CN103937751A - Colloidal gold immunochromatograohic assay detection test strip based on NDV (Newcastle Disease Virus) hemagglutinin protein monoclonal antibody - Google Patents
Colloidal gold immunochromatograohic assay detection test strip based on NDV (Newcastle Disease Virus) hemagglutinin protein monoclonal antibody Download PDFInfo
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Abstract
The invention relates to the field of bioengineering, and particularly relates to a newly prepared hybridoma cell strain for resisting NDV (Newcastle Disease Virus)hemagglutinin protein. A monoclonal antibody with a broad spectrum neutralization effect on NDV is obtained by using the cell strain, and an immune colloidal gold test strip for quickly detecting a newcastle disease virus is developed by using the antibody. By adopting the test paper disclosed by the invention, the result can be obtained within 5 minutes by sampling once, and the test paper has the characteristics of being simple and rapid, specific and sensitive. A more practical tool and method are provided for rapid diagnosis of a newcastle disease by development of the test strip, and the colloidal gold immunochromatograohic assay detection test strip is applicable to primary veterinary stations and small and medium-sized farms.
Description
Technical field
The present invention relates to the diagnostic reagent in veterinary biological product field, a kind of have NDV colloidal gold immunochromatographimethod technology and the test strip in wide spectrum, set up with the active grand antibody of monoclonal antibody with as antibody are specifically provided.
Background technology
Newcastle disease is height contact, the infectivity epidemic disease being caused by Avian pneumo-encephalitis virus, can cause the mortality of bird or the sharply decline of production performance, causes huge financial loss to world's aviculture.Chinese scholars has been carried out large quantity research to the diagnostic method of newcastle disease, and traditional diagnostic method comprises that virus separates, ELISA, agar diffusion experiment, neutralization test, blood clotting and hemagglutination-inhibition test etc.RT-PCR, quantitative fluorescent PCR, diagnostic nucleic acid technology, restriction endonuclease analysis equimolecular biology techniques are also widely applied in the diagnosis of ND in recent years, but aforesaid method is loaded down with trivial details time-consuming, need certain experiment condition and experimenter and special plant and instrument, inconvenience is promoted the use of in plant of basic unit.
For this area, can be fast, this disease of Accurate Diagnosis, to epidemic monitoring, control popularly, reduce aquaculture cost significant.And utilize the monoclonal antibody with wide spectrum neutralizing effect to meet above-mentioned requirements, and become on the basis of existing technology possibility, how to utilize this technology to become prior art problem demanding prompt solution.
Summary of the invention
The present invention is directed to the problem existing in the non-diagnosis detecting method of existing Avian pneumo-encephalitis virus, utilize the hybridoma cell strain of freshly prepd anti-newcastle disease hemagglutinin, obtain monoclonal antibody NDV to wide spectrum neutralizing effect, and utilized this antibody to develop the immune colloidal gold detection test paper strip of rapid detection Avian pneumo-encephalitis virus.Adopt test paper of the present invention only to need a step application of sample, 5m in is energy result of determination just, have feature simple and quick, special sensitivity, the quick diagnosis that the development of this test strip is newcastle disease provides a kind of more practical tool and method, is applicable to veterinary station of basic unit and medium and small plant and uses.
Concrete technical scheme of the present invention is:
First contriver provides a strain brand-new monoclonal antibody hybridoma cell strain, and called after hybridoma cell strain C3B3 has carried out biological preservation simultaneously, and its deposit number is: CCTCC NO:C2013174.
