CN107064514A - For detecting kit and its detection method with biological activity MBL in human blood - Google Patents
For detecting kit and its detection method with biological activity MBL in human blood Download PDFInfo
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- CN107064514A CN107064514A CN201710109471.XA CN201710109471A CN107064514A CN 107064514 A CN107064514 A CN 107064514A CN 201710109471 A CN201710109471 A CN 201710109471A CN 107064514 A CN107064514 A CN 107064514A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention discloses for detecting kit and its detection method with biological activity MBL in human blood, belong to the ELISA detection technique fields of mannose binding lectin.Technical scheme main points are：There is biological activity MBL kit for detecting, binding partner of the kit using mannosan as MBL is used as MBL detection antibody using anti-human MBL CLR mAb in human blood.The invention further particularly discloses the detection method that this is used to detect the kit with biological activity MBL in human blood.The present invention utilizes mannosan and high polymer MBL high-affinities and Ca2+The characteristics of highly relying on, the good MBL of bioactivity is captured first, in order to exclude the interference for the other non-destination proteins that can be combined in blood plasma with mannosan, the anti-human MBL CLR monoclonal antibodies of mouse are introduced again as detection antibody, the characteristic uniquely combined with the anti-human MBL CRD monoclonal antibodies of mouse using MBL, it is ensured that the specificity of detection.
Description
Technical field
The invention belongs to the ELISA detection technique fields of mannose binding lectin, and in particular to one kind is used to detect people
Kit and its detection method with biological activity MBL in blood.
Background technology
Mannose binding lectin（Mannan-binding lectin, MBL）It is that the main height by hepatocytes secrete is protected
The plasma protein kept, is collectin in c-type agglutinin superfamily（collectins）Family member.MBL is also a kind of acute stage
Reactive protein, stress such as pathogenic infection, surgical operation when, its plasma concentration can raise 2-3 times.With preceding antibody
（ante-antibody）The MBL of title pass through its carbohydrate recognition domain（Carbohydrate recongnition domain, CRD）
Identification is distributed in the sugared structure on the multiple pathogens such as surface such as bacterium, virus, parasite, fungi, including D-MANNOSE, L- extensively
Fucose, N-Acetyl-D-glucosamine, ManNAc etc., its collagen-like region（Collagen-like region, CLR）Knot
Close mannosan associated serine protease（MBL-associated serine protease, MBL-MASP）, activating complement
Lectin pathway and play molten broken and indirect conditioning functions；Or combined with phagocyte collectin acceptor, with independent of complement
Mode start opsonophagocytosis.Science once published the article（Thompson C. Protein proves to be a key link
in innate immunity[J]. Science, 1995,269(5222):301-302.）It is called " in natural immune system
Key molecule ".Available data is it has been shown that MBL is most important anti-infective innate immune molecule in not immune host.
With deepening continuously for studying MBL, it is found that it, except can recognize that and remove pathogen, is also equipped with many endogenous
Function（endongenous functions）, such as combine autoimmunity globulin；Recognize ischemical reperfusion injury in exposure from
The autoantigen modified under body antigen or morbid state；Participate in the phagocytosis to apoptotic cell；Can be thin with monocytic series THP1
Born of the same parents interact, and suppress the cytokine secretion of Calbicans inductions, while to bone-marrow-derived lymphocyte system Raji cells and T cell system
Jurkat cell is respectively provided with immunoregulation effect.Therefore, MBL is used as pattern-recognition having multi-functions in the innate immunity point
Son, is not only only involved in the innate immune defence of body, and its effect is also played between the innate immunity and acquired immunity.
Have now been found that 3 point mutation of MBL structural genes（CGT52TGG, GGC54GAC and GGA57GAA）And start
Opsonophagocytosis defect caused by blood plasma MBL caused by son and 5 ' non-translational regions are mutated is low is the most common something lost that is found so far
Transmissibility immunodeficiency disease.MBL defect persons among the infection risk in height, can suffer from various infection or even threaten life at any time throughout one's life
The infection of life.Recent researches show that MBL disease resistance mechanisms are related to its serum level, and low-level MBL is easily caused
The defense function of body substantially weakens, easily infect Crohn disease, Autoimmune neuropathies obstacle, primary immunodeficiency and
Invasive infections with fungi；On the contrary, recent studies have shown that high-caliber MBL can improve the survival of Patients with Chronic Obstructive Pulmonary Disease
Rate.Therefore, clinically MBL contents are for body health physical examination, judgement body immunity and predict certain in timely detection blood
A little autoimmune diseases are significant.
