CN1621415A - Immunoglobulin antibody against SARS-CoV and its preparing method - Google Patents
Immunoglobulin antibody against SARS-CoV and its preparing method Download PDFInfo
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Abstract
The present invention relates to anti-SARS-CoV immune globulin F(ab' )2 antibody for treatment, prevention and diagnosis of SARS-CoV. The present invention features that the antibody is horse anti-SARS-CoV immune globulin F(ab' )2 antibody. The present invention also discloses the preparation process of the antibody. The antibody of the present invention can kill SARS-CoV specifically, and has excellent effect of preventing and treating SARS.
Description
Technical field
The present invention relates to a kind of anti-SARS-CoV immune globulin antibody that is used for the treatment of, prevents and diagnose SARS-CoV, belong to field of biological pharmacy.
Background technology
The World Health Organization after sending global warning March 12 this year about atypical pneumonia (atypicalpneumonia), subsequently the called after severe acute respiratory syndrome (SevereAcute Respiratory Syndrome, SARS).Under great attention, coordination and the tissue of WHO, cause that the pathogenic agent of SARS is confirmed to be a kind of coronavirus new or variation very soon, and corresponding called after SARS-CoV.Propagate very soon after SARS outburst, now spread to more than 30 countries and regions, the world, seriously influence people's health and lives, become at the beginning of 21 century the No.1 formidable enemy of harm humans in the whole world.Horse anteserum (being the horse anti-immunoglobulin again) is a kind of effective emergence therapeutic measure that is used for important pathogenic infection since ancient times, has 7 kinds of antiserum(antisera)s so far at least in wide clinical application, brings into play important treatment and the effect that keeps off infection.Traditional whole antibody molecule has been experienced in the horse anteserum development, develops into and effectively removes Fc section antibody molecule and other blood plasma foreign protein molecule that can cause toxic side effect today, and acquisition has the high purity F (ab ') with the pathogen antigen specific combination
2Treat antibody, reach the effect of eliminating pathogen.The effect that has treatment SARS based on Hong Kong, Singapore and China mainland report at SARS convalescent's serum, but there is certain problem in the widespread use of SARS convalescent's serum, and people are also very superficial to the understanding of SARS-CoV, particularly there is certain problem, restricts its application and development at aspects such as securities.
Therefore, development has treatment and the anti-SARS-CoV immunoglobulin (Ig) of horse that prevents SARS, is very valuable therapeutic and preventative antibody drug.
Summary of the invention
One object of the present invention is to disclose a kind of novel anti-SARS-CoV IgF (ab ') 2 antibody that are used for the treatment of, prevent and diagnose SARS-CoV; Another object of the present invention is the open method for preparing the anti-SARS-CoV IgF of horse (ab ') 2 antibody.
A kind of anti-SARS-CoV immune globulin antibody that is used for the treatment of, prevents and diagnose SARS-CoV is the anti-SARS-CoV IgF of horse (ab ') 2 antibody.
The preparation of the anti-SARS-CoV IgF of horse of the present invention (ab ') 2 antibody may further comprise the steps:
1) deactivation purifying SARS-CoV adds incomplete freund adjuvant emulsification and prepares immunogen, adopt subcutaneous, muscle, the immunity of inguinal lymph nodes multiple spot to meet the horses of biological products requirement through quarantine, immunity four times, interval immunity in 10 days are once, examination blood survey antibody titer counterelectlophoresis countercurrent electrophoresis 1: 128, indirect enzyme-linked immunosorbent 1: 12800, survey antibody neutralization tire cytopathy political reform 1: 12800, plaque subtractive method 1: 12800, and the neutralizing antibody that different SARS-CoV type strain immunity horse produces all presents consistent neutralizing antibody activity with other SARS-CoV strain;
2) gather the anti-SARS-CoV blood plasma of horse, add 21% saturation ratio ammonium sulfate mixing, centrifugal precipitation scleroproein and the albumin of abandoning with 2 times of distilled water dilutings of no thermal source, supernatant adds that 9% saturation ratio ammonium sulfate mixing is centrifugal will to precipitate immunoglobulin G (IgG) again, with no thermal source distilled water recover original volume again with 30% saturation ratio ammonium sulfate mixing, centrifugally to precipitate IgG, add earlier a certain amount of no thermal source distilled water, back and add no thermal source 0.9% sodium chloride brine with 60,000 ultra-fine filter ultrafiltration desalinations, concentrated and foreigh protein removing, ultrafiltrated is the anti-SARS-CoV IgG of purifying horse;
3) the anti-SARS-CoV IgG of the horse of purifying transfers the stomach en-cutting of PH to 3.2 back with hydrochloric acid, transfers PH to 7.4 with sodium hydroxide again, heats 58 ℃, 30min, crosses elimination Fc section with canvas, and filtrate is the anti-SARS-CoV IgF of preliminary purification horse (ab ')
2Antibody;
4) the anti-SARS-CoV IgF of the horse of preliminary purification (ab ')
2Antibody is crossed phenyl vegetables water layer and is analysed (Phenyl Sepharose 6 Fast Flow), and with 200mmol/L pH7.0 phosphoric acid buffer, 1mol/L ammonium sulfate upper prop, 20mmol/L pH7.0 phosphoric acid buffer is collected F (ab ')
2The antibody effluent liquid with 60,000 ultra-fine filter ultrafiltration desalinations, concentrated, is the anti-SARS-CoV IgF of purifying horse (ab ') again
2Antibody.