Utilize this hybridoma cell strain, contriver has further obtained monoclonal antibody NDV to wide spectrum neutralizing effect, and concrete steps are as follows:
1. the preparation of monoclonal antibody hybridoma cell strain
The purifying of virus: by NDV LaSota standard virus inoculation 9-10 day instar chicken embryo, gather in the crops the allantoic fluid of dead chicken embryo, by allantoic fluid freeze thawing 1 time, the centrifugal 1h of 15000rpm/min, collect supernatant again through the centrifugal 2.5h of 30000rpm/min supercentrifuge, remove supernatant, precipitation suspends with PBS (pH7.4), with the centrifugal 2min of sucrose density gradient centrifugation 35000rpm/min, the NDV LaSota virus of results purifying;
Taking the NDV LaSota virus of purifying as immunogen, age 6 weeks age～8 weeks of immunity 6 of Balb/c mouse, the immunizing dose of each every is 50ug, immunization method is abdominal injection, has altogether immune four times; Be 2 weeks the interval of each immunity, and head exempts from the Freund's complete adjuvant ratio emulsified protein of 1:1 by volume, and two exempt to four to exempt from Freund's incomplete adjuvant 1:1 emulsified protein by volume; Three immunity are after one week, and mouse orbit blood sampling is surveyed it and tired, monitoring serum antibody titer; Four exempted from after two weeks, selected the highest mouse of tiring, with the albumen abdominal injection booster immunization that does not add adjuvant once, after 3d, get mouse spleen lymphocyte and SP2/0 cytogamy, be placed in 37 DEG C of 5%CO
2incubator is cultivated;
After 5d～7d with the HT 1/2HAT substratum that swaps out, after 7d～10d with HT whole HAT that swap out; Adopt indirect ELISA and hemagglutination-inhibition test (HI experiment) to screen all positive clone as alternative hybridoma cell strain;
Wherein said ELISA method is screened specifically: the virus after purifying is pressed to the coated elisa plate of 10ug/mL, by monoclonal antibody (the being cell culture supernatant) combination with it of hybridoma secretion, it is 2 positive that the ratio that detects the OD value in hole and the OD value of negative control is more than or equal to;
The concrete steps that described blood clotting suppresses experiment are: the nutritive medium that absorption 25 μ L ELISA detect positive hole adds in 96 first holes of hole V-type plate, then carry out doubling dilution to the 11 holes with 25 μ LPBS (pH7.4), the 12nd hole is PBS control wells, add the LaSota primary standard virus 25 μ L of 4HA unit, after room temperature effect 20min, add 1% chicken red blood cell 25 μ L, result of determination after 15-20min, suppresses valency taking the highly diluted multiple of not aggegation chicken red blood cell as blood clotting;
Positive by aforesaid method picking ELISA in the positive colony that have blood clotting inhibition is with limiting dilution assay subclone 3 times, finally select mono-clonal enlarged culturing the conduct that in ELISA detection, OD value reacting value is higher and build strain cell, and it is carried out to biological preservation, deposit number is: CCTCC NO:C2013174;
2, the preparation of odd contradictive hydroperitoneum
8 weeks age～10 week Balb/c mouse peritoneal injection in age sterile liquid paraffin oil 0.5mL/ only, 7d pneumoretroperitoneum inject positive hybridoma cell 500,000 in logarithmic phase/; Observe every day to mouse web portion and obviously expand, extract ascites and centrifugal after get supernatant, thereby obtain monoclonal antibody prepared by positive hybridoma cell C3B3;
By centrifugal fat and the hemocyte that can get rid of in ascites, and then can from ascites, obtain enough monoclonal antibodies for follow-up qualification and preparation.
3, monoclonal antibody CHARACTERISTICS IDENTIFICATION
3.1 monoclonal antibody specificity identification SPF chick embryo allantoic liquids, Avian pneumo-encephalitis virus, avian influenza virus, avian infectious bronchitis virus, infectious bursa of Fabricius virus, duck hepatitis virus, duck plague virus, flavivirus etc. are coated with 96 hole enzyme plates, adopt conventional indirect ELISA qualification, monoclonal antibody prepared by the positive hybridoma cell C3B3 obtaining in the upper step of final discovery only has specific reaction with NDV, with other viral no cross reactions;
3.2 monoclonal antibody indirect ELISA titers are measured
The every hole of enzyme plate of coated NDV virus adds respectively the odd contradictive hydroperitoneum obtaining in step, doubling dilution, and it is 2.56 × 10 that the ELISA that finally obtains monoclonal antibody C3B3 prepared by positive hybridoma cell C3B3 tires
5;
The blood clotting of 3.3 monoclonal antibodies suppresses (HI) characteristic
First with NDV LaSota, F48E8 and other different N DV strain are HI antigen, the HI characteristic of research monoclonal antibody, and result shows C3B3 and NDV standard strain LaSota, F48E8 separates poison reaction in HI test with 17 NDV all positive;
In 3.4 monoclonal antibodies and CHARACTERISTICS IDENTIFICATION (adopt cell neutralization test carry out) monoclonal antibody C3B3 and the strong toxicity of NDV standard (F48E8) and NDV Reference Strains neutralization reaction all positive, consistent with HI test-results;
The above-mentioned strain relating to is common strain, and the Reference Strains bacterium adopting in the present invention derives from national reference laboratory;
4, the purifying of monoclonal antibody and titration
Slightly carry the monoclonal antibody C3B3 obtaining in abdomen Overwater walking with caprylic acid-ammonium, the monoclonal antibody of purifying is purified with Protein G pillar affinity chromatography, detect the monoclonal antibody of purifying as two band (see figure 1)s taking SDS-PAGE, be respectively light chain and heavy chain, suppress experiment taking blood clotting and detect the tiring as 210 of monoclonal antibody of purifying.
The monoclonal antibody purity that obtains by the known purifying of above-mentioned qualification is high and have higher antibody titer and a good biologic activity.
