CN103901218A - Method for preparing positive serum of autoimmune antigen - Google Patents
Method for preparing positive serum of autoimmune antigen Download PDFInfo
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Abstract
The invention relates to a method for preparing positive serum of an autoimmune antigen. The method comprises the following sequentially-executed steps: (1) repeatedly immunizing a healthy animal with an autoimmune antigen, drawing blood from the healthy animal before every immunization, so as to assay an OD (Optical Density) value, ending immunization after the OD value is stable, and drawing blood, so as to obtain antiserum; (2) carrying out affinity purification on the antiserum obtained in the step (1), so as to obtain an IgG (Immunoglobulin G) antibody; (3) coupling the IgG antibody obtained in the step (2) and a human IgG antibody according to the mass ratio of 1: (1-5), then, separating and purifying, so as to obtain an IgG-IgG connective concentrated solution, and diluting the IgG-IgG connective concentrated solution until the concentration is 0.5-1 microgram per milliliter, thereby obtaining the positive serum. The method disclosed by the invention can be applied to the batch preparation of positive serums of various autoimmune antigens and is simple and easy.
Description
Technical field
The invention belongs to vitro diagnostic techniques field, be specifically related to a kind of preparation method of positive serum of autoimmunity antigen.
Background technology
Autoimmune disease is that autoimmune response acquires a certain degree and the disease that causes.The most basic function of immune system is understanding self and identification allosome, reaches protection self and the object of repelling allosome.In normal human's serum, can have the multiple antibody for autoantigen, but their level is extremely low, is not enough to destroy self component, can remove the autologous tissue of old and feeble regression, Here it is autoimmune response.Excessively strong when this reaction, cause serious tissue damage, while showing clinical symptoms, be just called autoimmunity disease.The autoimmunity antigen of this class autoimmune disease includes but not limited to following listed: AMA M2, P0, His, nucl, dsDNA, PCNA, CENP B, JO-1, PM-Scl, Scl-70, SSB, SSA, Sm, nRNP, GBM, MPO, PR3, SAL/LP, GP210, SP100, LC-1, LKM etc.
Common autoimmunity disease has following several: systemic loupus erythematosus, Sjogren syndrome, mixed connective tissue disease, chorionitis, polymyositis/dermatomyositis etc.
For example: systemic loupus erythematosus is a kind of diseases associated with inflammation of getting involved with closely-related chronic, the many internal organs of autoimmunity system.This disease is often occurred frequently in women population.Common clinical manifestation comprises haemocyte minimizing in pathology and the hematological system of dermexanthesis, pleurisy and pericarditis, kidney or central nervous system of migration arthralgia and arthritis, cheek and other skin part etc.The clinical diagnosis for SLE needs close combination with clinical performance and results of serological detection.For in active stage serious symptom patient clinical conventionally use corticosteroid, hydroxychloroquine and some immunodepressant to treat.
The patient of about 70-90% is women (taking child-bearing period women as main).The incidence of disease of SLE in Black people and Asian will be higher than white man.This disease can, in any age bracket morbidity, comprise neonate.Because clinician more and more payes attention to for the diagnosis of SLE, therefore the case load of annual report continues to rise.In some countries, the incidence of disease of SLE even exceedes rheumatoid arthritis (RA).The autoimmune response that environmental factor causes is a principal element of inheritance susceptible crowd morbidity.
Because having many internal organs, the clinical manifestation of SLE involves, therefore the clinical diagnosis of often obscuring SLE and other rheumatic disease.For example: possess in early days the SLE patient of obvious joint symptom, may be misdiagnosed as rheumatoid arthritis.The disease that may mistaken diagnosis occur with SLE also comprises, mixed connective tissue disease (MCTD), rheumatoid panarthritis, polymyositis/dermatomyositis, sarcoidosis and secondary knurl syndrome etc.It should be noted that some infectious diseases (such as bacterial endocarditis and histoplasmosis etc.) may mistaken diagnosis be also SLE, thereby cause unnecessary immunosuppressive therapy.Therefore, serology antibody test in laboratory is the important evidence of distinguishing SLE and other clinical illness.
But because the positive serum of autoimmunity antigen is rarer, clinical collection is feasible on a small quantity, serum a large amount of or high concentration can not be collected substantially.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of preparation method of the autoimmunity antigen positive serum can be mass.