The anti-SARS-CoV IgF of horse (ab ') of the purifying of the present invention's preparation
2Antibody can be prepared into horse anti-SARS-CoV Gammimune N type and mucous membrane aerosol finished product.
The deactivation SARS-CoV of the used purifying of the present invention can make as follows:
1) the frozen Vero cell strain of cell seed bank is got in recovery, the cultivation of green monkey kidney cell (Vero cell), with the DMEM substratum and add 10% calf serum and cultivate, provides the Vero cell of cultivating SARS-CoV;
2) sars coronavirus (SARS-CoV) titration adds through identifying frozen SARS-CoV the Vero cell of cultivating, and amplification cultivation SARS-CoV adopts tissue culture median infective dose (TCID
50) measure the virulence (10 of SARS-CoV
6-10
8), frozen to titrating SARS-CoV packing;
3) add titrating SARS-CoV with the Vero cell that covers with individual layer, cultivate SARS-CoV, produce SARS-CoV with serum-free DMEM;
4) collect the SARS-CoV nutrient solution after the complete pathology of Vero cell, the SARS-CoV cell culture fluid is collected in multigelation three (freezing-37 ℃) or not freeze thawing;
5) add the beta-propiolactone of 1/2000-1/8000 to the SARS-CoV nutrient solution, 4 ℃ spend the night, 37 ℃ of 2h deactivation SARS-CoV, the SARS-CoV nutrient solution of getting deactivation connects on the Vero cell and passes the three generations, detects the SARS-CoV inactivating efficacy;
6) the SARS-CoV nutrient solution of deactivation, 4 ℃, centrifugal 12000rpm/min 30min abandon the precipitation foreign protein, and supernatant is a SARS-CoV liquid;
7) with molecular weight 300,000 ultrafiltration post ultrafiltration SARS-CoV liquid, concentrate and foreigh protein removing, filtrate is rough deactivation SARS-CoV;
8) rough deactivation SARS-CoV is gone up Sepharose 4 Fast Flow molecular sieve columns, the beginning damping fluid is a 0.1MPBS PH7.0 damping fluid, with 100mM PBS PH7.0 wash-out, collects SARS-CoV, merges to concentrate and collects preliminary purification SARS-CoV;
9) to DE52 anion column chromatography on the SARS-CoV liquid of preliminary purification, use 100mM PB, PH7.2 liquid balance also is adsorbed on virus on the post, uses 100mM PB, PH7.2, and 0.35M NaCl wash-out is collected the SARS-CoV peak, merges to concentrate to collect purifying SARS-CoV;
10) to the effect analysis of SARS-CoV liquid behind the ultrafiltration and concentration, record antigen rate of recovery average out to 55%, record foreign protein clearance average out to 92% with improvement Lowry method with double antibody sandwich method;
11) to the effect analysis of gel-filtration purifying SARS-CoV, with double antibody sandwich method record antigen rate of recovery average out to 85%, adopt improvement Lowry method record foreign protein clearance average out to 94.5%, complete with the electron microscopic observation virion, measure Vero cell residue dna content less than 10.0ng/ml, suppress the remaining bovine serum content of measuring virus less than 12.5ng/ml with dot hybridization with blood clotting;
12) to the effect analysis of ion-exchange purification SARS-CoV, record the 9.4ng/U of proteantigen before with double antibody sandwich method by purifying, be reduced to 0.8ng/U, HPLC shows that viral purity is 97%; Adopt improvement Lowry method to record foreign protein clearance average out to 98.5%; Complete with the electron microscopic observation virion, measure Vero cell residue dna content less than 1.0ng/ml with dot hybridization; SDS-PAGE shows S, N, M, the major protein band of SARS-CoV, and reacts with decubation patient's SARS serum, and purifying deactivation SARS-CoV divides and puts-70 ℃ of preservations.