After having obtained above-mentioned monoclonal antibody, contriver further utilizes this antibody to obtain corresponding colloidal gold strip, and the concrete preparation method of this test strip is as follows:
1. the preparation of Radioactive colloidal gold
Trisodium citrate method of reducing after employing improvement is prepared the Radioactive colloidal gold of different diameter, and concrete steps are as follows: (related is all weight percentage)
Get 0.01% hydrochloro-auric acid l00mL and be placed on the inherent constant-temperature heating magnetic stirring apparatus of Erlenmeyer flask and heat, until boil;
Accurately draw 1% trisodium citrate of 2.0mL, add rapidly in Erlenmeyer flask, after rocking evenly, being put into immediately constant-temperature heating magnetic stirring apparatus continues to boil, rotating speed is controlled at 100r/min, until becoming after boiling 15min after redness again, takes out flavous hydrochloro-auric acid, cold filtration, and cryopreservation is to keep the state of colloid.Be limpid transparent orange red with the standby Radioactive colloidal gold outward appearance of this legal system, without the particle of aggegation, at electric Microscopic observation, diameter is about the colloid gold particle of 30nm, even particle size, is single dispersion state and suspends;
Determining of the labelled amount of 2 colloid gold particles and monoclonal antibody C3B3 the best
First by the colloidal gold solution of above-mentioned acquisition with 0.2mol/L K
2cO
3be adjusted to pH=8.3, get some small test tubes, add lmL colloidal gold solution; In each test tube, add afterwards the aqueous solution of the 1.0mg/mL monoclonal antibody C3B3 of different volumes, after 5min, in above-mentioned each pipe, add respectively 100 μ L10% sodium-chlor, mix rear standing 2h;
Afterwards in 4 DEG C of centrifugal 10min of 2000r/min, get supernatant ultra-violet and visible spectrophotometer (400-600nm) in visible-range and measure maximum absorption wavelength, light absorption value, protein consumption corresponding when there is maximum absorption band is as Minimal Protective dosage, adds on this basis the 20% antibody protein actual amount that is stable colloid gold again; Finally find in the time that in lmL Radioactive colloidal gold, monoclonal antibody addition is 30 μ g, corresponding absorption peak maximum, adds 20% on this basis again, when the suitableeest labelled amount is 36 μ g/mL, is the optimum mark amount of monoclonal antibody;
3. the colloid gold label of monoclonal antibody C3B3 and purifying
In 50mL small beaker, add the Radioactive colloidal gold of preparation in 20mL step 1, with 0.1mol/L K
2cO
3it is 8.3 that solution is adjusted to pH, under induction stirring, the 720 μ L1.0mg/mL C3B3 monoclonal antibody aqueous solution are joined in above-mentioned colloidal gold solution, continue to stir 30min, adding concentration is that 10% bovine serum albumin (BSA) to final concentration is 1%, as stablizer, continue to stir 30min, finally obtain the golden labeling antibody of 36 μ g/mL;
First, by 1500r/min low-speed centrifugal 1h for the 20mL gold labeling antibody of above-mentioned acquisition, get supernatant, then by supernatant with 15000r/min4 DEG C of centrifugal 1h, abandon supernatant; To precipitate with 0.02mol/L TBS pH8.3 and dissolve, repeated centrifugation 2-3 time, precipitation is dissolved in the TBS of 1mL0.02mol/L TBS pH8.3, and 4 DEG C save backup;
4. detect determining with the anti-NDV IgG of rabbit concentration
Golden labeling antibody concentration is fixed, with coating buffer (0.01MpH7.4PBS+0.01%Tween-20, Tween-20 is volume percent) anti-rabbit NDV IgG is carried out to 2 times of doubling dilutions, be marked on NC film by different quantity for sprays, glass fibre is pasted on PVC base plate with above-mentioned several NC films respectively, preparation test strip, and with they detect respectively positive and negative reference sample; Determine that according to detecting the band colour developing degree of depth and developing time it is 0.8mg/ml that detection band antibody uses best spraying concentration, quantity for spray is 1ul/cm;
5. determining of quality control band sheep anti-mouse igg antibody concentration
Golden labeling antibody and detection band spraying concentration are fixed, be added on quality control band goat-anti cavy IgG on NC film for antibody coating buffer (with step 4 coating buffer used) do successively after 2 times of doubling dilutions, be sprayed on NC film, glass fibre is pasted on PVC base plate with above-mentioned several NC films respectively, preparation test strip first product, detect positive reference sample, determine that the spraying concentration of quality control band goat-anti cavy IgG antibody is 1mg/ml according to detecting band with the colour developing situation of quality control band, quantity for spray is 1ul/cm;
6. the assembling of test strip and result are judged
First sample pad (glass fibre element film GF33) and gold-marking binding pad (glass fibre element film GF33) are processed with pretreatment fluid (20mMpH8.5Tris-HCl+1%Tween-20), dry in 37 DEG C of incubators;
The golden labeling antibody metal spraying work diluent (10mM pH7.4PB+1%BSA+20%sucrose) step 3 being prepared with a film instrument dilutes 10 times and is sprayed on glass fibre, and quantity for spray is 2ul/cm, puts into immediately frozen vacuum dryer and drains;