MBL alternatives recognize multiple pathogens surface with the sugared structure for terminal saccharide such as Man, ManNAc, GlcNAc,
By with 2 MBL associated serine protease 2s（MBL associated serine protease-1/-2、MASP-1、MASP-
2）With reference to the activating complement in the way of independent of antibody and C1q plays molten broken and indirect conditioning functions, moreover it is possible to phagocyte
Collectin acceptor（That is C1qR）With reference to and rise direct opsonic action.MBL peptide chains have 4 domains successively by N-terminal to C-terminal：N-terminal
Cys enrichment regions, the collagen-like region of the repetitive sequence of triplet containing Gly-X-Y（Collagen-like region, CLR）, curling
Spiral neck region and the carbohydrate recognition domain of C-terminal（Carbohydrate-recognition domain, CRD）.3 peptide chains pass through N ends
The Cys of petiolarea forms disulfide bond and connected, and CLR is then mutually wound in a- spirals, and 3 independent CRD folded are in the common shape of C-terminal
Into 1 spherical head, this structural units borrows disulfide bond or non-covalent bond to connect into oligomer, up to six aggressiveness again.MBL poly
Body form is the key of its Function, and polymer is greatly improved the affinity of MBL and glycan molecule.Only polymer MBL points
Son just has biological activity, therefore, and exploitation can detect that the polymer MBL with biological activity has important face in human body
Bed practice significance.
As collectin family member in c-type agglutinin superfamily, MBL has the denominator of c-type agglutinin, Neng Gouyu
Saccharide ligand high-affinity is combined and its binding activity is Ca2+Highly rely on.Present invention mannosan（mannan）Capture
There is the polymer MBL of biological activity in blood plasma, in order to increase MBL and mannosan Percentage bound, the sensitivity of detection is improved
Property, the present invention will add certain density Ca in reaction system2+.Consider the albumen of mannan-binding into the human body except relying on
Ca2+MBL outside, also have independent of Ca2+Can with mannose specifically bind IgA, IgM and IgG antibody-like, therefore in order to
The specificity of detection is improved, invention introduces anti-human MBL-CLR monoclonal antibodies, MBL and anti-human MBL-CLR monoclonals is utilized
The characteristic that antibody is uniquely combined, fully ensures that the specificity of detection.
The content of the invention
Present invention solves the technical problem that there is provided a kind of reagent for being used to detect and there is biological activity MBL in human blood
Box and its detection method, the kit is from anti-human MBL-CLR mAb as detection antibody, and MBL-CRD ends are poly- with reference to sweet dew
The active component of sugar, in order to avoid due to the follow-up anti-human MBL-CLR mAb and MBL of MBL-CRD and mannosan combination interference
Joint efficiency, MBL is combined as detection antibody from anti-human MBL-CLR mAb, because MBL-CLR ends are away from MBL-CRD
End, therefore be effectively prevent from anti-human MBL-CRD mAb as detection antibody because MBL-CRD and mannosan junction belt are next
Disturbing factor, substantially increase the sensitiveness of detection.
The present invention adopts the following technical scheme that to solve above-mentioned technical problem, has biological activity in human blood for detecting
MBL kit, it is characterised in that：Binding partner of the kit using mannosan as MBL, with anti-human MBL-CLR mAb
It is used as MBL detection antibody.
