash is a dissociation solution
The 6:M molecular weight marker is followed successively by 97,66,43,31,20,14.4KD from top to bottom
The result shows: contain a large amount of impurity (comprising LM, PIM etc.) the LAM(sample solution of chemical process purifying), handle through LCA-Sepharose 4B, swimming lane 10 almost completely is the LM that LAM(only contains minute quantity); COA-Sepharose 4B also can adsorb LAM, but effect is not as LCA, especially PIM.
Fig. 4 is that flat LcA sandwich assay detects LAM antigen.
Fig. 5 is LAM antibody test result.
Fig. 6 is a tubercule bacillus LAM antigen detecting agent preparation flow.
Embodiment
1. obtain the method for high purity tubercule bacillus fat AM (LAM):
The present invention has found that a class phytohemagglutinin has very high affinity to tubercule bacillus fat AM (LAM), set up the purification process of tuberculosis bacilipin AM (LAM) antigen lectin affinity chromatography by this, used this method can obtain purity easily in a large number and reach LAM antigen more than 95%.
Experiment material: ConA-dextrane gel 4B(COA-Sepharose 4B), flat LcA-dextrane gel 4B(LCA-Sepharose 4B) available from GE company (former Pharmacia), methyl glucoside is available from Sigma company, and other reagent is available from traditional Chinese medicines share (Shanghai) Co., Ltd..
Fat AM (LAM Crude) is from mycobacterial cell wall reference method extraction and purification, and fat AM (LAM Crude) is with 20mM Tris-HCl, pH7.6,0.15M NaCl, 1mM MgCl
2, 1mM MnCl
2Balance, total sugar content (1mg/ml).
The affinity purification process:
1. dress post: get COA-Sepharose 4B and LCA-each 1mL of Sepharose 4B centrifugal post of packing into respectively, with 20mM Tris-HCl, pH7.6,0.15M NaCl, 1mM MgCl
2, 1mM MnCl
2Balance.
2. go up sample: each 0.5ml of sample is added, and centrifugal behind the 5min, the centrifugal liquid that comes out was post liquid (Filtration).
3. washing: with 20mM Tris-HCl, pH7.6,0.15M NaCl, 1mM MgCl
2, 1mM MnCl
2Washing, centrifugal 2 times, obtain Elute 1 respectively successively, Elute 2.
4. dissociate: add 20mM Tris-HCl, pH7.6,0.15M NaCl, 0.5M methyl glucoside, 1mM MgCl
2, 1mM MnCl
2Centrifugal behind the 5min, the centrifugal liquid that comes out is dissociation solution (WASH).
5. electrophoretic separation and analysis: distinct portions in the affinity purification process is used 12%SDS-PAGE, alkali colour developing after silver dyes.
The swimming lane explanation:
1: for sample solution is fat AM (LAM Crude)/PBS, total sugar content (1mg/ml)
2,7:Filtration was a post liquid, promptly went up the sample effluent liquid,
3,4,8,9:Elute is a washings,
5,10:Wash is an elutriant
The 6:M molecular weight marker is followed successively by 97,66,43,31,20,14.4KD from top to bottom
Fig. 3 experimental result shows: contain a large amount of impurity (comprising LM, PIM etc.) the LAM(sample solution of chemical process purifying), handle through LCA-Sepharose 4B, swimming lane 10 almost completely is the LM that LAM(only contains minute quantity); COA-Sepharose 4B also can adsorb LAM, but effect is not as LCA, especially PIM.
General method obtains slightly carries fat AM LAM(LAM Crude) (Fig. 3. swimming lane 1, Crude),
LAM antigen behind the purifying (Fig. 3. swimming lane 10 WASH).Calculate purity through the scanning gray scale, slightly put forward fat AM LAM purity and only account for 30% of total reducing sugar amount, the LAM antigen purity can reach more than 95% behind this method purifying.
Embodiment 2.
Experiment material:: flat LcA (LCA) is from flat root of Szemao crotalaria self-control purifying, and other reagent is available from traditional Chinese medicines share (Shanghai) Co., Ltd..