At nitrocellulose filter (NCM-HF240) upper spraying detection line and nature controlling line, the anti-NDV antibody of detection line spraying rabbit, concentration is 0.8mg/ml, quantity for spray is 1ul/cm; Nature controlling line spraying sheep anti-mouse igg, concentration is 1mg/ml, and quantity for spray is 1ul/cm, and detection line and nature controlling line interval 5mm take out after dry 30min in 37 DEG C of incubators;
Finally sample pad, Radioactive colloidal gold pad, NC film and absorption pad are sticked on PVC base plate successively, test strip; (concrete structure is shown in Fig. 4); Described test strip comprises PVC end liner, on PVC end liner, be respectively arranged with from left to right sample pad, Radioactive colloidal gold pad, NC film and absorption pad, wherein NC film middle part is respectively arranged with a detection line and a control line from left to right, NC film left side upper surface is pasted with the Radioactive colloidal gold pad of coated with gold labeling antibody, and NC film right lateral surface is pasted with absorption pad; Radioactive colloidal gold pad left side upper surface is pasted with sample pad;
What on described Radioactive colloidal gold pad, apply is based on NDV hemagglutinin monoclonal antibody C3B3.
Result criterion: when macroscopic red-purple band appears in nature controlling line and the detection line of test strip simultaneously, result is judged to the positive; If only nature controlling line occur and there is not red-purple band in detection line, result is judged to feminine gender; Article two, all there is not being judged to test strip inefficacy in line.
The performance test results of the colloidal gold strip of above-mentioned acquisition is as follows:
1 susceptibility is done doubling dilution by the NDV of standard with containing virulent allantoic fluid, is diluted to 1:2 always
11, detecting with other domestic NDV immune colloidal gold detection test paper strips simultaneously, susceptibility result is as Fig. 2, and visible colloidal gold strip of the present invention is at blood clotting valency 1:2
10below all can detect.The parallel contrast and experiment of other domestic test paper row cultures is at 1:2
7-8can detect.
2 specificitys, by viruses such as AIV, IBV, IBDV, MDV, DPV, DHV, drip respectively in test strip, and every kind of virus detects 5 parts, and result all shows feminine gender.
3 repeatability are positive and negative allantoic fluid with different batches ELISA test strip ND, each sample duplicate detection 10 times, and result shows: positive coincidence rate is 98.3%, and negative match-rate is 100% (table 2).
The repeated detected result of table 2
5.5.4 stability is preserved test strip hermetically drying at room temperature, detects week about above-mentioned detection sample, and test strip is within half a year, and its detected result is 100% with the coincidence rate that detected in the past sample.
Visible, the monoclonal antibody C3B3 that the hybridoma obtaining by the present invention obtains, there is the monoclonal antibody of wide spectrum neutralizing effect, and utilize this antibody to develop the immune colloidal gold detection test paper strip of rapid detection Avian pneumo-encephalitis virus, adopt this test paper only to need a step application of sample, 5m in is energy result of determination just, there is feature simple and quick, special sensitivity, the quick diagnosis that the development of this test strip is newcastle disease provides a kind of more practical tool and method, is applicable to veterinary station of basic unit and medium and small plant and uses.
Contriver has carried out biological preservation to the monoclonal antibody hybridoma cell of restructuring disclosed in this invention, and concrete preservation information is as follows:
Preservation information
The preservation time: on December 25th, 2013
Depositary institution's title: Chinese Typical Representative culture collection center
Deposit number: CCTCC NO:C2013174
Depositary institution address: Wuhan University's Chinese Typical Representative culture collection center
Classification And Nomenclature: hybridoma cell strain C3B3
Brief description of the drawings
Fig. 1 is the C3B3 monoclonal antibody electrophoresis schematic diagram that SDS-PAGE detects purifying:
In figure, Far Left swimming lane is molecular weight of albumen Marker, the purified monoclonal antibody albumen that 1,2,3 swimming lanes are different applied sample amounts; Monoclonal antibody purity is higher as we know from the figure, presents 2 single bands after PAGE electrophoresis, and molecular weight is about respectively 25KD, 50KD, represents respectively light chain and the heavy chain of purified monoclonal antibody;
Fig. 2 is test strip sensitivity test result gray-scale map of the present invention;
Numeral in figure represents that respectively tested virus is with 1:2
1, 1:2
2... 1:2
11dilute, C line represents Quality Control first, and T line represents detection line, and viral dilution is to 1:2 as we know from the figure
10still can detect;
Fig. 3 is the partial detection gray-scale map of clinical sample in experimental example 1;
In experimental example 1 shown in figure, 37 clinical samples are that the cotton swab sample gathering is directly added drop-wise to the detected result presenting in test strip after PBS simple process;
Fig. 4 is the structural representation of test strip of the present invention,
In figure, 1 is sample pad, and 2 is Radioactive colloidal gold pad, and 3 is detection line, 4 control lines, and 5 is absorption pad, and 6 is PVC end liner, and 7 is NC film.
