Production Of Egg Yolk Antibody (IgY) Against Recombinant .
S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Protein.Acta Scientiae Veterinariae. 40(2): 1029.Acta Scientiae Veterinariae, 2012. 40(2): 1029.ORIGINAL ARTICLEISSN 1679-9216 (Online)Pub. 1029Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2ProteinShuizhong Han, Xiaoying Zhang & Jinzi ZhaoABSTRACTBackground: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 inthe past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment.CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa),while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technologyhas been recognized as a promising alternative to generate large amount of qualified high specific antibody for use inimmunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scaleproduction over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to therheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never beenreported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosisand treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF)was amplified by PCR and inserted into the pMD18-T clone vector. The isolate was defined as CPV-2a (JN403045) subtypeby sequencing. The VP2 ORF was inserted into pET-32a by T4 ligase and introduced into the E. coli Bal21. VP2 proteinwas produced by the induction of the E. coli Bal21 containing pET-32a-VP2 with isopropyl-β-D-1-thiogalactopyrannoside(IPTG). Expression of the recombinant VP2 protein (rVP2) fused with His-tag was analyzed by SDS-PAGE and detectedNby western blotting using anti-His monoclonal antibody. The rVP2 was purified by Ni -affinity purification chromatography under denature condition and dialyzed against PBS. The concentration of the rVP2 was determined by Bradfordmethod. After immunizing the chickens with rVP2, anti-VP2 IgY was isolated by PEG 6000 precipitation and analyzedby SDS-PAGE. The activity and specificity of the IgY antibody were analyzed by indirect ELISA and western blotting.SDS-PAGE analysis showed that the rVP2 fused with His-tag had a molecular of 85 kDa, accompanied with the low molecular fractions around 50 kDa and 40 kDa. The rVP2 could be recognized be the anti-His monoclonal antibody. The IgYantibody isolated by PEG 6000 method and analyzed by SDS-PAGE showed the IgY mainly contained two parts, 23 kDaand 67 kDa, which corresponded to light and heavy chain, respectively. In additional, some lower bands around 40 kDawere presented on the gel. The anti-VP2 IgY reached to 1:40960 after the fourth immunization. The anti-VP2-IgY couldrecognize the VP2 specially in Western blotting, while no reaction was seen with the low molecular.Discussion: The emergence of the low molecular proteins in 50 kDa and 40 kDa in the pellets of the bacterial lysates maybe due to the degradation of the rVP2 by endogenous proteases in E. coli. The fact that IgY could recognize the entire VP2fraction suggests the lost of the antigenic sites of the low molecular proteins.Keywords: canine parvovirus (CPV), VP2 protein, egg yolk antibody (IgY).Received: October 2011www.ufrgs.br/actavetAccepted: December 2011College of Veterinary Medicine, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi 712100, China. CORRESPONDENCE:X.Y Zhang [ zhang.xy@nwsuaf.edu.cn - Fax & Tel: 86-29-8709 1239].1
S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Protein.Acta Scientiae Veterinariae. 40(2): 1029.INTRODUCTIONDNA cloning of the VP2 geneFull length of VP2 ORF (1 1755nt) was amplified by PCR. The supernatant was boiled as template.The primers (F 5’-ATGGATCCCCAATGAGTGATGGAGCAG-3’; R 5’-CCGCTCGAGATATAATTTTCTAGGTGCTAGTTG-3’) including a BamH I and XhoI restriction site represented underline respectively,were designed using primer 5.0 based on the CPVreference strain (GenBank Accession No.M19296) andcustom-synthesized by the Genscript2. The templatewas denatured at 94oCfor 5 min, followed by 35 cyclesof 94oC for 30 s, 55oC for 30 s and 72oC for 1 min andfinal extension at 72oC for 10 min.The amplified fragments were purified andligated into pMD18-T