CN103983767B - A kind of kit and application thereof detecting α-1,3Gal antigen - Google Patents
A kind of kit and application thereof detecting α-1,3Gal antigen Download PDFInfo
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- CN103983767B CN103983767B CN201410187336.3A CN201410187336A CN103983767B CN 103983767 B CN103983767 B CN 103983767B CN 201410187336 A CN201410187336 A CN 201410187336A CN 103983767 B CN103983767 B CN 103983767B
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Abstract
The present invention relates to a kind of detection kit, be specifically related to a kind of kit detecting α-1,3Gal antigen.The present invention adopts Gal1, and the previously prepared solid phase antigen bag of 3Gal-BSA antigen is by plate, and the specific antibody M86 of combination α-1,3Gal antigen, is prepared into antibody competition ELISA and suppresses method detection kit.The quantitative and qualitative analysis that kit of the present invention can be used for the α-Gal antigen of various biomaterial detects; Residual α-the Gal being particularly useful for animal derived biomaterial detects.The specificity of kit of the present invention reaches 100%, and sensitivity is to detect the Gal antigen in the Gal antigen positive reference material of the rabbit haemocyte of 3.9x104/ml, 6.25 μ g/ml.
Description
Technical field
The present invention relates to a kind of detection kit, be specifically related to a kind of kit and the application thereof that detect α-1,3Gal antigen.
Background technology
The biogenic material be made up of mammalian extracellular matrix has because of it timbering material etc. that good biocompatibility is widely used in the trauma repair of surgery, tissue reconstruction and organizational project.Xenogeneic organ also becomes in solution organ transplant already for one of potential approach of organ famine.But animal derived biomaterial or Xenogeneic organ's organizations directly affect security and the validity of this kind of materials'use in the immunological problem that human body brings.The biggest obstacle of xenotransplant is activating complement system after being combined for organ heterogenetic antigen natural antibody in recipient's body, attack the Hyperacute immunological rejection (Hyperacuterejection produced for organ, HAR), its time of origin a few minutes after heterograft to a few hours, for organ at short notice generating function exhaustion make graft failure.Though animal derived biomaterial is through the process of various removal antigen, be difficult to thoroughly remove all heterogenetic antigens, residual antigen remains and causes Chronic immune rejection and immunotoxicity and the key factor that affects wound healing.
Existing research display heterogenetic antigen α-galactosyl antigen (α-1,3-galactosyle, α-Gal) is the main target antigen in animal derived biomaterial or xenotransplant Hyperacute immunological rejection.α-Gal is cell surface secreting type glycoprotein containing poly lactose amine core end residue or glycolipid, is extensively present in pig and other lower animal body.Due to human body and anthropoid cape, old century monkey galactoside transferase gene have 2 bases dislocation variations and do not express Gal antigen, but there is the human natural antibodies (accounting for 1% of total serum globulin) of high titre in human serum, therefore can cause Hyperacute immunological rejection and the reaction of chronic immunotoxicity when human body accepts biomaterial or the xenotransplant containing Gal antigen.In animal derived biomaterial or xenotransplant, the removal of Gal antigen becomes the key reducing immunological rejection and Chronic immune toxic reaction.At present, the immunotoxicity of removal and residual Gal antigen induction how to evaluate Gal antigen still also exists bottleneck.Due to wild type lower animal all express alpha-Gal antigen, therefore wild type lower animal cannot be utilized to go to evaluate the relevant immunotoxicity reaction of α-Gal antigen, can only investigate the immunotoxicity reaction of α-Gal antigen positive or allosome organ as acceptor with baboon, and this animal rareness is difficult to universal use.Therefore, the immunotoxicity how evaluating animal derived biomaterial or Xenogeneic organ's tissue become puzzled detect with regulator because of do not have method and standard can according to and effective security and quality monitoring cannot be carried out, also limit research and development and the industrialized development of this series products simultaneously.
In order to fill up the blank detecting α-Gal antigenic reagent box, special proposition the present invention.
Summary of the invention
Primary goal of the invention of the present invention is to propose a kind of kit detecting α-1,3Gal antigen.
In order to realize object of the present invention, the technical scheme of employing is:
The present invention relates to a kind of detection α-1, the kit of 3Gal antigen, contain in described kit: the antigen coated orifice plate of solid phase α-1,3Gal, confining liquid, mouse M86 antibody, secondary antibodies enzyme conjugates, TMB nitrite ion, colour developing stop buffer, Tissue lysates, concentrated washing lotion, sample diluting liquid, positive reference material and negative reference product.