Embodiment
The preparation of embodiment 1 monoclonal antibody hybridoma cell strain
The purifying of virus: by NDV LaSota standard virus inoculation 9-10 day instar chicken embryo, gather in the crops the allantoic fluid of dead chicken embryo, by allantoic fluid freeze thawing 1 time, the centrifugal 1h of 15000rpm/min, collect supernatant again through the centrifugal 2.5h of 30000rpm/min supercentrifuge, remove supernatant, precipitation suspends with PBS (pH7.4), with the centrifugal 2min of sucrose density gradient centrifugation 35000rpm/min, the NDV LaSota virus of results purifying;
Taking the NDV LaSota virus of purifying as immunogen, age 6 weeks age～8 weeks of immunity 6 of Balb/c mouse, the immunizing dose of each every is 50ug, immunization method is abdominal injection, has altogether immune four times; Be 2 weeks the interval of each immunity, and head exempts from the Freund's complete adjuvant ratio emulsified protein of 1:1 by volume, and two exempt to four to exempt from Freund's incomplete adjuvant 1:1 emulsified protein by volume; Three immunity are after one week, and mouse orbit blood sampling is surveyed it and tired, monitoring serum antibody titer; Four exempted from after two weeks, selected the highest mouse of tiring, with the albumen abdominal injection booster immunization that does not add adjuvant once, after 3d, get mouse spleen lymphocyte and SP2/0 cytogamy, be placed in 37 DEG C of 5%CO
2incubator is cultivated;
After 5d～7d with the HT 1/2HAT substratum that swaps out, after 7d～10d with HT whole HAT that swap out; Adopt indirect ELISA and hemagglutination-inhibition test (HI experiment) to screen all positive clone as alternative hybridoma cell strain;
Wherein said ELISA method is screened specifically: the virus after purifying is pressed to the coated elisa plate of 10ug/mL, by monoclonal antibody (the being cell culture supernatant) combination with it of hybridoma secretion, it is 2 positive that the ratio that detects the OD value in hole and the OD value of negative control is more than or equal to;
The concrete steps that described blood clotting suppresses experiment are: the nutritive medium that absorption 25 μ L ELISA detect positive hole adds in 96 first holes of hole V-type plate, then carry out doubling dilution to the 11 holes with 25 μ LPBS (pH7.4), the 12nd hole is PBS control wells, add the LaSota primary standard virus 25 μ L of 4HA unit, after room temperature effect 20min, add 1% chicken red blood cell 25 μ L, result of determination after 15-20min, suppresses valency taking the highly diluted multiple of not aggegation chicken red blood cell as blood clotting;
Positive by aforesaid method picking ELISA in the positive colony that have blood clotting inhibition is with limiting dilution assay subclone 3 times, finally select mono-clonal enlarged culturing the conduct that in ELISA detection, OD value reacting value is higher and build strain cell, and it is carried out to biological preservation, deposit number is: CCTCC NO:C2013174;
Acquisition and the purification Identification of embodiment 2 monoclonal antibodies
1, the preparation of odd contradictive hydroperitoneum
8 weeks age～10 week Balb/c mouse peritoneal injection in age sterile liquid paraffin oil 0.5mL/ only, 7d pneumoretroperitoneum inject positive hybridoma cell 500,000 in logarithmic phase/; Observe every day to mouse web portion and obviously expand, extract ascites and centrifugal after get supernatant, thereby obtain monoclonal antibody prepared by positive hybridoma cell C3B3;
By centrifugal fat and the hemocyte that can get rid of in ascites, and then can from ascites, obtain enough monoclonal antibodies for follow-up qualification and preparation;
2, monoclonal antibody CHARACTERISTICS IDENTIFICATION
2.1 monoclonal antibody specificity identification SPF chick embryo allantoic liquids, Avian pneumo-encephalitis virus, avian influenza virus, avian infectious bronchitis virus, infectious bursa of Fabricius virus, duck hepatitis virus, duck plague virus, flavivirus etc. are coated with 96 hole enzyme plates, adopt conventional indirect ELISA qualification, monoclonal antibody prepared by the positive hybridoma cell C3B3 obtaining in the upper step of final discovery only has specific reaction with NDV, with other viral no cross reactions;
2.2 monoclonal antibody indirect ELISA titers are measured
The every hole of enzyme plate of coated NDV virus adds respectively the odd contradictive hydroperitoneum obtaining in step, doubling dilution, and it is 2.56 × 10 that the ELISA that finally obtains monoclonal antibody C3B3 prepared by positive hybridoma cell C3B3 tires
5;
The blood clotting of 2.3 monoclonal antibodies suppresses (HI) characteristic
First with NDV LaSota, F48E8 and other different N DV strain are HI antigen, the HI characteristic of research monoclonal antibody, result shows C3B3 and NDV standard strain LaSota, F48E8 separates poison all positive (as shown in table 1) of reaction in HI test with 17 NDV.
