이 작품은 교육과 연구의 독일 연방 교육부에서 교부금에 의해 지원되었다 (프로젝트 BiGRUDI [동성 ologische G efahrenlagen : R isikobewertung, U ltraschnelle D etektion 깨물어 I dentifizierung 폰 bioterroristisch relevanten Agenzien, 13N9594]). 우리는 우수 기술 지원 B. Diemar을 (샤히떼 - Universitätsmedizin 베를린, 인스티투트 FÜR Pharmakologie) 감사합니다.

1. 프로토콜 - PEG - 강수량에 의해 IgY - 추출 그림. 1 IgY - 적출 수술의 구조 다이어그램을 (표 2 참조) 있습니다. 프로토콜이 처음 폴손 외에 설명되어되었습니다. 1980. 모든 단계 라텍스 장갑을 사용하여 수행되어야합니다. <ol><li> 달걀 껍질은 신중하게 금이되고 노른자는 가능한 한 많은 흰 계란으로 제거하기 위해 &#39;노른자 스푼 &quot;으로 전송됩니다. </li><li> 난황이 필터 종이로 전송하고 나머지 흰자위를 제거하는 압연입니다 노른자 피부는 첨두 또는 이와 유사한 악기 (피펫 팁)으로자를 수 있습니다. 난황은 50 ML 관에 부어 있으며, 계란 볼륨 (V1) 등록되어 있습니다. </li><li> 두 번 PBS의 계란 노른자 볼륨이 전체 볼륨의 노른자 (ΣV1 + V2), 이후 3.5 %의 PEG 6000 (g에 박살 나자)와 혼합이 추가되고 vortexed 10 분 롤링 믹서에서 굴러옵니다. 추출 절차의 단계는 두 단계의 현탁액을 분리합니다. 한 단계는 &quot;노른자의 고체 및 지방산 물질&quot;(폴손 외의 원래 견적. 1980)과 IgY 및 기타 단백질을 포함하는 물의 단계로 구성되어 있습니다. </li><li> 튜브는 4 centrifuged 아르 ° 20 분 (10,000 RPM 13,000 XG, Heraeus Multifuge 3SR + 고정 앵글 로터에 따라)를위한 C. 뜨는 (V3)이 접혀 필터를 통해 부어 새로운 튜브로 전송됩니다. </li><li> g (새 볼륨에 따라 계산)에 8.5 %의 PEG 6000은 튜브에 추가 vortexed과 스텝 3에서와 같이 회전 믹서에 압연 있습니다. </li><li> 뜨는이 삭제되는 차이와 4 단계를 반복합니다. </li><li> 펠렛은 신중하게 유리 막대와 vortexer에 의해 한 ML PBS에 용해됩니다. PBS 10 ML의 최종 볼륨 (V4)에 추가됩니다. 솔루션은 12 % PEG 6000 (W / V, 1.2 g)과 혼합하여 3 단계 (와동, 압연 믹서)와 같이 취급됩니다. </li><li> 6 단계를 반복하여 800 μl PBS에 신중 펠렛을 (유리 막대와 와동) 디졸브. 사라지고 다음 투석 캡슐을 (피펫) 추출을 전송하기 위해 공기 방울 나올 때까지 기다리는 것이 좋습니다. 400 μl PBS로 튜브를 씻어 투석 장치 (V5)에 볼륨을 추가합니다. (투석 장치와 점막의 준비에 대한 부록을 참조하십시오.) </li><li> 추출물은 0.1 %의 염분 (1,600 ML)에서 밤새도록 dialysed 부드럽게 자기 활동가에 의해 흔들 수 있습니다. 다음 아침, 호수는 PBS로 대체하여 다른 세 시간 동안 dialysed입니다. </li><li> 이후 IgY - 추출물은 피펫으로 투석 캡슐에서 가져온 및 2ml 튜브로 전송됩니다. 최종 볼륨은 2 ML (V6) 주변이다. </li><li> 샘플의 단백질 함량 (MG / ML)을 (그림을 참조하십시오. 2) 280 NM (PBS에 희석 1시 50분)에 photometrically 측정과 IgY에 대한 1.33의 흡광 계수와 램버트 - 비어 법칙에 따라 계산, 그림 . 4 준비의 품질 (순도 및 복구가 80 % 정도된다)를 보여줍니다. </li><li> 그것은 ° C (-70 ° C에서 샘플을 동결하지 않음) -20에서 aliquots에 샘플을 저장하는 것이 좋습니다. </li><li> 최종 준비의 품질로 조브 프로토콜에 설명된 간단한 SDS - PAGE로 분석됩니다 <a href="http:/www.jove.com/details.php?id=1916">http:/www.jove.com/details.php?id=1916</a> </li></ol> 2. 