plasmid vector3 by T-A clone.The resulted plasmid was designated pMD18-T-VP2and introduced into E. coli DH5α. The positive colonywas further confirmed by restriction enzyme digestionand sequencing.Canine parvovirus (CPV) disease is a seriousinfectious disease among canines featured by myocarditis and hemorrhagic enteritis [11]. CPV capsidconsists of 60 copies of a combination of viral coatproteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5kDa), while about 90% of the proteins in the capsidare VP2 protein [13]. A series of B cell epitopes havebeen found on the VP2 protein and could be used as avaccine for the prevention of CPV [3, 9].After first isolation of CPV-2 in the US in1978 [2], its variants, including CPV-2a, CPV-2b andCPV-2c, had emerged and replaced the CPV-2 in thepast decades worldwide [4, 7]. In China, most isolateswere identified as CPV-2a [18]. Although the antibodyinduced by the CPV-2/CPV-2b vaccine may offer certain protection effect, it may not be sufficient [5, 12,14]. The morbidity of CPV disease is considerable highamong canines and has leaded to great economic lossin China according to our previous survey [8]. Traditionally, monoclonal antibody has been used for CPVtreatment, here we present a relatively inexpensiveand simple method to develop high qualified antibodybased on chicken egg yolk antibody (IgY) technology.IgY antibody is recognized as an excellentsource of polyclonal antibodies with relatively simple, noninvasive method and large-scale productionadvantage compared with the mammalian antibodies[19]. IgY antibody was used for the passive protectionof the pathogen infections, especially the intestinalinfections, such as rotavirus [15], Salmonella [10],and exhibited good efficiencies.The present study was conducted to characterize the specific IgY antibody produced by immunizingthe hens with CPV-VP2 expressed in the E. coli.Expression of the His-tagged recombinant CPV-VP2proteinThe plasmids pMD18-T-VP2 and pET-32awere digested with BamH I and Xho I, respectively,eluted by gel purification, and then ligated with T4DNA ligase to yield pET-32a-VP2 recombinant plasmid. The plasmid was transferred into E. coli BL21(DE3), inoculated into LB agar medium containingampicillin (100 μg/mL) and grown overnight at 37oC.A single colony was obtained and further culturedat 37oC in LB liquid medium, supplemented withampicillin (100 μg/mL) until the OD630 reached 0.8,then induced by adding IPTG at a final concentrationof 1.0 mmol/L for 5 h. After harvesting the cells bycentrifugation (7,000 g, 20 min, 4oC), the pellets werelysed by sonication (10 s each, 10 cycles with 5 s interval) in iced water bath. After centrifugation (16,000g, 20 min), the supernatant and pellet of the lysatewere collected separately. Both of the supernatant andpellet were analyzed on 12% SDS-PAGE followed byWestern blotting to detect 6 His-tagged recombinantproteins. Diluted mouse anti-His monoclonal antibody4was used as primary antibody (1:1000, v/v), and HRPconjugated rabbit anti-mouse antibody4 was used assecondary antibody (1:5,000 v/v). The reactivity wasdetected using a chemiluminescent substrate withluminol and H2O25.MATERIALS AND METHODSMaterialsCPV strain was isolated from puppies thathad a history of vomiting and diarrhea in the Xi’anPet Hospital. The animals were confirmed with CPVinfections by a colloidal gold test kit1. Fecal sampleswere obtained by rectal swabs and homogenized(10%, w/v) in phosphate buffered saline (PBS, pH 7.2)containing streptomycin (100 μg/mL) and penicillin(100,000 IU/L).2