In 2.4 monoclonal antibodies and CHARACTERISTICS IDENTIFICATION (adopt cell neutralization test carry out)
Monoclonal antibody C3B3 and the strong toxicity of NDV standard (F48E8) and NDV Reference Strains neutralization reaction are all positive, consistent with HI test-results (as shown in table 1).
The reactivity of table 1 monoclonal antibody C3B3 strains different from NDV in HI and neutralization test
Note: 1. "-" represents reaction negative; 2. "+" represents reacting positive; The above-mentioned strain relating to is common strain, and the Reference Strains bacterium adopting in the present invention derives from national reference laboratory;
3, the purifying of monoclonal antibody and titration
Slightly carry the monoclonal antibody C3B3 obtaining in abdomen Overwater walking with caprylic acid-ammonium, the monoclonal antibody of purifying is purified with Protein G pillar affinity chromatography, detect the monoclonal antibody of purifying as two band (see figure 1)s taking SDS-PAGE, be respectively light chain and heavy chain, suppress experiment taking blood clotting and detect the tiring as 2 of monoclonal antibody of purifying
10.
The preparation of embodiment 3 test strip
1. the preparation of Radioactive colloidal gold
Trisodium citrate method of reducing after employing improvement is prepared the Radioactive colloidal gold of different diameter, and concrete steps are as follows: (related is all weight percentage)
Get 0.01% hydrochloro-auric acid l00mL and be placed on the inherent constant-temperature heating magnetic stirring apparatus of Erlenmeyer flask and heat, until boil;
Accurately draw 1% trisodium citrate of 2.0mL, add rapidly in Erlenmeyer flask, after rocking evenly, being put into immediately constant-temperature heating magnetic stirring apparatus continues to boil, rotating speed is controlled at 100r/min, until becoming after boiling 15min after redness again, takes out flavous hydrochloro-auric acid, cold filtration, and cryopreservation is to keep the state of colloid.Be limpid transparent orange red with the standby Radioactive colloidal gold outward appearance of this legal system, without the particle of aggegation, at electric Microscopic observation, diameter is about the colloid gold particle of 30nm, even particle size, is single dispersion state and suspends;
2. determining of the labelled amount of colloid gold particle and monoclonal antibody C3B3 the best
First by the colloidal gold solution of above-mentioned acquisition with 0.2mol/L K
2cO
3be adjusted to pH=8.3, get some small test tubes, add l mL colloidal gold solution; In each test tube, add afterwards the aqueous solution of the 1.0mg/mL monoclonal antibody C3B3 of different volumes, after 5m in, in above-mentioned each pipe, add respectively 100 μ L10% sodium-chlor, mix rear standing 2h;
Afterwards in 4 DEG C of centrifugal 10min of 2000r/min, get supernatant ultra-violet and visible spectrophotometer (400-600nm) in visible-range and measure maximum absorption wavelength, light absorption value, protein consumption corresponding when there is maximum absorption band is as Minimal Protective dosage, adds on this basis the 20% antibody protein actual amount that is stable colloid gold again; Finally find in the time that in lmL Radioactive colloidal gold, monoclonal antibody addition is 30 μ g, corresponding absorption peak maximum, adds 20% on this basis again, when the suitableeest labelled amount is 36 μ g/mL, is the optimum mark amount of monoclonal antibody;
3. the colloid gold label of monoclonal antibody C3B3 and purifying
In 50mL small beaker, add the Radioactive colloidal gold of preparation in 20mL step 1, with 0.1mol/L K
2cO
3it is 8.3 that solution is adjusted to pH, under induction stirring, the 720 μ L1.0mg/mL C3B3 monoclonal antibody aqueous solution are joined in above-mentioned colloidal gold solution, continue to stir 30min, adding concentration is that 10% bovine serum albumin (BSA) to final concentration is 1%, as stablizer, continue to stir 30min, finally obtain the golden labeling antibody of 36 μ g/mL;
First, by 1500r/min low-speed centrifugal 1h for the 20mL gold labeling antibody of above-mentioned acquisition, get supernatant, then by supernatant with 15000r/m in4 DEG C of centrifugal 1h, abandon supernatant; To precipitate with 0.02mol/L TBS pH8.3 and dissolve, repeated centrifugation 2-3 time, precipitation is dissolved in the TBS of 1mL0.02mol/L TBS pH8.3, and 4 DEG C save backup;
4. detect determining with the anti-NDV IgG of rabbit concentration