First optimal technical scheme of the present invention is: the antigen coated orifice plate of solid phase α-1,3Gal adopts Gal α-1,3Gal-BSA to carry out bag quilt in advance, and bag is buffered the carbonate buffer solution that liquid is pH9.5,0.2M, and package amount is preferably 50 ~ 100 μ l; Antigen concentration is 10 ~ 100ng/ml, is preferably 50 ~ 100ng/ml.
Second optimal technical scheme of the present invention is: described confining liquid is human serum albumins; Described sample diluting liquid is PBS; Described colour developing stop buffer is the concentrated sulphuric acid; Described concentrated washing lotion is Tween-20; Described secondary antibodies enzyme conjugates is horseradish peroxidase-sheep anti mouse IgM enzyme conjugates.
3rd optimal technical scheme of the present invention is: described positive reference material is the dried frozen aquatic products of animal tissue, is the freeze-dried powder of pig liver tissue; Negative reference product are the dried frozen aquatic products of human tissue, are the freeze-dried powder of human placenta.Described positive reference material and the preparation method of negative reference product for first flesh tissue is removed after 0 ~ 4 DEG C of physiological saline rinsing excessive moisture, then carry out homogenate under 0 ~ 4 DEG C of condition; Finally homogenised tissue is carried out freeze-drying, packing is kept at-20 DEG C.
The invention still further relates to the preparation method of the kit of this detection α-1,3Gal antigen: the Gal1 of preparation 50ng ~ 200ng/ml, 3Gal-BSA solution, damping fluid is 0.2M, pH9.5 carbonate buffer solution; Get 100 μ LGal1,3Gal-BSA solution joins orifice plate, 4 DEG C of overnight incubation; The secondary human serum albumins of daily 1% is closed, and the condition closed preferably 4 DEG C, 1 ~ 2 hour, then uses the Tween-20 of 0.05%/PBS washing lotion to carry out washing plate three times, add washing lotion 250 μ l/ hole, soaks 30s, cleans three times; Dry after removing washing lotion, pack, 4 DEG C of preservations.
The invention still further relates to the using method of the kit of this detection α-1,3Gal antigen, comprise the following steps:
(1) test specimen preparation: sample to be tested is put in lysate and carries out low-temperature homogenate; α-Gal antigen positive reference material and α-Gal antigen negative reference material respectively add in lysate carries out Tissue Lysis; Centrifugal, get supernatant for subsequent use;
(2) standard curve control product preparation: get rabbit blood 1.25 × 10
6individual/ml cell number or SP2/0 cell 2.5 × 10
6individual/ml, carries out doubling dilution, totally 5 ~ 7 concentration gradients;
(3) sample antigen-antibody response: each 100 μ l of dilution of the supernatant and control sample of getting test specimen put into centrifuge tube, add the M86 antibody-solutions after dilution (dilution ratio is 1:50 ~ 1:100) 100 μ l respectively, mix rear 4 DEG C of overnight incubation, or 37 DEG C hatch 1 ~ 3 hour with slight shake; Supernatant is got for subsequent use after centrifugal;
(4) competitive residue antibody-solid phase antigen reaction: get each 100 μ l of above-mentioned reaction supernatant and join in the antigen coated ELISA Plate of α-Gal, residue M86 antibody in reaction supernatant and the solid phase antigen in orifice plate are fully reacted, 4 DEG C of overnight incubation, or 37 DEG C hatch 1 ~ 3 hour; Plate is washed 3 times by washing lotion;
(5) secondary antibodies reaction and colour developing: every hole adds the enzyme labelled antibody 100 μ l of 1:1000 dilution, puts 37 DEG C and hatches 0.5 ~ 2 hour; Washing lotion washes plate 3 times, dries; Add chromogenic enzyme substrate agent TMB, after lucifuge colour developing, add stop buffer, utilize microplate reader to measure absorbance value A450nm at 450nm;
(6) experimental result is judged.
The invention still further relates to this detection α-1, application in the qualitative or quantitative detection of kit α-Gal antigen in animal derived biomaterial of 3Gal antigen, described animal derived biomaterial comprises derivant, the purified of the tissue of animal origin, organ, cell and animal tissue or organ.Described be applied as animal derived biomaterial residual α-Gal qualitative or quantitatively detect.