S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Protein.Acta Scientiae Veterinariae. 40(2): 1029.-PAGE under reducing conditions, and stained withCoomassie Brillant Blue.Purification of the His-tagged CPV-VP2 fusion proteinsAfter passing through 0.22 μm filter, the VP2protein was purified from the dissolved pellet usingHis Trap affinity column6 under denaturing conditions(8 mol/L Urea) as described in the manual instruction.After washing the column with the binding buffer (20mmol/L Tris-HCl, 500 mmol/L NaCl, 30 mmol/L imidazole, pH 7.4), the VP2 protein was eluted using elution buffer (20 mmol/L Tris-HCl, 500 mmol/L NaCl,500 mmol/L Imidazole, pH 7.4). 12% SDS-PAGE wasused to analyze the relative purification efficiency ofVP2 protein followed by Commassie blue staining.The concentration of the VP2 protein was determinedby Bradford method after dialyzing against PBS (100mmol/L, pH 7.4) containing different amounts of urea.Indirect Enzyme-linked immune sorbent assay (iELISA)Specific activity of IgY antibody against VP2protein was determined by iELISA. Briefly, a 96well microtiter plate8 was coated with 100 μL of VP2antigen (10 μg/mL) in sodium bicarbonate buffer (pH9.6), and incubated at 4oC overnight. After washingthree times with PBS containing 0.05% (v/v) Tween20 (PBST), the nonspecific binding sites were blockedwith 5% (w/v) nonfat milk powder in PBST for 4 h at37oC. After three times washing, 100 μL of a seriallydiluted IgY antibody in PBST was added to the wellsas the primary antibody and then incubated for 2 h at37oC. The plate was washed again and incubated with100 μL of HRP labeled goat-anti-chicken IgG (1:6000)as the secondary antibody for 1 h at 37oC. Finally, theactivity was detected with 100 μL of 3,3’,5,5’-tetramethylbenzidine (TMB, 1%, w/v) as substrate for 15min at room temperature, The reaction was stopped byadding 50 μL of 2 mol/L H2SO4 to the wells. Opticaldensity (OD) at 450nm was read on microtiter platereader9. While ODSample/ODNegative 2.1, the maximumdilution multiple of the sample was determined as IgYantibody titer.NImmunizationTwenty-week-old Hy-line Brown hens werekept in individual cages with food and water ad libitum.They were immunized intramuscularly with VP2 protein mixed with Freund’s adjuvant7 at different sites ofthe breast. 1000 μg of freeze-drying VP2 protein weresuspended in 500 μL of PBS and emulsified with anequal volume of complete Freund’s adjuvant for thefirst immunization. Three booster immunizations werefollowed up using incomplete Freund’s adjuvant withtwo weeks interval. Eggs were collected daily, markedand stored at 4oC until they were further processed.Western blottingThe purified VP2 protein was separated using12% SDS-PAGE and transferred onto polyvinylidenefluoride (PVDF) membrane using a semi-dry transferwestern blotting apparatus10. The unreacted sites wereblocked with 5% (w/v) nonfat milk power in TBST(20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween20 (v/v), (pH 8.0) buffer for 1 h and then incubatedwith anti-VP2 IgY antibody (1:1000) for 1 h at roomtemperature. After washing three times with TBST,HRP-conjugated goat-anti- chicken antibody11 dilutedin TBST (1:5000) was added and incubated for 1 h atroom temperature. The membrane was washed threetimes with TBST and the specific binding of IgY antibody to the VP2 protein was detected as describedabove.IgY antibody purificationIgY antibody was extracted and purified fromeggs using PEG precipitation procedure as describedbefore [19]. Briefly, the yolk of a single egg was separated from the white by an egg separator and diluted1:2 with sterile PBS with vortex. The PEG 6000 (3.5%,w/v) was added to precipitate abundance of lipids andlipoproteins. After gentle shaking at room temperaturefor 10 min followed by centrifugation (16,000 g, 20min, 4oC), the supernatant was harvested and filtered.PEG 6000 was added to a final concentration of 8%(w/v), the mixture was centrifuged again under theabove conditions after gentle shaking at room temperature for 10 min. The precipitation was dissolved in10 mL PBS and 12% (w/v) PEG 6000 was added andcentrifuged as described above. Finally, the pelletswere dissolved in 1.2 mL PBS and dialyzed againstbulks of PBS over night at 4oC. The purity of theisolated IgY antibody was determined by 12% SDS-RESULTSExpression of the recombinant VP2 proteinThe coding region (1-1755 nt) of the VP2protein was amplified by PCR and inserted into the3