Golden labeling antibody concentration is fixed, with coating buffer (0.01MpH7.4PBS+0.01%Tween-20, Tween-20 is volume percent) anti-rabbit NDV IgG is carried out to 2 times of doubling dilutions, be marked on NC film by different quantity for sprays, glass fibre is pasted on PVC base plate with above-mentioned several NC films respectively, preparation test strip, and with they detect respectively positive and negative reference sample; Determine that according to detecting the band colour developing degree of depth and developing time it is 0.8mg/ml that detection band antibody uses best spraying concentration, quantity for spray is 1ul/cm;
5. determining of quality control band sheep anti-mouse igg antibody concentration
Golden labeling antibody and detection band spraying concentration are fixed, be added on quality control band goat-anti cavy IgG on NC film for antibody coating buffer (with step 4 coating buffer used) do successively after 2 times of doubling dilutions, be sprayed on NC film, glass fibre is pasted on PVC base plate with above-mentioned several NC films respectively, preparation test strip first product, detect positive reference sample, determine that the spraying concentration of quality control band goat-anti cavy IgG antibody is 1mg/ml according to detecting band with the colour developing situation of quality control band, quantity for spray is 1ul/cm;
6. the assembling of test strip and result are judged
First sample pad (glass fibre element film GF33) and gold-marking binding pad (glass fibre element film GF33) are processed with pretreatment fluid (20mMpH8.5Tris-HCl+1%Tween-20), dry in 37 DEG C of incubators;
The golden labeling antibody metal spraying work diluent (10mM pH7.4PB+1%BSA+20%sucrose) step 3 being prepared with a film instrument dilutes 10 times and is sprayed on glass fibre, and quantity for spray is 2ul/cm, puts into immediately frozen vacuum dryer and drains;
At nitrocellulose filter (NCM-HF240) upper spraying detection line and nature controlling line, the anti-NDV antibody of detection line spraying rabbit, concentration is 0.8mg/ml, quantity for spray is 1ul/cm; Nature controlling line spraying sheep anti-mouse igg, concentration is 1mg/ml, and quantity for spray is 1ul/cm, and detection line and nature controlling line interval 5mm take out after dry 30min in 37 DEG C of incubators;
Finally sample pad, Radioactive colloidal gold pad, NC film and absorption pad are sticked on PVC base plate successively, test strip (as shown in Figure 4);
Described test strip comprises PVC end liner 6, on PVC end liner 6, be respectively arranged with from left to right sample pad 1, Radioactive colloidal gold pad 2, NC film 7 and absorption pad 5, wherein NC film 7 middle parts are respectively arranged with a detection line 3 and a control line 4 from left to right, NC film 7 left side upper surfaces are pasted with the Radioactive colloidal gold pad 2 of coated with gold labeling antibody, and NC film 7 right lateral surface are pasted with absorption pad 5; Radioactive colloidal gold pad 2 left side upper surfaces are pasted with sample pad 1;
What on described Radioactive colloidal gold pad 2, apply is based on NDV hemagglutinin monoclonal antibody C3B3.
Experimental example 1
(1) gather poultry throat swab, cloaca secretory product or ight soil with cotton swab;
(2) immediately cotton swab is inserted to the test tube that sample buffer (the PBS solution of pH7.4) is housed, cotton swab is repeatedly firmly rotated at least 10 times on test tube wall, and mix solution, sample is dissolved in solution as far as possible.On test tube wall, push cotton swab, liquid is extruded as far as possible, abandon cotton swab.
(3) from sealing bag, take out test strip.Test card is placed on to flat surface, and with suction pipe, from imbitition in vitro, accurate dropping 4 slowly, dropwise drips to and adds in sample pad;
(4) at room temperature place 5 minutes judged results (partial results as shown in Figure 3);
(5) above sample is inoculated 9-10 age in days SPF chicken embryo simultaneously, 3 chicken embryos of each sample inoculation, hatching in 37 DEG C of incubators, gets the allantoic fluid of dead chicken embryo (about 2-3d) after 24h and does blood coagulation tests (HA) and hemagglutination-inhibition test (HI), detection positive number.
(6) totally 53 parts, this cotton swab sample of result, utilizing colloidal gold strip of the present invention to detect positive number is 12 parts, and negative sample number is 41 parts, and positive rate is 22.6%; Utilizing egg inoculation and HA and HI test to detect positive number is 14 parts, 39 parts of negative sample numbers, and positive rate is 26.4%; Both coincidence rates are 96.2%.