The present invention utilizes isolating SARS-CoV immune health horses behind cultivation, deactivation, purifying, produce in high the tiring and the antiserum(antisera) of SARS-CoV, removal can cause the Fc fragment of side effect, the preparation anti-SARS-CoV IgF of horse (ab ') 2 antibody, can special elimination SARS-CoV, the treatment and the prevention of atypical pneumonia there is good effect; The anti-SARS-CoV immune globulin antibody of purifying horse has excellent specificity and susceptibility to SARS-CoV.
Following experimental example progress explanation the present invention.
The evaluation of the experimental example 1 refining anti-SARS-CoV IgF of horse (ab ') 2 antibody
1) the refining anti-SARS-CoV IgF of horse (ab ') 2 antibody and the anti-SARS-CoV immune globulin antibody of purifying horse adopt the counterelectlophoresis countercurrent electrophoresis antibody titer to reach 1: 128 above, indirect enzyme-linked immunosorbent antibody titer up to more than 1: 12800;
2) neutralization of the refining anti-SARS-CoV IgF of horse (ab ') the 2 antibody titration of tiring, be at first on the Vero cell to SARS-CoV titration determine that titre is 10
6-8, select 200TCID
50Detect the neutralization of the anti-SARS-CoV blood plasma of horse, the refining anti-SARS-CoV IgF of horse (ab ') 2 antibody tires by the antibody dilution method.Or with tiring more than 1: 10000 with the neutralization of the anti-SARS-CoV blood plasma of experimental technique detection horse, the refining anti-SARS-CoV IgF of horse (ab ') 2 antibody in the plaque subtrahend;
The evaluation of experimental example 2 refining anti-SARS-CoV immunoglobulin therapies of horse and preventive effect
1) sets up SARS-CoV and infect the Vero-6 cell model, add 4 * 10 at 16 porocyte culture plates
5/ hole Vero-6 cultivates 24h and adds 100CTID
50BJ01 SARS-CoV200ul, add the cell that the anti-SARS-CoV immunoglobulin therapy of 0.1mg/100mL horse SARS-CoV infects behind the 24h, observe 48h and detect the effect that the anti-SARS-CoV immunoglobulin (Ig) of horse has fine treatment SARS-CoV through cytopathy political reform, mtt assay and plaque subtractive method;
2) the anti-SARS-CoV immunoglobulin (Ig) of horse 0.1mg/100mL adds 4 * 10
5/ hole Vero-6 cell/16 porocyte culture plates, 12h 100CTID
50BJ01 SARS-CoV200ul infects the Vero-6 cell, observes 72h and detects the effect that the refining anti-SARS-CoV immunoglobulin (Ig) of horse has fine prevention SARS-CoV through cytopathy political reform, mtt assay and plaque subtractive method;
3) pass through every rhesus monkey 1 * 10
8Collunarium infects BJ01 strain SARS-CoV and has set up infection model, infects back 6h, 12h, 24h, 48h intramuscular injection 2mg/2mL (100IU) respectively, observes dissection in 15 days, is relieved to nothing, white corpuscle and immunocyte from symptom performance treatment group and recovers normal gradually.Observe treatment group lung, liver, kidney, brain, thymus gland, spleen, lymphoglandula, wipe away position bacterial isolate bodies such as rouge, blood, ight soil from etiology, and PCR detection S, N, M, E and X4 main pathogens protein gene, all negative through virus culture and pcr amplification pathogenic agent.Observe the improvement that treatment group lung, hepatic tissue pathology change to be had in various degree from pathological change, and produce effective anti-SARS-CoV antibody.Illustrate that the anti-SARS-CoV immunoglobulin (Ig) of horse has the effect of obvious treatment SARS in the SARS-CoV infected animals;
4) the anti-SARS-CoV immunoglobulin (Ig) of horse mucous membrane sprays 3mg/3mL (150IU) administration, every 12h once, shared 4 times to rhesus monkey prevention, use 1 * 10 again
8Collunarium infects BJ01 strain SARS-CoV and infects and attack rhesus monkey, observes that 30 days prevention group rhesus monkeies symptom do not occur, have no temperature, orthocytosis, falls ill.Observe prevention group lung, liver, kidney, brain, thymus gland, spleen, lymphoglandula, wipe away position bacterial isolate bodies such as rouge, blood, ight soil from etiology, and PCR detection S, N, M, E and X4 main pathogens protein gene, all negative through virus culture and pcr amplification pathogenic agent.Observe the improvement that treatment group lung, hepatic tissue pathology change to be had in various degree from pathological change, and produce and have the active anti-SARS-CoV antibody of neutralization.Illustrate that the anti-SARS-CoV immunoglobulin (Ig) of horse has the effect of obvious prevention SARS in the SARS-CoV infected animals.