S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Protein.Acta Scientiae Veterinariae. 40(2): 1029.cloning vector to construct pMD18-T-VP2. After restriction enzyme digestion analysis and sequencing, theclinical sample was defined as CPV-2a type (GenBankAccession No. JN403045). After 5 h induction withIPTG, a prominent band with the expected molecular weight of 85 kDa was observed in the insolublefraction of the bacterial containing the pET-32a-VP2plasmid, while no proteins bands at the approximatesize were observed in both none-IPTG inductionand IPTG-treated culture containing pET-32a only(Figure 1). It revealed that the recombinant proteinswere expressed in inclusion body format. In additionto the expected molecular weight proteins, two lowerbands, around 50 kDa and 40 kDa were expressed inthe insoluble fraction.Western blotting showed that the bands around85 kDa and 50 kDa were recognized by mouse-anti-Hismonoclonal antibody.that two bands, around 85 kDa and 50 kDa were elutedfrom the column.Purification of IgY antibodyIgY antibody was isolated and purified fromthe egg yolk by PEG 6000 precipitation method. Theeffectively was estimated by SDS-PAGE under reducing condition (Figure 3). It showed that IgY antibodycontained two major proteins 23 kDa and 67 kDa thatcorrespond to light and heavy chain, respectively. Inadditional, some lower bands around 40 kDa werepresented on the gel.Specific activity of IgY antibody against VP2 proteindetermined by ELISAThe levels of specific activities of anti-VP2IgY antibody were determined by iELISA. The IgYantibody titer increased from initial immunizationPurification of the His-tagged CPV-VP2 fusion proteinTo purify the CPV-VP2 protein, the crude extract was filtered and loaded onto the Ni - resin purifycolumn. The recombinant proteins eluted from thecolumn were analyzed by 12% SDS-PAGE. It showedFigure 1. SDS-PAGE analysis of the soluble and insoluble fractions from lysates for recombinant CPV-VP2 protein expression.Lane 1: pET-32a( ) without induction with IPTG; Lane 2: pET32a( ) induction with IPTG; Lane 3: solubility of pET-32a( )without induction with IPTG; Lane 4: solubility of pET-32a-VP2without induction with IPTG; Lane 5: solubility of pET-32a induction with IPTG; Lane 6: solubility of pET-32a-VP2 inductionwith IPTG; Lane 7: insolubility of pET-32a without induction withIPTG; Lane 8: insolubility of pET-32a-VP2 without inductionwith IPTG; Lane 9: insolubility of pET-32a induction with IPTG;Lane 10: insolubility of pET-32a-VP2 induction with IPTG; M: Marker.Figure 2. Western blotting analysis of the recombinant proteins using anti-His monoclonalantibody. Lane 1: Marker; Lane 2: insolubility ofpET-32a-VP2 induction with IPTG.4
S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Protein.Acta Scientiae Veterinariae. 40(2): 1029.Figure 3. SDS-PAGE analysis of the recombinant proteins purified using Ni affinity column. Lane 1: pET-32a-VP2 withoutinduction with IPTG; Lane 2: pET-32a-VP2 induction with IPTG;Lane 3: solubility of pET-32a-VP2 induction with IPTG; Lane 4:insolubility of pET-32a-VP2 induction with IPTG; Lane 5: flowthrough; Lane 6: elutes in 20 mmol/L imidazole; Lane 7: elutesin 500 mmol/L imidazole; M: marker.and arrived the peak (1:40960) after the fourth immunization.Detection of VP2 antigen with anti-VP2 IgYBinding activity of the IgY antibody to the VP2protein was further determined by western blotting. Asshown in Figure 5, isolated IgY antibody could bindthe rVP2 protein (85 kDa) at a dilution of 1:1000,while could not recognize the fraction around 50 kDa.NFigure 4. SDS-PAGE analysis of the isolated IgY antibody purified by PEG 6000.DISCUSSIONThe CPV isolated from Shaanxi was definedas New-CPV-2a subtype by sequencing, which posedan amino mutation at the position 297 (Ser-Ala) of theVP2 protein. While using the prokaryotic expressionvector pET-32a, the histidine tags at both amino andcarbon terminal of the VP2 protein will be added,which would increase its molecular mass to 85 kDa.SDS-PAGE analysis of the soluble and insoluble ofthe lysate, VP2 protein was found mainly as insolubleinclusion body (Figure 1, lane 10). In addition to theexpect VP2 