Egg inoculation and HA and HI test are the traditional methods of isolation identification Avian pneumo-encephalitis virus, are also the most reliable methods.With colloidal gold strip of the present invention, sample detected and make comparisons with traditional method, the coincidence rate of finding both is 96.2%, on detection time, be 5-10min detection time of the present invention, and traditional egg inoculation and HA and HI experiment are 2-3d, show that colloidal gold strip of the present invention has higher detection sensitivity, and have easy to detect fast, result is easy to the advantage of judging, is applicable to the rapid detection to clinical sample.
Embodiment 2
In order to evaluate pathogenic to pigeon of pigeon strain isolated SD03, attack poison experiment, after attacking poison, the experimental animal of survival was gathered to larynx, cloacal swabs in the 3rd, 10 days, the inoculation of 51 parts of employing cells of cotton swab, PCR and three kinds of methods of colloidal gold strip to experimental session collection detect simultaneously, result is as table 3, and the Positive rate of Radioactive colloidal gold and cell inoculation and Radioactive colloidal gold method is respectively 54.91%, 52.94%, 58.82%.
The detected result of the different detection methods of table 3 to sample
Result shows that Radioactive colloidal gold is lower than the sensitivity of PCR method, and cell is inoculated owing to being subject to the impact of virus titer and vigor, and Positive rate is lower.But the cost of this colloidal gold strip provided by the present invention is lower, and clinical application is simple, does not need special plant and instrument, is more applicable to the large-scale promotion and application of basic unit.
Claims (3)
1. the anti-newcastle disease hemagglutinin of strain hybridoma cell strain, called after hybridoma cell strain C3B3, its deposit number is: CCTCC NO:C2013174.
2. based on a NDV hemagglutinin monoclonal antibody C3B3, it is to be by deposit number: the hybridoma cell strain of CCTCC NO:C2013174 obtains.
3. the colloidal gold immunochromatographydetection detection test paper bar based on NDV hemagglutinin monoclonal antibody, comprise PVC end liner (6), it is characterized in that: on PVC end liner (6), be respectively arranged with from left to right sample pad (1), Radioactive colloidal gold pad (2), NC film (7) and absorption pad (5), wherein NC film (7) middle part is respectively arranged with a detection line (3) and a control line (4) from left to right, NC film (7) left side upper surface is pasted with the Radioactive colloidal gold pad (2) of coated with gold labeling antibody, and NC film (7) right lateral surface is pasted with absorption pad (5); Radioactive colloidal gold pad (2) left side upper surface is pasted with sample pad (1);
What described Radioactive colloidal gold pad (2) above applied is based on NDV hemagglutinin monoclonal antibody C3B3.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104459119A (en) * | 2014-11-14 | 2015-03-25 | 宁波大学 | Soft-shelled turtle systemic septicemia virus detection test strip and preparation method thereof |
| CN105486871A (en) * | 2015-11-25 | 2016-04-13 | 北京世纪元亨动物防疫技术有限公司 | Colloidal gold test strip for rapidly detecting hemagglutination inhibition titer of canine parvovirus antibody, and kit and detection method thereof |
| CN105759046A (en) * | 2016-04-08 | 2016-07-13 | 重庆理工大学 | Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof |
| CN109061140A (en) * | 2018-08-31 | 2018-12-21 | 广州华澳生物科技有限公司 | A kind of and peptide element immunochromatographiassay assay reagent card and preparation method thereof |
| CN109085334A (en) * | 2018-06-22 | 2018-12-25 | 东南大学 | A kind of colloidal gold fast detecting test paper strip and its preparation method and application of SFTSV nucleoprotein antigen |
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| CN104459119A (en) * | 2014-11-14 | 2015-03-25 | 宁波大学 | Soft-shelled turtle systemic septicemia virus detection test strip and preparation method thereof |
| CN104459119B (en) * | 2014-11-14 | 2016-04-13 | 宁波大学 | A kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips and preparation method thereof |
| CN105486871A (en) * | 2015-11-25 | 2016-04-13 | 北京世纪元亨动物防疫技术有限公司 | Colloidal gold test strip for rapidly detecting hemagglutination inhibition titer of canine parvovirus antibody, and kit and detection method thereof |
| CN105759046A (en) * | 2016-04-08 | 2016-07-13 | 重庆理工大学 | Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof |
| CN109085334A (en) * | 2018-06-22 | 2018-12-25 | 东南大学 | A kind of colloidal gold fast detecting test paper strip and its preparation method and application of SFTSV nucleoprotein antigen |
| CN109061140A (en) * | 2018-08-31 | 2018-12-21 | 广州华澳生物科技有限公司 | A kind of and peptide element immunochromatographiassay assay reagent card and preparation method thereof |
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