Following embodiment all can reach the effect of above-mentioned experimental example.
Embodiment 1The anti-SARS-CoV IgF of horse (ab ') 2 Antibody Preparation
1, the cultivation of SARS-CoV, deactivation and purifying:
1) the frozen Vero cell strain of cell seed bank is got in recovery, the cultivation of green monkey kidney cell (Vero cell), with the DMEM substratum and add 10% calf serum and cultivate, provides the Vero cell of cultivating SARS-CoV;
2) sars coronavirus (SARS-CoV) titration adds through identifying frozen SARS-CoV the Vero cell of cultivating, and amplification cultivation SARS-CoV adopts tissue culture median infective dose (TCID
50) measure the virulence (10 of SARS-CoV
6-10
8), frozen to titrating SARS-CoV packing;
3) add titrating SARS-CoV with the Vero cell that covers with individual layer, cultivate SARS-CoV, produce SARS-CoV with serum-free DMEM;
4) collect the SARS-CoV nutrient solution after the complete pathology of Vero cell, the SARS-CoV cell culture fluid is collected in multigelation three (freezing-37 ℃) or not freeze thawing;
5) add 1/4000 beta-propiolactone (1/2000-1/8000 all can) to the SARS-CoV nutrient solution, 4 ℃ spend the night, 37 ℃ of 2h deactivation SARS-CoV, the SARS-CoV nutrient solution of getting deactivation connects on the Vero cell and passes the three generations, detects the SARS-CoV inactivating efficacy;
6) the SARS-CoV nutrient solution of deactivation, 4 ℃, centrifugal 12000rpm/min 30min abandon the precipitation foreign protein, and supernatant is a SARS-CoV liquid;
7) with molecular weight 300,000 ultrafiltration post ultrafiltration SARS-CoV liquid, concentrate and foreigh protein removing, filtrate is rough deactivation SARS-CoV;
8) rough deactivation SARS-CoV is gone up Sepharose CL-2B molecular sieve purification, rough deactivation SARS-CoV is gone up Sepharose CL-2B column chromatography, 0.01Mtris-HCl the PH7.4 buffer solution elution is collected SARS-CoV, merges to concentrate to collect preliminary purification SARS-CoV;
9) to DE52 anion column chromatography purification on the SARS-CoV liquid of preliminary purification, with 0.03MNacl, 50mMTris PH8.0 buffer solution elution, collect the SARS-CoV peak, merge to concentrate and collect purifying SARS-CoV;
10) to the effect analysis of SARS-CoV liquid behind the ultrafiltration and concentration, record antigen rate of recovery average out to 55%, record foreign protein clearance average out to 92% with improvement Lowry method with double antibody sandwich method;
11) to the effect analysis of gel-filtration purifying SARS-CoV, with double antibody sandwich method record antigen rate of recovery average out to 85%, adopt improvement Lowry method record foreign protein clearance average out to 94.5%, complete with the electron microscopic observation virion, measure Vero cell residue dna content less than 10.0ng/ml, suppress the remaining bovine serum content of measuring virus less than 12.5ng/ml with dot hybridization with blood clotting;
12) to the effect analysis of ion-exchange purification SARS-CoV, record the 9.4ng/U of proteantigen before with double antibody sandwich method by purifying, be reduced to 0.8ng/U, HPLC shows that viral purity is 97%; Adopt improvement Lowry method to record foreign protein clearance average out to 98.5%; Complete with the electron microscopic observation virion, measure Vero cell residue dna content less than 1.0ng/ml with dot hybridization; SDS-PAGE shows S, N, M, the major protein band of SARS-CoV, and reacts with decubation patient's SARS serum, and purifying deactivation SARS-CoV divides and puts-70 ℃ of preservations.