부록 - 전에 투석 가방이 제조 업체의 권장 사항에 따라 다음과 같은 방법으로 준비를해야합니다 사용 : <ol><li> 20 투석 가방은 30cm의 조각으로 절단하고 2000 ML 유리 비커에 부여됩니다. </li><li> 5 MM - EDTA (에틸렌 다이아 민 테트라 초산) 솔루션의 1750 ML이 추가됩니다. </li><li> 유리 깔때기는 가방은 EDTA (에틸렌 다이아 민 테트라 초산) 솔루션이 적용되는 것을 보장하기 위해 가방 위에 배치됩니다. 솔루션은 온수와 5 분 (철판)에 삶은입니다. 솔루션은 decanted되고 가방은 증류수로 세 번 씻어 있습니다. </li><li> 다시 한번 1750 ML - EDTA (에틸렌 다이아 민 테트라 초산) 솔루션은 추가 5 분 삶은 이상과 같은 증류수로 세 번 씻어 있습니다. </li><li> 마지막으로 투석 가방은 증류수에서 10 분 끓인 4에 저장됩니다 ° C. 멸균 핀셋 통해 투석 가방을 가져가라. </li></ol> 3. 대표 결과 <img src="/files/ftp_upload/3084/3084fig1.jpg" alt="그림 1" /> 그림 1. 폴손 외의 원래 설명에 따라 폴리에틸렌 글리콜 석출에 의해 IgY - 추출의 도식 다이어그램. 1980. <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">큰 이미지를 보려면 여기를 클릭하십시오.</a> <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="그림 2" /> 그림 2. 시대의 의존에서 예방 접종을 암탉의 계란 노른자에서 총 - IgY 개발. 총 IgY / 계란 및 SD (N = 4 누워있는 암탉)의 주간 의미는 주어진. [폴리 외. 2009]에서). <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="그림 3" /> 그림 3. Layi다른 항원과 예방 접종을 사 암탉의 NG 용량 모니터링. 화살표는 예방 접종 날짜를 ([폴리 외. 2009]에서)을 나타냅니다. <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">여기 큰 이미지를 클릭 볼 수 있습니다.</a> <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="그림 4" /> 그림 4. 조건을 감소에 따라 다른 계란에서 개별 IgY의 준비 Polyacrylamide - Gelelectrophoresis 레인 1 - 분자량 마커, 레인 2 - IgY 표준, 레인 3-5 - 프로토콜에서 설명한대로 다른 IgY 샘플 준비. 이것은 프로토콜의 10 단계에 따라 최종 준비하고 있습니다. HC - 무거운 체인, LC - 라이트 chaines? - 35 KDA (아마도 vitellogenin II 전구체의 C - 말단 조각, [. Klimentzou 외, 2006]) 주변의 분자량에 해당하는 경미한 불순물. <table border="1"><tbody><tr><td>&nbsp;</td><td> 치킨 21 (항원) </td><td> 치킨 22 일 (항원) </td><td> 치킨 19 일 (항원 B) </td><td> 치킨 23 조 (항원 C) </td></tr><tr><td> 에그 번호 (총 IgY) 1, 2 년 기간 </td><td> 625 (38 G) </td><td> 626 (38 G) </td><td> 545 (33 G) </td><td> 608 (37 G) </td></tr><tr><td> 에그 번호 (총 IgY), 첫해 </td><td> 345 (20 G) </td><td> 304 (16g) </td><td> 326 (18 G) </td><td> 308 (17 G) </td></tr><tr><td> 에그 번호 (총 IgY), 두번째 년 </td><td> 280 (18 G) </td><td> 322 (21 G) </td><td> 219 (15 G) </td><td> 300 (20g) </td></tr><tr><td> 최대의 %. 가능한 계란 번호 (첫 해에) 2 </td><td> 106 </td><td> 93 </td><td> 100 </td><td> 95 </td></tr><tr><td> 최대의 %. 가능한 계란 번호 (두 번째 년) </td><td> 86 </td><td> 99 </td><td> 67 </td><td> 92 </td></tr><tr><td> 가공 계란의 수를 </td><td> 565 </td><td> 620 </td><td> 283 </td><td> 401 </td></tr></tbody></table> 총 IgY 1 금액은 그림 2의 데이터에 따라 계란 60 MG 단백질의 의미 값으로 달걀의 개수를 곱하여 계산됩니다. 