Fat AM (LAM Crude) is with 20mM Tris-HCl, pH7.6,0.15M NaCl, 1mM MgCl
2, 1mM MnCl
2The balance liquid balance;
Flat LcA (LCA) gold sol mark: conventional citrate three sodium method prepares Radioactive colloidal gold, and selects the optimum mark substrate concentration, carries out flat LcA mark, obtains flat LcA (LCA) gold sol marker (not containing sucrose);
Flat LcA (LCA) is fixing: with flat LcA (LCA) 1mg/mL point on nitrocellulose filter (NC) bar in 0.6um aperture (5mm * 3mm), after the drying, with 20mM Tris-HCl, pH7.6,0.15M NaCl, 1mM MgCl
2, 1mM MnCl
23%BSA, the 0.05%TWEEN-20 sealing;
Sandwich assay detects:
On 96 hole microwell plates, the flat LcA of 20ul (LCA) gold sol marker is mixed with 20ul balance liquid, 20ul fat AM (LAM Crude) total sugar content 0.1mg/ml, 20ul fat AM (LAM Crude) 1mg/ml respectively, (5mm * 3mm) insert in the hole respectively utilizes the chromatography colour developing with three nitrocellulose filters (NC) bar.
Fig. 4 experimental result shows: fat AM 0.1, the equal display dot of 1mg/ml, presentation of results owing to exist 3 seminose branched structures to have two in the LAM antigen, can form sandwich structure in conjunction with 2 flat LcAs.Although have non-specific combination (containing seminose in the lipopolysaccharides of many mycetocyte walls) because above LCA detects,
The experimental result prompting: but use single flat LcA sandwich assay can be used for the LAM detection of antigens.
2. serve as the mycobacterium tuberculosis antibody detection reagent of basis preparation with high purity LAM antigen:
Detection reagent is formed: confining liquid, washings, gold sol marker (goat anti-human igg's gold sol marker), diafiltration Sptting plate (containing NC film, thieving paper, LAM antigen, human IgG).
The principal character of reagent is to use in the diafiltration Sptting plate LAM of 95% above purity antigen coated, and LAM antigen consumption is adjusted according to the gold sol marker concentrations, is advisable with 0.01-2mg/mL concentration bag, and is preferably good for 0.05-1 mg/mL.
Phosphoric acid or TRIS damping fluid in confining liquid, the washings, its PH value scope is advisable with 6.5-8.5, between the best 7.0-8.0, best 7.2.
Contain an amount of tensio-active agent tween 20 in confining liquid, the washings and reach the sealing purpose, consumption between 0.01-0.1%, best 0.05%.
Also containing an amount of polyoxyethylene glycol (PEG) in confining liquid, the washings increases the permeability of film, improves antigen antibody reaction speed, the PEG molecular weight between 2000-40000, best 10000-20000, PEG consumption between 0.1-2%, best 1%.
The gold sol marker can be by general preparation method's preparation.
The using method of reagent is, adds 4 of confining liquids in the diafiltration Sptting plate, and increase serum 50uL adds 4 of washingss, adds 3 of gold sols, adds 4 of washingss, observed result.
Because the LAM antigen of general method purifying only has 50% purity, contain assorted a large amount of (near 30-50%) fat mannosans (LM), phosphatidylinositol mannosidase (PIM), LM, PIM are as non-genus-specific antigen meeting disturbance reponse, therefore, the specificity reduction that the LAM Detection of antigen can make the LAM antibody test is slightly proposed in use.Reagent has used this 95% above purity LAM antigen that utilizes example 1 to obtain as the antigen that detects antibody the specificity of detection to be improved.
Experiment material: 184 parts of serum, wherein, confirm 78 parts of serum lungy, 106 parts of non-tuberculosis patient serum.
The goat anti-human igg is available from Sigma company; reagent such as trisodium citrate, hydrochloro-auric acid, salt of wormwood, sodium-chlor, bovine serum albumin (BSA), tween 20, polyoxyethylene glycol are available from traditional Chinese medicines Shanghai chemical reagent company limited, and plain film NC is available from AMERSHAM company for 0.65um nitric acid thousand dimension.Gold sol, goat anti-human igg's gold sol marker diafiltration reaction box are provided by Xiamen Bo Sheng Bioisystech Co., Ltd, and experimentation is:
1. confining liquid preparation: 0.1%BSA/ 0.05% tween 20/1% polyoxyethylene glycol PH7.2 phosphoric acid buffer (PBS),
2. washings preparation: 0.05% tween 20/1% polyoxyethylene glycol PH7.2 phosphoric acid buffer (PBS),
3. gold sol preparation, goat anti-human igg's gold sol marker compound method: 0.01% hydrochloro-auric acid 100mL is boiled, add 1% trisodium citrate 1mL, cooling.Salt of wormwood is regulated pH according to a conventional method, adds the 10%NaCl judgement and selects antibody optimum mark concentration, determines that 10ug/mL is advisable, and behind the adding salt of wormwood, adds the 1mg goat anti-human igg in the 100mL gold sol, adds 10mg/mL BSA 5mL after mixing.