2, the anti-SARS-CoV IgF of horse (ab ') 2 Antibody Preparation
1) SARS-CoV that selects for use is the standard strain that we separate and are identified through national biological product calibrating, and used Vero cell is from cells produce factory, carries out SARS-CoV cultivation, deactivation and purifying, preparation deactivation purifying SARS-CoV antigen;
2) deactivation purifying SARS-CoV adds incomplete freund adjuvant emulsification and prepares immunogen, adopt subcutaneous, muscle, the immunity of inguinal lymph nodes multiple spot to meet the horses of biological products requirement through quarantine, immunity four times, interval immunity in 10 days are once, examination blood survey antibody titer counterelectlophoresis countercurrent electrophoresis 1: 128, indirect enzyme-linked immunosorbent 1: 12800, survey antibody neutralization tire cytopathy political reform 1: 12800, plaque subtractive method 1: 12800, and the neutralizing antibody that different SARS-CoV type strain immunity horse produces all presents consistent neutralizing antibody activity with other SARS-CoV strain;
3) gather the anti-SARS-CoV blood plasma of horse, add 21% saturation ratio ammonium sulfate mixing, centrifugal precipitation scleroproein and the albumin of abandoning with 2 times of distilled water dilutings of no thermal source, supernatant adds that 9% saturation ratio ammonium sulfate mixing is centrifugal will to precipitate immunoglobulin G (IgG) again, with no thermal source distilled water recover original volume again with 30% saturation ratio ammonium sulfate mixing, centrifugally to precipitate IgG, add earlier a certain amount of no thermal source distilled water, back and add no thermal source 0.9% sodium chloride brine with 60,000 ultra-fine filter ultrafiltration desalinations, concentrated and foreigh protein removing, ultrafiltrated is the anti-SARS-CoV IgG of purifying horse;
4) the anti-SARS-CoV IgG of the horse of purifying transfers the stomach en-cutting of PH to 3.2 back with hydrochloric acid, transfers PH to 7.4 with sodium hydroxide again, heats 58 ℃, 30min, crosses elimination Fc section with canvas, and filtrate is the anti-SARS-CoV IgF of preliminary purification horse (ab ')
2Antibody;
5) the anti-SARS-CoV IgF of the horse of preliminary purification (ab ')
2Antibody is crossed phenyl vegetables water layer and is analysed (Phenyl Sepharose 6 Fast Flow), and with 200mmol/L pH7.0 phosphoric acid buffer, 1mol/L ammonium sulfate upper prop, 20mmol/L pH7.0 phosphoric acid buffer is collected F (ab ')
2The antibody effluent liquid with 60,000 ultra-fine filter ultrafiltration desalinations, concentrated, is the anti-SARS-CoV IgF of purifying horse (ab ') again
2Antibody;
6) the anti-SARS-CoV IgF of the horse of purifying (ab ')
2Antibody adds an amount of Thiomersalate sanitas, with physiological saline dilution desired content 0.22um degerming packing, prepares horse anti-SARS-CoV Gammimune N type and mucous membrane aerosol finished product respectively;
7) the anti-SARS-CoV immunoglobulin (Ig) of horse finished product is done the agarose electrophoresis detection with 2% protein concentration, does not have the albumin composition, detects F (ab ') by 10%SDS-PAGE
2Purity is more than 90%, antibody neutralization tire cytopathy political reform, plaque subtractive method 1: 2000/mg albumen, is 1 treatment unit (1IU), the then anti-SARS-CoV immunoglobulin (Ig) of horse finished product 50IU/mg by existing country to the anti-SARS-CoV Imnunoglobulin injection 1 of people: 40/mg;
8) physico-chemical property of the anti-SARS-CoV immunoglobulin (Ig) of horse finished product detects, protein content 3.0g/L, pH value 6.8, sodium chloride content 8.5g/L, ammonium sulfate content 0.5g/L, Thiomersalate 0.06g/L, freeze-dried preparation moisture content 2.0% all meet the regulation of biological products;
9) the anti-SARS-CoV immunoglobulin (Ig) of horse finished product carries out thermal source in rabbit and mouse body, undue toxicity is observed, and the anti-SARS-CoV immunoglobulin (Ig) of experiment confirm horse does not have thermal source, undue toxicity;
10) the anti-SARS-CoV immunoglobulin (Ig) of horse finished product is put 4 ℃, 37 ℃ observation stability, and the anti-SARS-CoV immunoglobulin (Ig) of experiment confirm horse is placed to neutralize to tire more than 6 months and do not subtracted, and good stability is arranged;
11) the anti-SARS-CoV immunoglobulin (Ig) of horse finished product, confirm do not have cross reaction by immunohistochemical methods with the normal popular feeling, liver, lung, kidney, brain, spleen, lymphoglandula, intestinal tissue organ, confirm not anti-people's structural constituent of the anti-SARS-CoV immunoglobulin (Ig) of horse, the control that is used for the people is safe.