protein, two other bands, around 50 kDaand 40 kDa existed in the insoluble fractions (Figure1, lane 10). Parvovirus capsids had a tendency to becleaved in vivo infection as described in the Aleutiandisease virus (ADV) infection [1]. The nature cleavage of VP2 to VP3 was observed in the full CPVparticles only [16]. Therefore, the emergence of thelower proteins may be not due to the degradation ofthe VP2 protein. The second possibility may be therare codons in the amino terminal of VP2 ORF, whichmay be likely to hamper the expression efficiency in E.coli. However, elimination of the rare codons at boththe amino and carbon terminal would not avoid theemergence of the lower molecular fractions [17]. Thethird possibility of the emergence of the lower bandsmay be the degradation of the high molecular weightproteins by endogenous proteases in E. coli. Overexpression of proteins with high molecular weight ina high rate may have an effect on the protein folding.Improper folding of the proteins may be degraded bythe endogenous proteases to conserve cellular resources. Analysis of the recombinant proteins by westernblotting showed that 87 kDa and 50 kDa proteins couldbe recognized by the anti-His monoclonal antibody,5
S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Protein.Acta Scientiae Veterinariae. 40(2): 1029.Figure 5. Development of IgY antibody titer against CPV-VP2 protein.lumn, two proteins including the 85 kDa and 50 kDawere purified together, which was in accordance withthe result analyzed by the western blotting (Figure 3).The chickens were first immunized with rVP2protein in the dosage of 300 μg per immunization, yetit produced a poor antibody titers response in iELISAanalysis (data not shown). Thus, the antigen dosageof 1mg rVP2 protein per injection was chosen as described above. The antibody titers reached the peak of1:40560 after the fourth immunization (Figure 5). Thisrevealed that dose of the antigen had an effect on theproduction of antibody in chicken. It had been reportedthat antigen among 10 1000 μg may more efficientlyinduce the antibody response [6].The VP2 inclusion body washed by 0.05%Tween-20 was applied on the SDS-PAGE and wasanalyzed by western blotting using the purified IgYantibody as the primary antibody. It revealed that the85 kDa band was recognized by the anti-VP2-IgY antibody, however, the 50 kDa was not (Figure 6). Most ofthe antigen sites were presented in the amino terminalregion of VP2 [9]. Anti-VP2-IgY antibody may notrecognize the degraded production if they have lostthe required N-terminal sequences from VP2 protein.The entire virus particles may often induceto considerable protection efficiency in differentIgY antibody studies [15]. However, choosing targetvirus protein as antigen could lead to better effect onpathogen detection. Comparison of the sequences ofVP2 ORF revealed that CPV isolated in China hadFigure 6. Western blotting analysis of thebinding activity of the anti-VP2 IgY. Lane 1:Marker; Lane 2: insolubility of pET-32a-VP2induction with IPTG.revealing that His-tag existed at the terminal of theproteins, while the 40 kDa one lost the tag and couldnot be recognized (Figure 2). Purification of the rVP2protein from the insoluble fraction by Ni affinity co-6
S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Protein.Acta Scientiae Veterinariae. 40(2): 1029.Genscript, Nanjing, China.TakaRa, Dalian, China.4ZSGB-BIO, Beijing, China.5Thermo scientific, Waltham, Massachusetts, USA.6GE Healthcare, Wauwatosa, Wisconsin, USA.7Sigma-Aldrich Co., St. Louis, Missouri, USA.8NUNC, Roskilde, Denmark.9Biotek, Winooski, Vermont, USA.10Junyi Company, Beijing, China.11Abcam, Cambridge, Massachusetts, USA.Acknowledgements. This work was supported by Ministry ofEducation and State Administration of Foreign Experts Affairs“overseas teacher” project (No. MS2011XBNL057) and agrant (No. 01140407) for returned oversea Chinese Scholarsof Northwest A & F University, China.2high identities (98.3%) [18]. In this study, Anti-VP2IgY antibody was produced by immunized the chickenwith