Below technical scheme of the present invention is made further explanation.
Object of the present invention is for providing the immue quantitative detection reagent box of a kind of direct-detection with α-Gal antigen in the animal derived biomaterial of evaluation or Xenogeneic organ's tissue and derivant thereof.The present invention adopts the previously prepared solid phase antigen bag of Gal α-1,3Gal-BSA by plate, and then the specific antibody M86 of combination α-Gal antigen, is prepared into antibody competition ELISA and suppresses method detection kit.
Kit of the present invention provides a kind of direct-detection and evaluates the quantitative detecting method of Gal antigen in animal derived biomaterial or Xenogeneic organ's tissue and derivant thereof.The present invention uses the specificity M86 antibody of Gal α-1,3Gal antigen, detects α-Gal antigen in animal derived biomaterial.This kit comprises: the antigen coated orifice plate (preferred 96-orifice plate) of solid phase Gal α-1,3Gal, confining liquid, mouse M86 antibody, secondary antibodies enzyme conjugates, TMB nitrite ion, colour developing stop buffer, concentrated washing lotion, sample diluting liquid, Tissue lysates, positive reference material and negative reference product.Wherein, ELISA Plate is wrapped by Gal α-1,3Gal-BSA in advance, and it is pH9.5 carbonate buffer solution (0.2M) that bag is buffered liquid, and package amount is 100 μ l (50ng/ml antigen concentrations); Confining liquid is the human serum albumins of mass concentration 1%; Sample diluting liquid is PBS; Nitrite ion is TMB; Colour developing stop buffer is the concentrated sulphuric acid (H of 10 times of dilutions (1mol/L)
2sO
4); Concentrated washing lotion is the Tween-20 (diluting 100 times during use) of 5%; Secondary antibodies enzyme conjugates is horseradish peroxidase (HRP)-sheep anti mouse IgM enzyme conjugates; Tissue lysates; Positive reference material and negative reference product are placed in box.
Kit of the present invention can detect the antigen number in sample quantitatively, can detect the residual α-Gal antigen of trace in testing sample, have very high sensitivity and specificity.Also be combined with all reagent of other sample preparations and follow-up ELISA detection in kit of the present invention, thus reactive system and integration can be made.
Whether meanwhile, this kit contains positive reference material and negative reference product, can directly validation test system set up, and can judge and eliminating false positive and false negative, guarantee the reliability detected.
This kit can be used for various biomaterial, animal organ, tissue, cell and derivant, purified, detects the quantitative and qualitative analysis that it carries out α-Gal antigen; Be particularly useful for the detection of the residual α-Gal antigen of animal derived biomaterial.The specificity of kit of the present invention reaches 100%; Sensitivity is for detecting 3.9 × 10
4α-Gal antigen in the rabbit haemocyte of individual/ml, the α-Gal antigen positive reference material of 6.25 μ g/ml.
Accompanying drawing explanation
Fig. 1 is the process chart of kit of the present invention;
Fig. 2 is the canonical plotting prepared for reference substance with rabbit blood;
Fig. 3 is that the M86 of positive reference material is in conjunction with inhibiting rate curve.
The specific embodiment of the present invention is only limitted to explain further and the present invention is described, does not limit Composition of contents of the present invention.
Embodiment
Embodiment 1: α-1,3Gal antigen detection kit
Kit of the present invention includes:
1. the 96-orifice plate that solid phase α-Gal is antigen coated: wrap by Gal α-1,3Gal-BSA antigen (DextraLaboratories, UK), it is 0.2M, pH9.5 carbonate buffer solution that bag is buffered liquid, and package amount is 100 μ l (50ng/ml);
2. confining liquid: the human serum albumins of mass concentration 1%;
3. mouse M86 antibody: commercially available prod (Enzo, ALX-801-090-1, α-GalEpitope, mAb)
4. secondary antibodies enzyme conjugates: the sheep anti mouse IgM antibody of horseradish peroxidase-labeled;
5.TMB nitrite ion: 1%TMB;
6. develop the color stop buffer: the 1mol/l concentrated sulphuric acid (diluting 10 times during use);
7. Tissue lysates: directly use commercially available prod, the green skies " P0013BRIPA lysate (by force) ";
8. concentrated washing lotion: the Tween-20 (diluting 100 times during use) of 5%;
9. sample diluting liquid: PBS;
10. positive reference material: the freeze-dried powder of pig liver tissue;
11. negative reference product: the freeze-dried powder of human placenta.