4. the plain film of 0.65um nitric acid thousand dimension is cut into 1.5 * 1.5cm square, during production method is packed the diafiltration reaction box that contains thieving paper into routinely, on the plain film intermediate point of diafiltration reaction box nitric acid thousand dimension, mix or purifying LAM antigen and positive control point (purifying human IgG).
5. testing process: add 4 of confining liquids, increase serum 50uL adds 4 of washingss, adds 3 of gold sols, adds 4 of washingss, observed result.
6. detected result: to confirming 78 parts of serum lungy, detection such as table 1 that non-tuberculosis patient serum is 106 parts:
Table 1 is confirmed 78 parts of serum lungy, the detected result that non-tuberculosis patient serum is 106 parts
。
Slightly carry the purified back of fat AM LAM the sensitivity that positive serum detects is not reduced (74.3%), but 106 parts of non-tuberculosis patient serum detection specificity bring up to 94.3% from 89.7%.Illustrate and slightly put forward fat AM LAM can obviously improve diagnostic reagent through purifying specificity (Fig. 5, partial L AM antibody test result).
3. serve as the tubercule bacillus LAM antigen detecting agent of basis preparation with high purity LAM antigen:
The essentially consist of reagent: LCA purifying/enrichment pipe and sandwich assay immunity detection reagent.
LCA purifying/enrichment pipe (immune magnetic particle that contains LCA-Sepharose 4B or LCA bag quilt) is used for the pre-treatment of sample, and the LAM that contains in the urine is carried out enrichment or concentrated, reaches and improves the sensitivity that detects.
Among LCA-Sepharose 4B on the Sepharose 4B crosslinked LCA amount be not less than 1mg/mL, more than best 2 mg/mL; Use immune magnetic particle to contain the LCA amount and be not less than 0.01mg/mg, be preferably in more than 0.03 mg/mg.
The sandwich assay immunity detection reagent is made up of enzymic-labelled antibody, reaction substrate, washings and microdetermination Sptting plate.
The sandwich assay immunity detection reagent is characterised in that the antibody (antibody of enzymic-labelled antibody, microdetermination Sptting plate bag quilt) of use must obtain by affinitive layer purification, promptly utilize affinitive layer purification antibody, the antibody that obtains through affinity chromatography has higher specificity and avidity to LAM antigen, can reach the sensitivity that LAM sandwich assay immunoreagent detects.
LAM is coupled to solid phase carrier and obtains affinity column, the principal character that is the affinity chromatography preparation is that the high purity LAM that embodiment 1 obtains is coupled to solid phase carrier obtains affinity column (LAM-beads), crosslinkedly select to use methods such as cyanogen bromide-activated, sodium periodate oxidation, carbodiimide coupling to connect, the best lower concentration sodium periodate method for oxidation that adopts.
Described lower concentration sodium periodate oxidation style refers to that used sodium periodate concentration is lower than conventional amount used, is advisable with 1mM-10mM, preferably 5mM.
?
Tubercule bacillus LAM antigen detecting agent preparation process as shown in Figure 6.The preparation of tubercule bacillus LAM antigen detecting agent, application are divided into 5 parts:
A.LCA purifying/enrichment pipe (containing LCA-Sepharose 4B) preparation: comprise LCA purifying and crosslinked.
B.LAM antigen purification and antibody affinity chromatography preparation: comprise bacterium cultivation, chemical purification, antigen affinity purification, antigen cross-linking.
C.LAM Antibody Preparation and purifying: comprise bacterium cultivation, immune animal, antibody affinity purification.
D. sandwich assay immunologic function test reagent preparation: comprise preparations such as antibody labeling, antibody sandwich substrate, washings.
E. fat AM LAM Detection of antigen in the urine: require to select to use detection method after directly detection or the enrichment according to sensitivity.
Claims (8)
1. a tubercule bacillus fat AM (LAM) purification process is the method that adopts existing affinity purification, and its basic step comprises: the dress post, go up sample, wash, dissociate, electrophoretic separation and analysis; It is characterized in that adopting lectin as affinity purification reagent.
2. tubercule bacillus fat AM purification process according to claim 1 is characterized in that described lectin is ConA or concanavalin A, lens culinaris agglutinin, pisum sativum agglutinin, DBL, sophora japonica lectin, peanut agglutinin, wheat germ element or soybean agglutinin.
3. tubercule bacillus fat AM purification process according to claim 1, it is characterized in that described lectin be meant can with the lectin of seminose, glucose specific combination, comprising: ConA or concanavalin A, flat LcA or pisum sativum agglutinin.