For solving above technical matters, the present invention takes following technical scheme:
A preparation method for autoimmunity antigen positive serum, comprises the following steps of carrying out successively:
Step 1, healthy animal is carried out repeatedly to immunity with autoimmunity antigen, before each described immunity, described healthy animal is got to hematometry OD value, when after OD value stabilization, immunity finishes, and gets blood, acquisition antiserum;
Step 2, the antiserum of step 1 gained is carried out to affinity purification obtain IgG antibody;
Step 3, the IgG antibody of step 2 gained and human IgG antibody are 1:1～5 coupling in mass ratio after, obtain IgG-IgG connector strong solution through separation and purification, it is 0.5～1 μ g/ml that described IgG-IgG connector strong solution is diluted to concentration, obtains described positive serum.
Preferably, in step 1, the assay method of OD value is enzyme-linked immunosorbent assay (ELISA).
Preferably, described healthy animal is rabbit.
Preferably, in step 1, the quality of the described autoimmunity antigen adopting when each immunity is 2～60mg.
Preferably, in step 1, first described autoimmunity psma ligand being set to the autoimmunity antigenic solution that concentration is 4～8mg/ml, is that 1～1.5:1 is configured to emulsion and carries out first immunisation by the volume ratio of described autoimmunity antigenic solution and Freund's complete adjuvant; Be that 1～1.5:1 is configured to emulsion and carries out follow-up immunization by described autoimmunity antigenic solution and the volume ratio of formula Freund's incomplete adjuvant not.
Preferably, described immune time is 6～8 times, and be 12～36 days the interval time of every twice immunity.
Preferably, in step 1, while getting blood, adopt auricular vein to get blood.
Preferably, adopt albumin A sepharose CL-4B affinity column to carry out described affinity purification.
Preferably, in step 3, described IgG antibody and described human IgG antibody by after described 2-imines thiophane coupling agent and the activation of 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester coupling agent, carry out coupling respectively at 2～8 DEG C.
Preferably, in step 3, adopt Supperdex200 gel column to carry out described separation and purification.
Preferably, in step 3, adopt containing the TRIS buffer of the mass ratio bovine serum albumin(BSA) that is 0.4～6%, pH7.5～8.5,0.09～0.11mol/L described IgG-IgG connector strong solution is carried out to described dilution.
Due to the enforcement of above technical scheme, the present invention compared with prior art tool has the following advantages:
The inventive method can be prepared the positive serum of various autoimmunity antigens in batches, and this preparation method is simple; The positive serum that the method obtains can be used for the positive quality control in detection kit preparation process, also can be used as the raw materials for production of calibration object.
Brief description of the drawings
Accompanying drawing 1 is the anti-JO-1IgG protein band of rabbit through albumin A sepharose CL-4B affinity column purifying.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following examples.The implementation condition adopting in embodiment can require to do further adjustment according to the difference of concrete use, and not marked implementation condition is the normal condition in the industry.
Embodiment 1
(1) immunity of autoimmunity antigen and rabbit anti-serum titration
Material and instrument
1, autoimmunity antigen: autoimmunity antigen freeze-dried powder, autoimmunity antigen is JO-1;
2, adjuvant: commercial Freund's complete adjuvant and incomplete Freund's adjuvant;
3, animal: the healthy new zealand white rabbit that selects 3 (3 rabbits of every kind of autoimmunity antigen immune) 2 monthly ages, body weight 1.5～2.0kg;
4, two is anti-: the goat anti-rabbit igg of HRP mark;
5, consumptive material: three-way device, disposable syringe, pipettor etc.
Immunity step
1, immune autoimmunity antigen is prepared: determine that by BCA method freeze-dried powder dissolves the concentration of rear autoimmunity antigen, get 4mg autoimmunity antigen PBS and be diluted to 600ul, with three-way device by autoimmunity antigen and Freund's complete adjuvant or Freund's incomplete adjuvant (autoimmunity antigen V: adjuvant V=6:5) emulsification, until an emulsion is splashed into and presents the spherical autoimmunity antigen that do not disperse in water and be ready to.First immunisation Freund's complete adjuvant, follow-up immunization is all used incomplete Freund's adjuvant.