The preparation of embodiment 2 kit
1. the preparation of the antigen coated ELISA Plate of α-Gal: preparation Gal α-1,3Gal-BSA (DextraLaboratories, UK) solution, 50ng/ml, damping fluid is pH9.5 carbonate buffer solution (0.2M); Get 100 μ l and join 96-orifice plate (Nunc.Rochester, NY), 4 DEG C of overnight incubation; Human serum albumins with 1% carries out closing (4 DEG C, 2 hours), uses the Tween-20 of 0.05%/PBS washing lotion to carry out washing plate three times afterwards, adds washing lotion 250 μ l/ hole, soak 30s, cleaning is jolted three times, for the first time 5min, rear twice 3min with microwell plate oscillator; After removing washing lotion, bag being placed in Biohazard Safety Equipment by plate exposure of ventilating makes it air-dry in 2 minutes, packs together with drying agent, 4 DEG C of preservations (using in a week).
2, other solution preparations:
2.1 sample diluting liquids: the phosphate buffer of pH7.2 ~ 7.5;
2.2 concentrated washing lotions: concentrate washing lotion (diluting 100 times during use) with the Tween-20 of PBS preparation 5%;
2.3 confining liquids: with the human serum albumins of PBS preparation 1%;
The preparation of 2.4 nitrite ion TMB: accurately take TMB100mg, adds in 10ml dimethyl sulfoxide (DMSO) (DMSO), makes it the TMB concentrate obtaining 100 times after dissolving completely;
2.5 colour developing stop buffers: get the concentrated sulphuric acid that 54.3ml concentration is 95%, adding distil water can obtain the concentrated sulphuric acid of 10mol/L to 1000ml; 10 times of uses are diluted again during use;
2.6 preparation Tissue lysates: directly can use commercially available product, the green skies " P0013BRIPA lysate (by force) ";
2.7M86 antibody: with PBS dilution, dilution ratio is: 1:50;
2.8 secondary antibodies enzyme conjugates: with PBS dilution, dilution ratio is: 1:1000;
2.9 other reagent are equipped with: the preparation of the animal derived biomaterial reference material of α-Gal antigen positive and α-Gal antigen negative biomaterial reference material: first flesh tissue is removed excessive moisture after 4 DEG C of physiological saline rinsings, then 4 DEG C are carried out homogenate; Finally homogenised tissue is carried out freeze-drying, packing is kept at-20 DEG C.
Said method is adopted to prepare kit disclosed in embodiment 1.
Embodiment 3: kit quality testing
1, check that bag is by effect: M86 antibody (1:100 is as initial concentration) is carried out gradient dilution, detects OD after being reacted with solid phase antigen bag by plate solid phase antigen, mapping obtains regression curve equation, and analysis package is by effect.Recommendation qualification determination standard is: R
2>=0.95.
2. specific detection: detect α-Gal antigen positive and α-Gal antigen negative reference material respectively with the kit of embodiment 1
2.1 α-Gal antigen positive reference materials and α-Gal antigen negative reference material (freeze-dried powder) each 2mg add in 1ml lysate and carry out Tissue Lysis 5 ~ 10min; 14000rpm, 4 DEG C, centrifugal 30min, positive reference material gets supernatant, and to carry out serial doubling dilution 5 concentration for subsequent use; Negative reference product are got supernatant and are directly used.
2.2, standard curve control product prepare: get rabbit blood 1.25 × 10
6individual/ml cell number or SP2/0 cell 2.5 × 10
6individual/ml, carries out doubling dilution, totally 5 ~ 7 concentration gradients;
2.3, sample antigen-antibody response: each 100 μ l of supernatant getting positive reference material and negative reference product put into 1.5ml centrifuge tube respectively, add the M86 antibody-solutions (final concentration is that 1:100 doubly dilutes) that 100 μ l dilution ratios are 1:50 respectively, mix rear 4 DEG C of overnight incubation, it is for subsequent use that 14000rpm gets supernatant after centrifugal 5 minutes;
2.4, competitive residue antibody-solid phase antigen reaction: get each 100 μ l of above-mentioned reaction supernatant and join in the antigen coated ELISA Plate of α-Gal, residue M86 antibody in reaction supernatant and the solid phase antigen in coated elisa plate are fully reacted, 37 DEG C, with shaking gently, are reacted 1 ~ 2 hour; Washing lotion washes plate 3 times, dries;
2.5, secondary antibodies reaction and colour developing: every hole adds enzyme labelled antibody 100 μ l, and dilution ratio is 1:1000, puts 37 DEG C and hatches 1 hour; Washing lotion washes plate 3 times, dries; Add chromogenic enzyme substrate agent TMB, every hole 100 μ l, lucifuge colour developing 5 ~ 10min, adds stop buffer, every hole 50 μ l.Microplate reader is utilized to measure absorbance value A450nm at 450nm.