Claims (3)
1, a kind of anti-SARS-CoV immune globulin antibody that is used for the treatment of, prevents and diagnose SARS-CoV is characterized in that the preparation method that this antibody is comprises the steps:
1) deactivation purifying SARS-CoV adds incomplete freund adjuvant emulsification and prepares immunogen, adopt subcutaneous, muscle, the immunity of inguinal lymph nodes multiple spot to meet the horses of biological products requirement through quarantine, immunity four times, interval immunity in 10 days are once, examination blood survey antibody titer counterelectlophoresis countercurrent electrophoresis 1: 128, indirect enzyme-linked immunosorbent 1: 12800, survey antibody neutralization tire cytopathy political reform 1: 12800, plaque subtractive method 1: 12800, and the neutralizing antibody that different SARS-CoV type strain immunity horse produces all presents consistent neutralizing antibody activity with other SARS-CoV strain;
2) gather the anti-SARS-CoV blood plasma of horse, add 21% saturation ratio ammonium sulfate mixing, centrifugal precipitation scleroproein and the albumin of abandoning with 2 times of distilled water dilutings of no thermal source, supernatant adds 9% again saturation ratio ammonium sulfate mixing is centrifugal and will precipitate immunoglobulin G, with no thermal source distilled water recover original volume again with 30% saturation ratio ammonium sulfate mixing, centrifugally to precipitate IgG, add earlier a certain amount of no thermal source distilled water, back and add no thermal source 0.9% sodium chloride brine with 60,000 ultra-fine filter ultrafiltration desalinations, concentrated and foreigh protein removing, ultrafiltrated is the anti-SARS-CoV IgG of purifying horse;
3) the anti-SARS-CoV IgG of the horse of purifying transfers the stomach en-cutting of PH to 3.2 back with hydrochloric acid, transfers PH to 7.4 with sodium hydroxide again, heats 58 ℃, 30min, crosses elimination Fc section with canvas, and filtrate is the anti-SARS-CoV IgF of preliminary purification horse (ab ')
2Antibody;
4) the anti-SARS-CoV IgF of the horse of preliminary purification (ab ')
2Antibody is crossed phenyl vegetables water layer and is analysed, and with 200mmol/L pH7.0 phosphoric acid buffer, 1mol/L ammonium sulfate upper prop, 20mmol/L pH7.0 phosphoric acid buffer is collected F (ab ')
2The antibody effluent liquid with 60,000 ultra-fine filter ultrafiltration desalinations, concentrated, is the anti-SARS-CoV IgF of purifying horse (ab ') again
2Antibody.