2, animal immune: 3 new zealand white rabbits buying are cultivated to 1 week at Animal House, animal is conformed; By the emulsion preparing, carry out the injection of neck or back subcutaneous inoculation with 1ml syringe, every new zealand white rabbit is injected 3 points, every some injection 300ul; The immunity cycle is 14 days.
3, sero-fast preparation: extract 2mL blood (as blank) prior to a side auricular vein before every animal immune, each immunity afterwards, after 14 days, is carried out auricular vein and got blood for antiserum evaluation before next immunity; After getting blood, take off syringe needle, the blood in syringe is slowly transferred in centrifuge tube, in 4 DEG C of refrigerator overnight, get supernatant and be serum, separate out faint yellow antiserum; Antiserum is transferred in another pipe, and blood clot is with the centrifugal 10min of 1500xg.Sucking-off supernatant merges in the antiserum pipe of collecting; Antiserum packing is stored in-70 DEG C.
4, the mensuration of antiserum titre: adopt enzyme linked immunosorbent assay (ELISA) (enzyme linked immunosorbent assay, ELISA), with coated damping fluid by autoimmunity antigen diluent to 10ug/ml, except negative hole, to add 100ul be 1ug in every hole; The each coated 1ug BSA of negative control 1 and negative control 2 and human hemoglobin, 4 DEG C of coated spending the night; Blank 1: get blood before immune autoimmunity antigen; Blank 2: primary antibodie is hatched confining liquid, does not wherein have an antiantibody; The goat anti-rabbit igg of blank 3:HRP mark is hatched confining liquid; All the other holes are by the antiserum of 1:100,1:200 doubling dilution, carry out titration.Parallelly when each titration carry out collecting last time sero-fast titration, after twice immunity in front and back, the anti-blood difference of tiring is little, can stop immunity and kill rabbit and get blood, is also significantly improved if tired, and continues immunity.
With antiserum evaluation result table 1 after JO-1 immunize rabbit:
Table 1
Note: A column data is the antiserum titre that immunity is measured for 6 times afterwards; B column data is the antiserum titre that immunity is measured for 7 times afterwards
From above table, can find out after rabbit immunity 6 times and 7 times, antiserum titre tends towards stability substantially, be 1:100-1:1600000 according to the serum gradient of experimental design evaluation, wherein in the time that serum is diluted to 1:51200, OD value is 2 times of left and right of blank well, be judged as the positive and tire, but OD value equals blank value or the twice less than blank value substantially in the time diluting again, be judged as without tiring.The negative contrast of A1-A2 and B1-B2, can find out that from numerical value the rabbit anti-serum and other autoimmunity antigen that obtain do not have reactivity; A3-A5 and B3-B5 are blank, can find out rabbit anteserum before autoimmunity antigen and immunity, with the two anti-non-specific respondings that all can not produce; The reaction in other holes is the specific reaction of JO-1 autoimmunity antigen and the anti-JO-1 autoimmunity of rabbit antigen.
(2) sero-fast affinity purification
Material and instrument
1, albumin A sepharose CL-4B affinity column; Peristaltic pump; Centrifuge tube; Hydro-extractor; Filtrator; Glass column;
2, TBS buffer solution: 6.06g Tris (50mM), 8.78g NaCl (150mM) and 0.5g sodium azide (0.05%) are dissolved in 1L distilled water, and regulate pH7.4 with HCl;
3, neutralization buffer solution: 121.2g Tris (1M), 87.8g NaCl (1.5M), 0.37g EDTA (1mM) and 5g sodium azide (0.5%) are dissolved in 1L distilled water, and regulate pH8.0 with HCl;
4, elution buffer solution (pH2.7): 3.75g glycocoll (50mM) is dissolved in 1L distilled water, regulates pH2.7 with HCl;
5, elution buffer solution (pH1.9): 3.75g glycocoll (50mM) is dissolved in 1L distilled water, regulates pH1.9 with HCl.
Operation steps
1, prepare albumin A sepharose CL-4B affinity column: prepare 10ml albumin A sepharose CL-4B filler, in Dewar bottle, isopyknic filler and TBS buffer solution are mixed, stir.Vacuumize approximately 15 minutes to remove the bubble in filler, otherwise capacity and the separating effect of the aeration pillar forming in post.Albumin A sepharose CL-4B filler is slowly added in glass column, utilize pump control filling speed to divide for divide～2ml/ of 1ml/, avoid post dry, utilize 10 times to bed volume and through the TBS buffer solution balance pillars of precooling.