the rVP2 protein, which had never been reportedbefore according to our survey. Compared the antibody production between chicken and mammalian,an extraordinary amount of antibody (approximately17-35 g of total IgY/chicken/year) can be producedfrom only one hen and almost 10% is antigen-specific[19]. Anti-VP2 IgY antibody may be applied into theimmunotherapy and immunoprophylaxis for the CPVinfection. Furthermore, IgY antibody doesn’t reactwith mammalian IgG nor binding to the rheumatoidfactor, which may reduce the false positive resultsin immunoassays [19]. Therefore, the anti-VP2-IgYcould be developed into the diagnosis and treatmentreagents for CPV infection, but the efficiency shouldbe investigated further.3Ethical approval. All of the experiments were performed according to Experimental Animal Management Law of Chinaand approved by Animal Ethics Committee of Northwest SciTech University of Agriculture and Forestry.Declaration of interest. The authors report no conflicts ofinterest. The authors alone are responsible for the content andwriting of the paper.SOURCES AND MANUFACTURERS1Bioland, Chungnam, Korean.REFERENCES1 Aasted B., Race R.E. & Bloom M.E. 1984. Aleutian disease virus, a parvovirus, is proteolytically degraded duringin vivo infection in mink. Journal of Virology. 51(1): 7-13.N2 Appel M.J., Cooper B.J., Greisen H., Scott F. & Carmichael L.E. 1979. Canine viral enteritis. I. Status report oncorona- and parvo-like viral enteritides. Cornell Veterinarian. 69(3): 123-133.3 Bizhanov G. & Vyshniauskis G. 2000. A comparison of three methods for extracting IgY from the egg yolk of hensimmunized with sendai virus. Veterinary Research Communications. 24(2): 103-113.4 Decaro N. & Buonavoglia C. 2011. Canine parvovirus-A review of epidemiological and diagnostic aspects, withemphasis on type 2c. Veterinary Microbiology. [in press / Epub ahead of print].5 Decaro N., Desario C., Elia G., Campolo M., Lorusso A., Mari V., Martella V. & Buonavoglia C. 2007. Occurrenceof severe gastroenteritis in pups after canine parvovirus vaccine administration: a clinical and laboratory diagnosticdilemma. Vaccine. 25(7):1161-1166.6 Erhard M.H., Ozpinar H. Bilal T., Abas I., kutay C., Eseceli H. & Stangassinger M. 2000. The humoral immuneresponse and the productivity of laying hens kept on the ground or in cages. Atla-alternatives to Laboratory Animals.29(2): 699-705.7 Hoelzer K., Shackelton L.A., Parrish C.R. & Holmes E.C. 2008. Phylogenetic analysis reveals the emergence,evolution and dispersal of carnivore parvoviruses. Journal of General Virology. 89(9): 2280-2289.8 Hu J.J., Zhang X.Y., Han S.Z., Zhao J.Z. & Tian Z.H. 2011. A survey of diagnosis and treatment of pet canineparvovirus disease in China. Journal of Animal and Veterinal Advance. 10(16): 2058-2060.9 Langeveld J.P.M., Casal J.I., Vela C., Dalsgaard K., Smale S.H., Puijk W.C. & Meloen R.H. 1993. B-Cell epitopesof Canine Parvovirus - Distribution on the primary structure and exposure on the viral surface. Journal of Virology.67(2): 765-772.10 Lee E.N., Sunwoo H.H., Menninen K. & Sim J.S. 2002. In vitro studies of chicken egg yolk antibody (IgY) againstSalmonella enteritidis and Salmonella typhimurium. Poultry Science. 81(5): 632-641.11 Moon H.S., Lee S.A., Lee S.G., Choi R., Jeoung S.Y., Kim D. & Hyun C. 2008. Comparison of the pathogenicityin three different Korean canine parvovirus 2 (CPV-2) isolates. Veterinary Microbiology.131(1-2): 47-56.7
S. Han, X. Zhang & J. Zhao. 2012. Production of Egg Yolk Antibody (IgY) against Recombinant Canine Parvovirus VP2 Protein.Acta Scientiae Veterinariae. 40(2): 1029.12 Pratelli A., Cavalli A., Martella V., Tempesta M., Decaro N., Carmichael L.E. &Buonavoglia C. 2001. Canineparvovirus (CPV) vaccination: Comparison of neutralizing antibody responses in pups after inoculation with CPV2 orCPV2b modified live virus vaccine. Clinical and Diagnostic Laboratory Immunology. 8(3): 612-615.13 Rhode, S.L. 1985. Nucleotide sequence of the coat protein gene of canine parvovirus. Journal of Virology. 