4. tubercule bacillus fat AM antigen that is obtained by claim 1,2 or 3 described method purifying, its concentration is more than 90 %.
One kind as LAM antigen as described in the claim 4 in preparation LAM antibody test with the application in the reagent.
6. application according to claim 5 is characterized in that the essentially consist of described reagent comprises: confining liquid, washings, gold sol marker, diafiltration Sptting plate; Wherein:
Use the LAM of 90% above purity antigen coated in the diafiltration Sptting plate, LAM antigen consumption is adjusted according to the gold sol marker concentrations, and the concentration bag is 0.01-2mg/mL;
Phosphoric acid or TRIS damping fluid in confining liquid, the washings, its PH value scope is 6.5-8.5;
Contain an amount of tensio-active agent tween 20 in confining liquid, the washings, the tween 20 weight content is 0.01-0.1%;
Also contain polyoxyethylene glycol in confining liquid, the washings, molecular weight polyethylene glycol is between 2000-40000, and the polyoxyethylene glycol consumption is 0.1-2% by weight.
One kind as LAM antigen as described in the claim 4 in the enrichment of preparation urine LAM antigen, LAM antibody purification and urine LAM Detection of antigen with the application in the reagent.
8. application according to claim 7 is characterized in that the essentially consist of described reagent comprises: LCA enriching column or LCA enrichment pipe, and sandwich assay immunity detection reagent, and here, LCA is a lens culinaris agglutinin; Wherein:
LCA enriching column or LCA enrichment pipe are used for the pre-treatment of sample, and the LAM that contains in the urine is carried out enrichment or concentrated;
In the LCA enriching column on the Sepharose 4B crosslinked LCA amount be not less than 1mg/mL; The immune magnetic particle that uses in the LCA enrichment pipe contains the LCA amount and is not less than 0.01mg/mg;
The sandwich assay immunity detection reagent is made up of enzymic-labelled antibody, reaction substrate, washings and microdetermination Sptting plate;
The antibody that the sandwich assay immunity detection reagent uses comprises: the antibody of enzymic-labelled antibody, microdetermination Sptting plate bag quilt, obtain by affinitive layer purification, promptly utilize affinitive layer purification antibody, the antibody that obtains through affinity chromatography has higher specificity and avidity to LAM antigen, can reach the sensitivity that LAM sandwich assay immunoreagent detects; Wherein, the affinity column used of affinitive layer purification is coupled to solid phase carrier by the described high purity LAM of claim 4 and obtains.
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| CN116120392A (en) * | 2023-04-18 | 2023-05-16 | 上海健士拜生物科技有限公司 | Method for purifying polymer protein |
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| CN102707053A (en) * | 2012-06-07 | 2012-10-03 | 上海交通大学 | Carbohydrate chip for quickly diagnosing tuberculosis, preparation method thereof and tuberculosis diagnostic kit adopting carbohydrate chip |
| CN104133027A (en) * | 2014-06-24 | 2014-11-05 | 江南大学 | Method for separating and identifying glycoprotein N-sugar chain structure |
| CN105137072A (en) * | 2015-04-30 | 2015-12-09 | 广东国际旅行卫生保健中心（广东出入境检验检疫局口岸门诊部） | Mycobacterium tuberculosis LAM (lipoarabinomannan) detection kit, preparation and use method thereof |
| CN110501492A (en) * | 2019-08-06 | 2019-11-26 | 上海荣盛生物药业有限公司 | Fat arabian mannan detects sensitizer and combinations thereof and kit |
| CN110568186A (en) * | 2019-08-06 | 2019-12-13 | 上海荣盛生物药业有限公司 | Component for detecting lipoarabinomannan and kit thereof |
| CN110568185A (en) * | 2019-08-06 | 2019-12-13 | 上海荣盛生物药业有限公司 | Lipoarabinomannan detection aid, composition and kit thereof |
| CN110501492B (en) * | 2019-08-06 | 2022-07-01 | 上海荣盛生物药业股份有限公司 | Lipo-arabinomannan detection sensitizer, composition and kit thereof |
| CN111337665A (en) * | 2020-01-16 | 2020-06-26 | 卢氏实验室公司 | Immunochromatographic test strip for detecting tuberculosis infection and preparation method thereof |
| CN116120392A (en) * | 2023-04-18 | 2023-05-16 | 上海健士拜生物科技有限公司 | Method for purifying polymer protein |
| CN116120392B (en) * | 2023-04-18 | 2023-08-01 | 上海健士拜生物科技有限公司 | Method for purifying polymer protein |
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