2, the antiserum preparing is put into frozen water or 4 DEG C of refrigerators and is slowly thawed to avoid the gathering of protein.The gathering occurring in protein course of defrosting can be dissolved by 37 DEG C of preheatings.Adding solid sodium azide to concentration is 0.05%, 4 DEG C, centrifugal 5 minutes of 15000xg, and the antiserum that shifts out clarification is filtered again removes unnecessary fat.
3, the antiserum after dissolving is diluted with the ratio of 1:5 with TBS buffer solution, then filter with filtrator.With the speed of per minute 0.5ml by antiserum to post, be the combination that ensures antiserum and filler, need continuous upper prop 2 times also retains loading efflux.Clean pillar with TBS buffer solution and add pH2.7 elution buffer solution to A λ 280nm<0.008, be eluted to all albumen with the speed of 0.5ml/min and all flow down.Be in charge of collection eluent with the 1.5ml EP pipe that adds 100ul neutralization buffer solution, mix the rear pH that checks eluent with pH test paper, if pH can utilize neutralization buffer to be adjusted to about pH7.4 to prevent the sex change of antibody lower than 7;
In post, add 10ml, pH1.9 elution buffer solution, collect as stated above eluent to A λ 280nm<0.008;
Utilize the content of protein in the each pipe of spectrophotometric determination.If protein concentration can add 10% glycerine to preserve lower than 0.5mg/ml, by after the IgG antibody packing of purifying 2 DEG C～8 DEG C preservations;
After cleaning pillar with the TBS buffer solution containing 0.05% sodium azide, pillar is stored in to 2 DEG C～8 DEG C environment.
(3) IgG antibody and human IgG antibody's coupling
Material and instrument
1, human IgG antibody, by the self-service preparation in Hao Oubo biological medicine company limited cooperation unit Medical University Of Tianjin immunization experiment chamber, purity is 95%, concentration is 1mg/ml, preserves with phosphate buffer;
2, coupling agent 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC), 2-imines thiophane (2-IT) is purchased from THERMO company, and the chemical reagent such as trishydroxymethylaminomethane (TRIS) all reach chemical pure;
3, G-25 gel column and Supperdex200 gel column are GE company product.
Operation steps
1, get 1mgIgG antibody, add the coupling agent 2-IT solution 3 μ l of 10mg/ml, room temperature leaves standstill 20min, adds the glycine solution 10 μ l of 0.1mol/L, and room temperature leaves standstill 5min.With G-25 gel column desalination, collect the rear antibody of activation, 5 DEG C save backup;
2, get human IgG antibody's solution of 1.5mg, add the SMCC solution 15 μ l of 5mg/ml, room temperature leaves standstill 30min, with G-25 gel column desalination, collects the rear antibody of activation, and 5 DEG C save backup;
3, the IgG antibody of above-mentioned activation is mixed with the human IgG antibody of activation, under 2-8 DEG C of condition, leave standstill 20h, purify conjugate with Supperdex200 gel column, acquisition connector strong solution, 5 DEG C save backup;
4, IgG-IgG connector strong solution is diluted to 0.5 μ g/ml with the TRIS damping fluid that contains 0.5% bovine serum albumin(BSA), pH8.0,0.1mol/L, obtains the positive serum of JO-1 autoimmunity antigen.
Embodiment 2
Material and instrument
1, autoimmunity antigen: adopt the autoimmunity antigen of the same batch of preparation of immune animal, dissolve rear portion for animal immune, a part detects for serum evaluation;
2, primary antibodie: Tu Kang JO-1IgG(Medical University Of Tianjin's immunization experiment chamber preparation); The positive serum that embodiment 1 prepares; Human IgG albumen is purchased from Sigma company;
3, two is anti-: the goat anti-human igg of the goat anti-rabbit igg of AP mark and AP mark is purchased from Sigma;
4, cleaning fluid is 0.1mol PBST, and luminous substrate is that the great Ou Bo in Suzhou produces;
5, instrument: wash plate machine and microplate reader
Operation steps
1, by autoimmunity antigen diluent in coated damping fluid (0.1mol/LNa
2cO
3, pH9.6) in, final concentration is 10ng/ μ L, every hole adds 100 μ L, and sets up blank (only adding coated damping fluid), and after 4 DEG C of coated spending the night, coated 3 groups altogether.