54(2): 630633.14 Spibey N., Greenwood N.M., Sutton D., Chalmers W.S. & Tarpey I. 2008. Canine parvovirus type 2 vaccine protectsagainst virulent challenge with type 2c virus. Veterinary Microbiology. 128(1-2): 48-55.15 Vega C., Bok M., Chacana P., Saif L., Fernandez F. & Parreno V. 2011. Egg yolk IgY: protection against rotavirusinduced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves. VeterinaryImmunology and Immunopathology. 142(3-4): 156-169.16 Weichert W.S., Parker J.S., Wahid A.T., Chang S.F., Meier E. & Parrish C.R. 1998. Assaying for structural variation in the parvovirus capsid and its role in infection. Virology. 250(1): 106-117.17 Yi L., Cheng S.P., Wang J.K., Chai X.L., Luo B., Zhang H.L., Wu W. & Yan X.J. 2010. Expression of VP2 geneof canine parvovirus virus and development of a recombinant protein-based indirect ELISA for antibodies detection.Chinese Journal of Veterinary Science. 30(8): 1038-1042.18 Zhang R., Yang S., Zhang W., Zhang T., Xie Z., Feng H., Wang S. & Xia X. 2010. Phylogenetic analysis of the VP2gene of canine parvoviruses circulating in China. Virus Genes. 40(3): 397-402.19 Zhang W.W. 2003. The use of gene-specific IgY antibodies for drug target discovery. Drug Discovery Today. 8(8):364-371.Pub. 1029www.ufrgs.br/actavet8
proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the proteins in the capsid are VP2 protein [13]. A series of B cell epitopes have been found on the VP2 protein and could be used as a vaccine for the prevention of CPV [3, 9]. After first isolation of CPV-2 in the U
3. Gently keep the egg moving in a clockwise direction, this helps keep the yolk in the middle. 4. Cook for 5 minutes for semi firm yolk or 6 minutes for a firm yolk. 5. Remove pan from heat. Using a slotted spoon, transfer an egg cup, carefully remove the top of egg and serve with toast, cut into strips for easy dipping. Serves 1
the egg production facility. This processing method is the most efficient egg collection and processing of eggs available. Eggs are delivered from the egg production facility to the egg processing facility by an enclosed and refrigerated conveyor system. Egg handling and processing is performed with automated equipment.
Crack an egg into the bread hole without breaking the yolk. 5. Season with salt and black pepper. 6. Cook egg for two minutes and then flip, season with Italian seasoning and cook for one minute. 7. Remove and serve while yolk is a little soft. Serve with bread cut outs. INGREDIE
Coronavirus Antibodies Catalog# Size Anti-Human IgG, Unconjugated Antibody 100736 100 µg 500 µg Anti-Human IgM, Unconjugated Antibody 100737 100 µg 500 µg Anti-Nucleocapsid Antibody (SARS-CoV-2) 100861 20 µg 100 µg Anti-Spike S1 Monoclonal Antibody (SARS-CoV-2) 100715 20 µg 100 µg Spike Neutralizing Antibody (Clone G10xA1) (SARS-CoV-2 .
TYPES OF PLACENTA BASED ON THE SOURCE OF VASCULAR SUPPLY From the Vitellinecirculation of the yolk sac or Allantoiccirculation provided by the allantois: 1. Chorio-vitelline/ Yolk-sac placenta -Highly vascular yolk sac fuses with the chorion eg. Metatherian mammals -Marsupials , Didelphis and Macropus 2.
Egg yolk consumption and carotid plaque J. David Spencea,*, David J.A. Jenkinsb, Jean Davignonc,d,e aStroke Prevention & Atherosclerosis Research Centre, Robarts Research Institute, 1400 Western Road, London, ON N6G 2V2, Canada b Canada Research Chair in Nutrition and Metabolism, Risk Factor Modific
oldest known paint formulations; for example the so-called tempera paints, used in the earliest European civilizations, consisted of egg yolk (binder), pigments and water as a thinner. In the middle ages, slow-dryingand curing (i.e. plastic-forming) vegetable oils, such as linseed oil, replaced egg yolk as a binder. Synthetic (man-
creating a usable specific antibody, including steps of immunogen preparation, immunization, hybridoma creation, collection, screening, isotyping, purification, and labeling for direct use in a particular method. The more restricted sense, antibody production refers to the steps leading up to antibody generation but does not