Experimental result is as shown in table 1, Fig. 2 and Fig. 3, and wherein Fig. 2 is rabbit haemocyte canonical plotting, and Fig. 3 is that positive reference material M86 is in conjunction with inhibiting rate curve map.
The M86 of table 1. negative reference product is in conjunction with inhibiting rate
Result shows: the 50%M86 antibody of α-Gal antigen positive reference material is 42.86 μ g/ml in conjunction with concentration during inhibiting rate.α-Gal antigen negative reference material does not detect α-Gal antigen, and specificity is 100%.
3, sensitivity technique: the kit manufactured with the present invention detects different dilution α-Gal antigen positive reference material, operation and judge to carry out (experimental procedure with 2, specific detection) according to the step in the embodiment in α-1,3Gal antigen ELISA detecting kit.
Result: minimumly detect 3.9 × 10
4gal antigen in the rabbit haemocyte of individual/ml, the positive reference material of 6.25 μ g/ml.
Embodiment 4: adopt the kit of embodiment 1 to carry out the standard practice instructions detected:
1, test specimen prepares: α-Gal antigen positive reference material and α-Gal antigen negative reference material (freeze-dried powder) each 2mg add in 1ml lysate and directly carry out Tissue Lysis 5 ~ 10min; 14000rpm, 4 DEG C, centrifugal 30min, positive reference material is got supernatant and is first carried out 10 times of dilutions, then it is for subsequent use to carry out serial doubling dilution 5 concentration; It is directly for subsequent use that negative reference product get supernatant.
Note: as being solid test sample (as: pig acellular matrix), can take 2mg put in 1ml lysate carry out low-temperature homogenate after 3 ~ 5 minutes room temperature place 5 ~ 10min, the abundant cracking of warranty test sample.14000rpm, 4 DEG C, centrifugal 30min, getting supernatant, to carry out serial doubling dilution 5 concentration for subsequent use;
2, standard curve control product prepare: getting 200 μ l concentration is 1.25 × 10
6the rabbit haemocyte of individual/ml carries out doubling dilution, totally 5 ~ 7 concentration gradients;
3, sample antigen-antibody response: the supernatant and each 200 μ l of control sample dilution that get test specimen put into 1.5ml centrifuge tube respectively, add the M86 antibody-solutions (final concentration is 1:100) that 200 μ l dilution ratios are 1:50 respectively, mix rear 4 DEG C of overnight incubation, or 37 DEG C hatch 2 hours; It is for subsequent use that 14000rpm gets supernatant after centrifugal 5 minutes;
4, competitive residue antibody-solid phase antigen reaction: get each 100 μ l of above-mentioned reaction supernatant and join (three multiple holes) in the antigen coated ELISA Plate of α-Gal, residue M86 antibody in reaction supernatant and the solid phase antigen in coated elisa plate are fully reacted, hatches 1 ~ 2 hour for 37 DEG C; Washing lotion washes plate 3 times, dries;
5, with " M86/ rabbit blood supernatant ", " M86/ Tissue lysates " that Tissue lysates preparation final concentration cleer and peaceful on rabbit blood is 1:100, association reaction as 100%M86 antibody contrasts (in order to get rid of the interference to antigen-antibody reaction and optical detecting of rabbit blood supernatant or Tissue lysates), directly join in (three multiple holes) in the antigen coated ELISA Plate of α-Gal, solid phase antigen in M86 antibody and coated elisa plate is fully reacted, hatches 1 ~ 2 hour for 37 DEG C; Washing lotion washes plate 3 times, dries; Meanwhile, contrast using cleer and peaceful Tissue lysates on 100 μ L rabbit blood as blank well.
5, secondary antibodies reaction and colour developing: every hole adds enzyme labelled antibody 100 μ l, and dilution ratio is 1:1000, puts 37 DEG C and hatches 1 hour; Washing lotion washes plate 3 times, dries; Add chromogenic enzyme substrate agent TMB, every hole 100 μ l, lucifuge colour developing 5 ~ 10min, adds stop buffer, every hole 50 μ l.Microplate reader is utilized to measure absorbance value A450nm at 450nm.