The detection method of the present invention for being used to detect the kit with biological activity MBL in human blood, its feature
It is to concretely comprise the following steps：It is 5mg/L, 100 μ L/ holes coating elisa plate, 4 with the carbonate buffer solution dilution mannosan of pH=9.6
DEG C overnight；1wt% BSA, 37 DEG C of closing 2h are added, is added after washing and contains CaCl2Fresh plasma to be checked, 100 μ L/ holes, 37 DEG C
Act on 1h；1 is added after washing:The anti-human MBL-CLR monoclonal antibodies of Biotin- of 400 dilutions, 37 DEG C of effect 1h；After washing, plus
Enter 1:The HRP-Avidin of 1000 dilutions, 100 μ L/ holes, 37 DEG C of effect 45min；Washing, adds the colour developing of TMB nitrite ions, with
2mol/L H2SO4Solution terminating reaction, is determined on automatic enzyme-linked instrumentA 450nmValue.
Further preferably, it is described to contain CaCl2Fresh plasma to be checked in Ca2+Molar concentration be 20mmol/L.
Further preferably, described carbonate buffer solution is 0.05mol/L Na2CO3-NaHCO3Buffer solution.
Further preferably, the specific preparation process of the anti-human MBL-CLR monoclonal antibodies of described Biotin- is：
（1）The anti-human μ L of MBL-CLR monoclonal antibodies 500 of mouse of 7.8mg/mL after purification are taken, 1 is diluted with 0.01mol/L PBS
It is dialysed after times, 4 DEG C overnight；
（2）Weigh 0.3mg Biotin to be dissolved in 20 μ L DMSO, and be added to step（1）The anti-human MBL-CLR of mouse dialysed is mono-
In clonal antibody, 4 DEG C of stirring 12h；
（3）By step（2）The anti-human MBL-CLR monoclonal antibodies of mouse marked are placed in PBS in 4 DEG C of dialysed overnights, change liquid two
It is secondary to finally give the anti-human MBL-CLR monoclonal antibodies of Biotin-.
The present invention has the advantages that compared with prior art：
1st, the present invention has founded antibody-part Sandwich ELISA detection human blood MBL albumen first, with preferable specificity
And sensitiveness；
2nd, the present invention captures the MBL albumen in blood plasma from mannosan as MBL binding partner, because mannosan is only
It is that there is biology with reference to the MBL albumen that the kit of the polymer MBL albumen with biological activity, therefore the present invention is detected
Destination protein；
3rd, the present invention selects anti-human MBL-CLR monoclonal antibodies as detection antibody, with destination protein calmodulin binding domain CaM away from MBL-
CRD ends, effectively prevent due to the disturbing factor that MBL-CRD and mannosan junction belt are come, substantially increase the sensitivity of detection
Property；
4th, Ca in blood plasma to be detected in the present invention2+Concentration is defined as 20mmol/L, imitates the combination of binding partner and destination protein
Rate reaches most preferably.
In summary, the present invention utilizes mannosan and high polymer MBL high-affinities and Ca2+The characteristics of highly relying on, it is first
The good MBL of bioactivity is first captured, in order to exclude the other non-destination proteins that can be combined in blood plasma with mannosan
Interference, introduces the anti-human MBL-CLR monoclonal antibodies of mouse as detection antibody, utilizes MBL and the anti-human MBL-CRD monoclonals of mouse again
The characteristic that antibody is uniquely combined, it is ensured that the specificity of detection, so that establishing in a kind of effective detection human blood has biology
Active MBL antibody-part sandwich ELISA system, with important clinical practice meaning.
Brief description of the drawings
Fig. 1 is the sensitivity curve of MBL albumen in antibody-part Sandwich ELISA detection human blood.
Embodiment
The above to the present invention is described in further details by the following examples, but this should not be interpreted as to this
The scope for inventing above-mentioned theme is only limitted to following embodiment, and all technologies realized based on the above of the present invention belong to this hair
Bright scope.
Embodiment
1st, key instrument and equipment
Title specification and the model place of production
Low-temperature and high-speed centrifuge 4K15 Sigma
Praise roc in nucleic acid-protein detector HD-97-1 Shanghai
The enzyme-linked U.S. BIO-RAD of instrument Model 450
Micro sample adding appliance 10-1000mL Eppendorf
Electric heating constant-temperature water-bath tank WMK-02 Wujin is automatically controlled
Electronic balance BP61 Germany sartorius
Electrophoresis apparatus DYY-III-5 Beijing 61
Ultraviolet specrophotometer DU®520 U.S. Beckman
Time constant-temperature magnetic stirrer ML-902 Shaoxing satellite
Freeze drier ALPHA1-2 Germany CHRIST
Flow cytometer FACSCalibur U.S. company BDs
2nd, materials and methods
2.1 main agents and animal
（1）The anti-human MBL-CLR monoclonal antibodies of mouse are prepared by this laboratory.