2, under room temperature, wash 3 times each 5min with 1 × PBST ［ 1 × PBS+ (0.1%) Tween20 ］.
3, every hole adds 200 μ L confining liquids (10% calf serum is dissolved in 1 × PBS), 37 DEG C of sealing 90min.
4,1 × PBST washing 3 times.Add primary antibodie, by 1:200,1:400 doubling dilution in 1 × PBS, every hole 100 μ L, negative hole adds the primary antibodie of 100 μ L1:200 dilutions, hatches 2h for 37 DEG C.In 3 groups, outside first negative hole, every kind of primary antibodie is hatched 15 holes.
5,1 × PBST washing 3 times.
6, add two to resist, two anti-by specifications, 1:1000 is diluted in 1 × PBS, every hole 100 μ L, 37 DEG C, 1h.
7, substrate colour developing;
8, stop.(its testing result is in table 2)
Table 2
| Title | A | B | C | D | E | F |
| Negative control | 0.066 | 0.071 | 0.073 | 0.069 | 0.067 | 0.072 |
| 1:100 | 3.297 | 0.11 | 3.271 | 3.229 | 0.073 | 0.069 |
| 1:200 | 3.154 | 0.097 | 3.229 | 3.357 | 0.069 | 0.073 |
| 1:400 | 3.079 | 0.092 | 2.973 | 3.272 | 0.065 | 0.076 |
| 1:800 | 2.995 | 0.085 | 2.655 | 3.073 | 0.067 | 0.067 |
| 1:1600 | 1.872 | 0.079 | 1.237 | 2.977 | 0.077 | 0.065 |
| 1:3200 | 1.037 | 0.071 | 0.701 | 1.934 | 0.074 | 0.07 |
| 1:6400 | 0.662 | 0.073 | 0.395 | 1.002 | 0.072 | 0.068 |
Coated: the first row (A group-F group) is negative control, coated irrelevant albumen; All the other are coated immune autoimmunity antigen all;
A group: primary antibodie is the anti-JO-1IgG of rabbit, and two resist the goat anti-rabbit igg for AP mark, as can be seen from the results, react;
B group: primary antibodie is the anti-JO-1IgG of rabbit, and two resist the goat anti-human igg for AP mark, as can be seen from the results, reactionless;
C group: primary antibodie is the positive serum that embodiment 1 prepares, two resist the goat anti-rabbit igg for AP mark, as can be seen from the results, react;
D group: primary antibodie is the positive serum that embodiment 1 prepares, two resist the goat anti-human igg for AP mark, as can be seen from the results, react;
E group: primary antibodie is human IgG albumen, two resist the goat anti-rabbit igg for AP mark, as can be seen from the results, reactionless;
F group: primary antibodie is human IgG albumen, two resist the goat anti-human igg for AP mark, as can be seen from the results, reactionless.
Can find out from the above results, autoimmunity antigen can react with the anti-JO-1IgG of rabbit, reactionless with human IgG albumen, so ensured the reaction capacity of autoimmunity antigen and rabbit anti-serum after the reactivity of the IgG-IgG proof anti-JO-1IgG of rabbit and human IgG albumen coupling, can react with anti-human IgG as similar human IgG again.
Above the present invention is described in detail; its object is to allow the personage who is familiar with this art can understand content of the present invention and be implemented; can not limit the scope of the invention with this; the equivalence that all Spirit Essences according to the present invention are done changes or modifies, and all should be encompassed in protection scope of the present invention.
Claims (10)
1. a preparation method for autoimmunity antigen positive serum, is characterized in that: comprise the following steps of carrying out successively:
Step 1, healthy animal is carried out repeatedly to immunity with autoimmunity antigen, before each described immunity, described healthy animal is got to hematometry OD value, when after OD value stabilization, immunity finishes, and gets blood, acquisition antiserum;
Step 2, the antiserum of step 1 gained is carried out to affinity purification obtain IgG antibody;
Step 3, the IgG antibody of step 2 gained and human IgG antibody are 1:1 ~ 5 coupling in mass ratio after, obtain IgG-IgG connector strong solution through separation and purification, it is 0.5 ~ 1 μ g/ml that described IgG-human IgG connector strong solution is diluted to concentration, obtains described positive serum.