6, result judges:
1) test specimen α-Gal antigen computing method:
1. the drafting of reference substance typical curve: with the concentration of rabbit haemocyte (cell number/ml) for horizontal ordinate, M86's is ordinate curve plotting figure in conjunction with inhibiting rate (%).Wherein, the M86 of the different cell concentration of reference substance is in conjunction with inhibiting rate %=[on (on M86/ rabbit blood clearance response OD value average-reference substance reaction OD value average)/M86/ rabbit blood clearance response OD value average × 100] %; Reference substance 50%M86 calculates from the equation of typical curve in conjunction with the cell concentration that inhibiting rate is corresponding, and then calculates control sample 50%M86 in conjunction with cell number corresponding to inhibiting rate.
2. the M86 of test specimen is in conjunction with inhibiting rate Drawing of Curve: drafting and the computing method of curve are the same, calculates test specimen 50%M86 in conjunction with mass concentration corresponding to inhibiting rate, and then calculate the quality of test specimen according to the equation in inhibiting rate curve.
3., in same reaction system, following identity relation is set up, that is: test specimen 50% in conjunction with antigen number=rabbit blood during inhibiting rate 50% in conjunction with antigen number during inhibiting rate.
The computing method of test specimen α-Gal antigen number are:
Test specimen 50% in conjunction with unit mass α-Gal epitope number=rabbit blood during inhibiting rate 50% in conjunction with cell number x2 × 10 during inhibiting rate
6antigen number/test specimen 50% in conjunction with quality during inhibiting rate;
Wherein, the epitope number of reference substance (rabbit haemocyte) is: 2 × 10
6antigen number/cell (from document: Transplantation.65 (8): 1129-1132, April27,1998).
Note: if in test specimen α-Gal antigen trace or trace, the valid data number of numerical value can be obtained lower than 3 in series of diluted samples, existing valid data then can be utilized to be updated in the inhibiting rate curve of positive reference material, to ask and calculate the antigenic content number relative with positive reference material.
2) experimental result judges:
A, the α-Gal antigen that can detect in α-Gal antigen positive reference material; Positive reference material is 42 μ g/ml at 50%M86 antibody in conjunction with the mass concentration of inhibiting rate, corresponding α-Gal antigen number 6.2 × 10
12individual/mg tissue;
B, α-Gal antigen negative reference material does not detect α-Gal antigen.
7, the testing result of this kit is used
1) typical curve prepared for reference substance with rabbit blood is shown in Fig. 2, and drafting and the computing method of curve are the same.
2) M86 of positive reference material is shown in Fig. 3 in conjunction with inhibiting rate curve, and drafting and the computing method of curve are the same.
3) M86 of negative reference product in conjunction with inhibiting rate in table 1.
4) positive reference material α-Gal antigen computing method: with reference to 6-1)-3. the formula that provides of test specimen α-Gal antigen computing method.
Result shows, the test positive of positive reference material, and its 50%M86 antibody is 42 μ g/ml in conjunction with the mass concentration of inhibiting rate, corresponding α-Gal antigen number 6.2 × 10
12individual/mg tissue; The M86 of negative reference product is 0 in conjunction with inhibiting rate, confirms negative reference product not containing α-Gal antigen.
Claims (7)
1. one kind is detected α-1, the kit of 3Gal antigen, it is characterized in that, contain in described kit: orifice plate, confining liquid, mouse M86 antibody, secondary antibodies enzyme conjugates, TMB nitrite ion, colour developing stop buffer, Tissue lysates, concentrated washing lotion, sample diluting liquid, the positive reference material of α-Gal and α-Gal negative reference product that solid phase α-Gal is antigen coated;
Wherein, the antigen coated orifice plate of solid phase α-Gal adopts Gal α-1,3Gal-BSA to carry out bag quilt in advance, and bag is buffered the carbonate buffer solution that liquid is pH9.5,0.2M, and package amount is 100 μ l, and antigen concentration is 50ng/ml;
Described confining liquid is the human serum albumins of mass concentration 1%; Described sample diluting liquid is PBS; Described colour developing stop buffer is the concentrated sulphuric acid; Described concentrated washing lotion is the Tween-20 of 5%; Described secondary antibodies enzyme conjugates is horseradish peroxidase-sheep anti mouse IgM enzyme conjugates; Described positive reference material is the freeze-dried powder of pig liver tissue; Negative reference product are the freeze-dried powder of human placenta.