（2）Biotin（Biotin）Sigma companies are purchased from DMSO.
（3）Mannan standard items are purchased from Sigma companies.
（4）HRP-Avidin is purchased from Beijing Ding Guo biotech firms.
（5）TMB shows that liquid is purchased from Jing Mei companies.
2.2 main solution
（1）0.01mol/L PBS（PH 7.4, NaCl containing 0.5mol/L）：
Na2HPO4·12H2O 1.451g
NaH2PO4·2H2O 0.148g
NaCl 14.61g
With dH2500mL is settled to after O dissolvings
（2）It is coated with buffer solution：0.05mol/L Na2CO3-NaHCO3Buffer solution（pH9.6）
Na2CO3 1.59g
NaHCO3 2.93g
With dH21000mL is settled to after O dissolvings
（3）Confining liquid：
PBS（pH7.4） 100mL
BSA 1g
（4）1‰ Tween 20-PBS（PBST）Cleaning solution：
PBS（pH7.4） 500mL
Tween 20 0.5mL
（5）Antibody diluent：0.1wt% BSA-PBST
（6）Terminate liquid：2mol/L H2SO4Solution.
The anti-human MBL-CLR monoclonal antibodies of 2.3 biotin labeling mouse
（1）Take the anti-human MBL-CLR monoclonal antibodies of mouse after purification（7.8mg/mL）500 μ L, 1 is diluted with 0.01mol/L PBS
It is dialysed after times, 4 DEG C overnight；
（2）Weigh 0.3mg Biotin to be dissolved in 20 μ L DMSO, and add in the antibody dialysed, 4 DEG C of stirring 12h；
（3）The antibody marked is placed in 4 DEG C of dialysed overnights in PBS, liquid is changed twice.
The processing of 2.4 blood plasma
（1）Fresh plasma 10mL is obtained from peripheral blood；
（2）Add 1mol/L CaCl2Solution makes its final concentration of 20mmol/L.
2.5 antibody-part sandwich ELISA system
It is 5mg/L with the carbonate buffer solution dilution mannosan of pH=9.6,100 μ L/ holes are coated with elisa plate, and 4 DEG C overnight；Add
1wt% BSA, 37 DEG C of closing 2h, the addition that the doubling dilution since 20mg/L is added after washing contains CaCl2Solution it is to be checked fresh
Blood plasma, 100 μ L/ holes, 37 DEG C of effect 1h；1 is added after washing:The anti-human MBL-CLR monoclonal antibodies of Biotin- of 400 dilutions, 37
DEG C effect 1h；After washing, 1 is added:The HRP-Avidin of 1000 dilutions, 100 μ L/ holes, 37 DEG C of effect 45min；Washing, is added
TMB nitrite ions develop the color, with 2mol/L H2SO4Solution terminating reaction, is determined on automatic enzyme-linked instrumentA 450nmValue.The detection architecture
Sensitivity is higher, can detect the MBL albumen that concentration is 0.16mg/L（Fig. 1）.
Embodiment above describes general principle, principal character and the advantage of the present invention, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, the original for simply illustrating the present invention described in above-described embodiment and specification
Reason, under the scope for not departing from the principle of the invention, various changes and modifications of the present invention are possible, and these changes and improvements are each fallen within
In the scope of protection of the invention.
Claims (5)
1. there is biological activity MBL kit for detecting in human blood, it is characterised in that：The kit is made with mannosan
For MBL binding partner, MBL detection antibody is used as using anti-human MBL-CLR mAb.