2 최대. 연간 가능한 달걀 번호는 개종의 정보에 따라 325입니다. 표 1. 다른 항원과 예방 접종을 닭이 계란 누워있는 용량을 2 년 통계. (그림도 참조하십시오. 3) 2 년 동안 서로 다른 항원뿐만 아니라 IgY - 결과와 예방 접종 후 네 암탉의 최대 비율의 용량을 누워, [폴리 외. 2009]). <table border="1"><tbody><tr><td> 에그 넘버. </td><td> 헨 넘버. 날짜 누워 </td><td> V1 [ML] 난황 </td><td> V2 [ML] PBS </td><td> 1. PEG prec. [G] </td><td> 권 [ML] 후 </td><td> 2. PEG prec. [G] </td><td> 작은 공 </td><td> 3. PEG prec. [G] </td><td> 작은 공 </td><td> 권 [ML] </td><td> 총 단백질 MG / ML </td><td> 수정된 총 단백질 MG / ML </td></tr><tr><td></td><td></td><td></td><td> 2xV1 ΣV1가 + V2 </td><td> 3,5 % [W / V] ΣV1 + V2 </td><td> prec., centrif. 및 여과 V3 </td><td> 8,5 % [W / V] V3 </td><td> V4 ML PBS에 용해 </td><td> 12% [W / V] V4 </td><td> ML PBS V5에 녹아있는 </td><td> PBS V6에 대한 투석 후 </td><td> A280/ml </td><td> A280/ml : 1.33 </td></tr><tr><td> 1 </td><td> H293 / 22.10. </td><td> 15 </td><td> 30 45 </td><td> 1.58 </td><td> 31 </td><td> 2.63 </td><td> 10 </td><td> 1.2 </td><td> 1.2 </td><td> 1.7 </td><td> 35.0 </td><td> 26.3 </td></tr><tr><td> 2 </td><td> H293 / 23.10. </td><td> 13 </td><td> 26 39 </td><td> 1.37 </td><td> 25 </td><td> 2.12 </td><td> 10 </td><td> 1.2 </td><td> 1.2 </td><td> 1.7 </td><td> 28.8 </td><td> 21.6 </td></tr></tbody></table> 표 2. PEG 침전의 전형적인 프로토콜

<ol>
	<li>Akita, E. M., Nakai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+purification+methods+for+the+production+of+immunoglobulins+from+eggs+laid+by+hens+immunized+with+an+enterotoxigenic+E.+coli+strain.">Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain.</a> J. Immunol. Methods. 160, 207-214 (1993).</li><li>De Meulenaer, B., Huyghebaert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+chicken+egg+yolk+immunoglobulins:+a+review.">Isolation and purification of chicken egg yolk immunoglobulins: a review.</a> Food Agricult. Immunol. 13, 275-288 (2001).</li><li>Klimentzou, P., Paravatou-Petsotas, M., Zikos, C. h., Beck, A., Skopeliti, M., Czarnecki, J., Tsitsilonis, O., Voelter, W., Livaniou, E., Evangelatos, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+immunochemical+evaluation+of+antibodies+Y+for+the+poorly,+immunogenic+polypeptide+prothymosin+alpha.">Development and immunochemical evaluation of antibodies Y for the poorly, immunogenic polypeptide prothymosin alpha.