2. detection α-1 according to claim 1, the kit of 3Gal antigen, it is characterized in that, described positive reference material and the preparation method of negative reference product are: first flesh tissue is removed excessive moisture after 0 ~ 4 DEG C of physiological saline rinsing, then carry out homogenate under 0 ~ 4 DEG C of condition; Finally homogenised tissue is carried out freeze-drying, packing is kept at-20 DEG C.
3. one kind is detected α-1 as claimed in claim 1, the preparation method of the kit of 3Gal antigen, is characterized in that, the preparation method of the described antigen coated orifice plate of α-Gal is: the Gal α-1 of preparation 50ng/ml, 3Gal-BSA solution, damping fluid is 0.2M, pH9.5 carbonate buffer solution; Get 100 μ LGal α-1,3Gal-BSA solution and join orifice plate, 4 DEG C of overnight incubation; The secondary human serum albumins of daily 1% is closed, and then uses the Tween-20 of 0.05%/PBS washing lotion to carry out washing plate three times, adds washing lotion 250 μ L/ hole, soaks 30s, cleans three times; Dry after removing washing lotion, pack, 4 DEG C of preservations.
4. preparation method according to claim 3, is characterized in that, the condition closed is 4 DEG C, 1 ~ 2 hour.
5. one kind is detected the non-diseases treatment of the kit of α-1,3Gal antigen or the using method in diagnosing as claimed in claim 1, it is characterized in that, comprises the following steps:
(1) test specimen preparation: sample to be tested is put in lysate and carries out low-temperature homogenate; α-Gal antigen positive reference material and α-Gal antigen negative reference material respectively add in lysate carries out Tissue Lysis; Centrifugal, get supernatant for subsequent use;
(2) standard curve control product preparation: get rabbit blood 1.25 × 10
6individual/ml cell number or SP2/0 cell 2.5 × 10
6individual/ml, carries out doubling dilution, totally 5 ~ 7 concentration gradients;
(3) sample antigen-antibody response: each 100 μ l of dilution of the supernatant and control sample of getting test specimen put into centrifuge tube, and add the M86 antibody-solutions 100 μ l after dilution respectively, dilution ratio is 1:50 ~ 1:100; Mix rear 4 DEG C of overnight incubation, or 37 DEG C hatch 1 ~ 3 hour with slight shake; Supernatant is got for subsequent use after centrifugal;
(4) competitive residue antibody-solid phase antigen reaction: get each 100 μ l of above-mentioned reaction supernatant and join in the antigen coated ELISA Plate of α-Gal, residue M86 antibody in reaction supernatant and the solid phase antigen in orifice plate are fully reacted, 4 DEG C of overnight incubation, or 37 DEG C hatch 1 ~ 3 hour; Plate is washed 3 times by washing lotion;
(5) secondary antibodies reaction and colour developing: every hole adds the enzyme labelled antibody 100 μ l of 1:1000 dilution, puts 37 DEG C and hatches 0.5 ~ 2 hour; Washing lotion washes plate 3 times, dries; Add chromogenic enzyme substrate agent TMB, after lucifuge colour developing, add stop buffer, utilize microplate reader to measure absorbance value A450nm at 450nm;
(6) experimental result is judged.
6. one kind is detected α-1 as claimed in claim 1, application in the non-diseases treatment of kit α-Gal antigen in animal derived biomaterial of 3Gal antigen or the qualitative or quantitative detection of diagnosis, described animal derived biomaterial comprises derivant, the purified of the tissue of animal origin, organ, cell and animal tissue or organ.
7. application according to claim 6, is characterized in that, described be applied as animal derived biomaterial residual α-Gal antigen qualitative or quantitatively detect.
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| CN108872584A (en) * | 2018-05-09 | 2018-11-23 | 北京三药科技开发公司 | A kind of α-Gal rapid antigen detection kit and its application method and application |
| CN108646029B (en) * | 2018-05-14 | 2021-07-02 | 中国食品药品检定研究院 | Human anti-Gal-IgG antibody detection kit and preparation method and application thereof |
| CN113267624A (en) * | 2021-01-13 | 2021-08-17 | 广东菲鹏生物有限公司 | Method and product for detecting RBD and NTD neutralizing antibody |
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