2, immune globulin antibody as claimed in claim 1 is characterized in that the deactivation SARS-CoV for preparing the used purifying of this antibody is made by following method:
1) the frozen Vero cell strain of cell seed bank is got in the recovery of green monkey kidney cell, cultivation, with the DMEM substratum and add 10% calf serum and cultivate, provides the Vero cell of cultivating SARS-CoV;
2) sars coronavirus titration adds through identifying frozen SARS-CoV the Vero cell of cultivating, and amplification cultivation SARS-CoV adopts the tissue culture median infective dose to measure the virulence 106-108 of SARS-CoV, and is frozen to titrating SARS-CoV packing;
3) add titrating SARS-CoV with the Vero cell that covers with individual layer, cultivate SARS-CoV, produce SARS-CoV with serum-free DMEM;
4) collect the SARS-CoV nutrient solution after the complete pathology of Vero cell, multigelation three times, the SARS-CoV cell culture fluid is collected in freezing-37 ℃ or not freeze thawing;
5) add the beta-propiolactone of 1/2000-1/8000 to the SARS-CoV nutrient solution, 4 ℃ spend the night, 37 ℃ of 2h deactivation SARS-CoV, the SARS-CoV nutrient solution of getting deactivation connects on the Vero cell and passes the three generations, detects the SARS-CoV inactivating efficacy;
6) the SARS-CoV nutrient solution of deactivation, 4 ℃, centrifugal 12000rpm/min 30min abandon the precipitation foreign protein, and supernatant is a SARS-CoV liquid;
7) with molecular weight 300,000 ultrafiltration post ultrafiltration SARS-CoV liquid, concentrate and foreigh protein removing, filtrate is rough deactivation SARS-CoV;
8) rough deactivation SARS-CoV is gone up Sepharose CL-2B molecular sieve purification, rough deactivation SARS-CoV is gone up Sepharose CL-2B column chromatography, 0.01Mtris-HCl the PH7.4 buffer solution elution is collected SARS-CoV, merges to concentrate to collect preliminary purification SARS-CoV;
9) to DE52 anion column chromatography purification on the SARS-CoV liquid of preliminary purification, with 0.03MNacl, 50mMTris PH8.0 buffer solution elution, collect the SARS-CoV peak, merge to concentrate and collect purifying SARS-CoV;
10) to the effect analysis of SARS-CoV liquid behind the ultrafiltration and concentration, record antigen rate of recovery average out to 55%, record foreign protein clearance average out to 92% with improvement Lowry method with double antibody sandwich method;
11) to the effect analysis of gel-filtration purifying SARS-CoV, with double antibody sandwich method record antigen rate of recovery average out to 85%, adopt improvement Lowry method record foreign protein clearance average out to 94.5%, complete with the electron microscopic observation virion, measure Vero cell residue dna content less than 10.0ng/ml, suppress the remaining bovine serum content of measuring virus less than 12.5ng/ml with dot hybridization with blood clotting;
12) to the effect analysis of ion-exchange purification SARS-CoV, record the 9.4ng/U of proteantigen before with double antibody sandwich method by purifying, be reduced to 0.8ng/U, HPLC shows that viral purity is 97%; Adopt improvement Lowry method to record foreign protein clearance average out to 98.5%; Complete with the electron microscopic observation virion, measure Vero cell residue dna content less than 1.0ng/ml with dot hybridization; SDS-PAGE shows S, N, M, the major protein band of SARS-CoV, and reacts with decubation patient's SARS serum, and purifying deactivation SARS-CoV divides and puts-70 ℃ of preservations.
3, immune globulin antibody as claimed in claim 1 is characterized in that the anti-SARS-CoV IgF of horse (ab ') of purifying
2Antibody can be prepared into horse anti-SARS-CoV Gammimune N type and mucous membrane aerosol finished product.
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| CN106860862A (en) * | 2017-01-18 | 2017-06-20 | 江苏安泰生物技术有限公司 | A kind of composition of anti-infectious disease and atomization spray and preparation method thereof |
| CN111333704A (en) * | 2020-02-24 | 2020-06-26 | 军事科学院军事医学研究院微生物流行病研究所 | Novel coronavirus COVID-19 vaccine, preparation method and application thereof |
| CN113388027A (en) * | 2020-03-12 | 2021-09-14 | 中国科学院武汉病毒研究所 | Antibody against novel coronavirus, and preparation method and application thereof |
| CN113388027B (en) * | 2020-03-12 | 2022-07-22 | 中国科学院武汉病毒研究所 | Antibody against novel coronavirus, and preparation method and application thereof |
| CN111471103A (en) * | 2020-03-20 | 2020-07-31 | 唐山怡安生物工程有限公司 | Heterologous antibody of new coronavirus (2019-nCOV) and preparation method thereof |
| CN111592595A (en) * | 2020-04-27 | 2020-08-28 | 南京医科大学 | Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof |
| CN111647076A (en) * | 2020-04-27 | 2020-09-11 | 南京医科大学 | Neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof |
| CN112979796A (en) * | 2021-04-27 | 2021-06-18 | 军事科学院军事医学研究院军事兽医研究所 | Horse anti-A H5N1 tiger source influenza virus immunoglobulin and specific immunoglobulin and refining method thereof |
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