</a> Peptides. 27, 183-193 (2006).</li><li>Pauly, D., Dorner, M., Zhang, X., Hlinak, A., Dorner, B., Schade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+laying+capacity,+immunoglobulin+Y+concentration,+and+antibody+titer+development+in+chickens+immunized+with+ricin+and+botulinum+toxins+over+a+two-year+period.">Monitoring of laying capacity, immunoglobulin Y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period.</a> Poultry Science. 88, 281-290 (2009).</li><li>Polson, A., von Wechmar, M. B., van Regenmortel, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+viral+IgY+antibodies+from+yolks+of+immunized+hens.">Isolation of viral IgY antibodies from yolks of immunized hens.</a> Immunol. Commun. 9, 475-493 (1980).</li><li>Schade, R., Behn, I., Erhard, M., Hlinak, A., Staak, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chicken egg yolk antibodies, production and application : IgY-Technology. , (2001).</li><li>Schade, R., Calzado, E. G., Sarmiento, R., Chacana, P. A., Porankiewicz-Asplund, J., Terzolo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+egg+yolk+antibodies+(IgY-technology):+a+review+of+progress+in+production+and+use+in+research+and+human+and+veterinary+medicine.">Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine.</a> Altern. Lab Anim. 33, 129-154 (2005).</li><li>Witkowski, P. T., Bourquain, D. R., Hohn, O., Schade, R., Nitsche, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+gun-supported+DNA+immunisation+of+chicken+for+straightforward+production+of+poxvirus-specifiv+IgY+antibodies.">Gene gun-supported DNA immunisation of chicken for straightforward production of poxvirus-specifiv IgY antibodies.</a> J. Immunol. Methods. 341, 146-153 (2009).</li></ol>

동물 실험 Landesamt 모피 아퍼 놀이 Soziales 베를린 (H 0069/03)에 의해 명시된 지침과 규정에 따라 수행되었다.

적당한 IgY 정화 방법의 선택은 정화 (연구소 또는 산업), 비용 효율성, 기술 (실험실 장비)와 환경 (폐기물 관리)에 영향을 미치는 규모에 의해 영향을받습니다. 다양한 IgY 추출 방법은 데 Meulenaer &amp; Huyghebaert (2001, 검토도 쉐이드 외를 참조하십시오. 2005)에 의해 상세하게 검토했다. 일반적으로 이러한 방법의 세 주요 그룹으로 나눌 수 있습니다 : <ol><li> 강수량 방법 : 암모니아 또는 나트륨 황산염, polyethyleneglycol (PEG), caprylic 산성 및 caragenean를 포함. </li><li> 색층 방법 : 친화도 크로마 토그래피, 이온 교환 크로마 토그래피, 소수성 상호 작용 크로마 토그래피, thiophylic 상호 작용 크로마 토그래피 및 겔 - 여과 크로마 토그래피. </li><li> Ultrafiltration. </li></ol> IgY 준비 순도는 방법의 조합에 의해 증가시킬 수 있으며, 예를 들어, PEG 강수량은 친화도 크로마 토그래피와 함께하실 수 있습니다. 