2. being used for described in claim 1 detects the detection method of the kit with biological activity MBL in human blood, its feature
It is to concretely comprise the following steps：It is 5mg/L, 100 μ L/ holes coating elisa plate, 4 with the carbonate buffer solution dilution mannosan of pH=9.6
DEG C overnight；1wt% BSA, 37 DEG C of closing 2h are added, is added after washing and contains CaCl2Fresh plasma to be checked, 100 μ L/ holes, 37 DEG C
Act on 1h；1 is added after washing:The anti-human MBL-CLR monoclonal antibodies of Biotin- of 400 dilutions, 37 DEG C of effect 1h；After washing, plus
Enter 1:The HRP-Avidin of 1000 dilutions, 100 μ L/ holes, 37 DEG C of effect 45min；Washing, adds the colour developing of TMB nitrite ions, with
2mol/L H2SO4Solution terminating reaction, is determined on automatic enzyme-linked instrumentA 450nmValue.
3. the detection method according to claim 2 for being used to detect the kit with biological activity MBL in human blood, its
It is characterised by：Described contains CaCl2Fresh plasma to be checked in Ca2+Molar concentration be 20mmol/L.
4. the detection method according to claim 2 for being used to detect the kit with biological activity MBL in human blood, its
It is characterised by：Described carbonate buffer solution is 0.05mol/L Na2CO3-NaHCO3Buffer solution.
5. the detection method according to claim 2 for being used to detect the kit with biological activity MBL in human blood, its
The specific preparation process for being characterised by the described anti-human MBL-CLR monoclonal antibodies of Biotin- is：
（1）The anti-human μ L of MBL-CLR monoclonal antibodies 500 of mouse of 7.8mg/mL after purification are taken, 1 is diluted with 0.01mol/L PBS
It is dialysed after times, 4 DEG C overnight；
（2）Weigh 0.3mg Biotin to be dissolved in 20 μ L DMSO, and be added to step（1）The anti-human MBL-CLR of mouse dialysed is mono-
In clonal antibody, 4 DEG C of stirring 12h；
（3）By step（2）The anti-human MBL-CLR monoclonal antibodies of mouse marked are placed in PBS in 4 DEG C of dialysed overnights, change liquid two
It is secondary to finally give the anti-human MBL-CLR monoclonal antibodies of Biotin-.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109813881A (en) * | 2019-02-15 | 2019-05-28 | 无锡市妇幼保健院 | A kind of hormone test reagent and kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1646916A (en) * | 2002-04-18 | 2005-07-27 | 安蒂博迪肖普股份有限公司 | Compositions, methods and kits for immunochemical determination of mannan-binding lectin (MBL) |
| CN1837238A (en) * | 2005-03-25 | 2006-09-27 | 中国药品生物制品检定所 | Monoclonal antibody against MBL and its use |
| WO2007111496A1 (en) * | 2006-03-28 | 2007-10-04 | Universiteit Utrecht Holding B.V. | Improved carbohydrate recognition domains |
| CN103808927A (en) * | 2014-01-27 | 2014-05-21 | 武汉中博生物股份有限公司 | Enzyme linked immunosorbent assay kit for detecting porcine reproductive and respiratory syndrome virus |
-
2017
- 2017-02-27 CN CN201710109471.XA patent/CN107064514A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1646916A (en) * | 2002-04-18 | 2005-07-27 | 安蒂博迪肖普股份有限公司 | Compositions, methods and kits for immunochemical determination of mannan-binding lectin (MBL) |
| CN1837238A (en) * | 2005-03-25 | 2006-09-27 | 中国药品生物制品检定所 | Monoclonal antibody against MBL and its use |
| WO2007111496A1 (en) * | 2006-03-28 | 2007-10-04 | Universiteit Utrecht Holding B.V. | Improved carbohydrate recognition domains |
| CN103808927A (en) * | 2014-01-27 | 2014-05-21 | 武汉中博生物股份有限公司 | Enzyme linked immunosorbent assay kit for detecting porcine reproductive and respiratory syndrome virus |
Non-Patent Citations (2)
| Title |
|---|
| 李瑞芳: "MBL高表达细胞株筛选及其产物生物学特性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 蔡学敏 等: "人MBL-CLR表达载体的构建及其在大肠杆菌中的表达", 《细胞与分子免疫学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109813881A (en) * | 2019-02-15 | 2019-05-28 | 无锡市妇幼保健院 | A kind of hormone test reagent and kit |
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