2. the preparation method of autoimmunity antigen positive serum according to claim 1, is characterized in that: described healthy animal is rabbit.
3. the preparation method of autoimmunity antigen positive serum according to claim 1, is characterized in that: in step 1, the quality of the described autoimmunity antigen adopting when each immunity is 2 ~ 60mg.
4. the preparation method of autoimmunity antigen positive serum according to claim 1, it is characterized in that: in step 1, first described autoimmunity psma ligand being set to the autoimmunity antigenic solution that concentration is 4 ~ 8mg/ml, is that 1 ~ 1.5:1 is configured to emulsion and carries out first immunisation by the volume ratio of described autoimmunity antigenic solution and Freund's complete adjuvant; Be that 1 ~ 1.5:1 is configured to emulsion and carries out follow-up immunization by described autoimmunity antigenic solution and the volume ratio of formula Freund's incomplete adjuvant not.
5. according to the preparation method of the autoimmunity antigen positive serum described in claim 1 or 4, it is characterized in that: described immune time is 6 ~ 8 times, be 12 ~ 36 days the interval time of every twice immunity.
6. the preparation method of autoimmunity antigen positive serum according to claim 1, is characterized in that: in step 1, adopt auricular vein to get blood while getting blood.
7. the preparation method of autoimmunity antigen positive serum according to claim 1, is characterized in that: adopt albumin A sepharose CL-4B affinity column to carry out described affinity purification.
8. the preparation method of autoimmunity antigen positive serum according to claim 1, it is characterized in that: in step 3, described IgG antibody and described human IgG antibody by after described 2-imines thiophane coupling agent and the activation of 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester coupling agent, carry out coupling respectively at 2 ~ 8 DEG C.
9. the preparation method of autoimmunity antigen positive serum according to claim 1, is characterized in that: in step 3, adopt Supperdex200 gel column to carry out described separation and purification.
10. the preparation method of autoimmunity antigen positive serum according to claim 1, it is characterized in that: in step 3, adopt containing the TRIS buffer of the mass ratio bovine serum albumin(BSA) that is 0.4 ~ 6%, pH7.5 ~ 8.5,0.09 ~ 0.11mol/L described IgG-IgG connector strong solution is carried out to described dilution.
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| CN201410553407.7A CN104977419A (en) | 2014-04-01 | 2014-10-17 | Purifying preparation method of autoimmuno antigen positive serum |
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| WO2016061861A1 (en) * | 2014-10-20 | 2016-04-28 | 苏州浩欧博生物医药有限公司 | Method for preparing and purifying positive serum of an autoimmune antigen |
| CN107490680A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin enzyme-linked immune detection method |
| CN111163786A (en) * | 2017-07-14 | 2020-05-15 | 徐民桢 | Methods and pharmaceutical compositions for inducing a M1 dominant immune response |
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| CN1176377C (en) * | 2002-12-30 | 2004-11-17 | 陕西超英生物医学研究开发有限公司 | Composite chip for detecting autoimmune antibody of diabetes mellitus, and preparation and detection method thereof |
| CN101215564B (en) * | 2003-11-07 | 2011-06-22 | 首都医科大学附属北京朝阳医院 | M2-vagusstoff receptor mutation gene of dilatant cardiomyopathy patient |
| CN1924579A (en) * | 2005-07-04 | 2007-03-07 | 上海富纯中南生物技术有限公司 | Cross-linked composite used as standard diagnosing reagent replacing positive serum and method for use as standard reagent |
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2014
- 2014-04-01 CN CN201410127283.6A patent/CN103901218A/en active Pending
- 2014-10-17 CN CN201410553407.7A patent/CN104977419A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016061861A1 (en) * | 2014-10-20 | 2016-04-28 | 苏州浩欧博生物医药有限公司 | Method for preparing and purifying positive serum of an autoimmune antigen |
| CN107490680A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin enzyme-linked immune detection method |
| CN111163786A (en) * | 2017-07-14 | 2020-05-15 | 徐民桢 | Methods and pharmaceutical compositions for inducing a M1 dominant immune response |
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| Publication number | Publication date |
|---|---|
| CN104977419A (en) | 2015-10-14 |
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