어떤 경우에는 최종 응용 프로그램에 따라 IgY의 물 추출물은 좋은 결과를 (아키타 &amp; 나카이 1993) 달성을 충분합니다. 우리의 경험에 따르면, 우리는 PEG - 석출에 의해 얻을 IgY - 샘플은 다양한 면역 assays 많은 매우 잘했습니다. PEG - 석출 방법을 표준화하여 한 기술 비서가 4 7g 주당 사이에 총 IgY의 금액에 해당하는 약 84 알을 주당을 처리할 수 있습니다. 단순 계산 : 누워있는 암탉은 항원과 예방 접종을합니다. 몇 부스트 예방 접종 후 암탉 30 일 기간 동안 1:50,000의 titre로 특정 항체 (AB)를 생산하고 있습니다. 30 달걀 X 2 ML IgY 추출물 (프로토콜과 그림. 3 참조) 3,000리터와 의미 차례로 3,000리터의 AB - 희석 (통의 용량이 10리터입니다)에 해당하는 60 ML 총 IgY, 해당 AB 솔루션 300 버킷이 채워져 수 있습니다. 그래서, 같은 거대한 AB - 수량의보기에 그것은 지속적인 품질과 특이성의 AB - 수영장 저장 아무런 문제가 없습니다. 이 기술은 별도 polyspecific AB에서 관찰 충전 / 많은 변화를 줄여줍니다. 포유류의 보완 시스템의 아니오 활성화, 하마 (인간의 안티 마우스 항체), 류마티스 요인이나 인간의 피를 그룹 항원 (heteroagglutinins 부족)와 노 교차 반응성 : 수량의 측면의에도 불구하고, IgY AB는 포유류의 IgG에 비해 더 장점이있다. IgY - AB의 뛰어난 장점은 소위 phylogenetic 거리입니다. Phylogenetic 거리가 동일하게 예방 접종을 때에도, 포유류와 닭의 순이 specificities 사이에 자주 설명한 차이에 대한 이유입니다. 포유 동물과 조류 자신의 면역 시스템 사이의 차이 이외에,이 두 동물 수업 phylogenetic 개발의 차이는 서로 다른 순이 specificities에 기여하고 있습니다. 몇몇 저자는 닭은 종종 더 효율적으로 토끼 (검토 쉐이드 동부 표준시 A. 2005 참조)보다 phylogenetically 고도 보존 포유 동물 단백질이나 펩티드에 대한 애비를 만들어보고있다. 그 결과, 보존 항원은 토끼 면역 체계에 &quot;가면을 쓴&quot;남아 있으므로에서만 약한 또는 &quot;자동&quot;응답을 일으킬 수 있습니다. 병아리와 토끼가 같은 포유류의 항원과 예방 접종을하는 경우뿐만 아니라, 매우 자주 닭이 거의 토끼에서 얻을 수 없습니다 AB - 특이성과 반응한다. AB는 난황에서 추출한 비 invasively 있기 때문에 또한 동물 복지의 측면이 중요합니다. 조합의 유전자 군 (플라스미드) 예방 접종과 IgY 생산은 (Witkowski 외. 2009) 완전히 비침습입니다. 결론적으로, 암탉과 PEG - 석출에 의해 IgY - 추출에서 AB의 생산은 매우 비용 효과적인 최대 1:1,000,000으로 안정 titres와 매우 구체적인 AB 결과. 에이브스와 포유류 사이 phylogenetic 거리로 인해, 닭고기 예 토끼와는 대조적으로 매우 보존 포유류의 항원에 대한 구체적인 AB 생산 수 있습니다. IgY 기술 얻은 AB의 특별한 금액은 치료 / 예방 목적으로 인간 및 동물용 의약품의 IgY - AB를 사용에도 문을 엽니다.

<ol><li> -14 KD, 칼 로스 GmbH를, 카를 스루에, 독일 잘라 가방 Visking, 유형 32분의 27을 Dialyse </li><li> 팔콘 튜브 블루 맥스 50 ML 폴리 프로필렌 코니컬 튜브, 백톤 디킨슨 Labware, 프랭클린 레이크스 NY, 미국 </li><li> 접힌 필터 MN 615 ¼, ∅ 150mm, 칼 로스 GmbH를, 카를 스루에, 독일 Heraeus </li><li> 자기 활동가, Variomag 모노, H + P Labortechnik AG, Oberschleißheim, 독일 </li><li> 마이크로 Dialyse - 캡슐 5,0 ML, 칼 로스 GmbH를, 카를 스루에, 독일 </li><li> Multifuge 3SR +, 써모 과학, Langenselbold, 독일 </li><li> 광도계 UV - 160A, Shimadzu 독일 GmbH를, 뒤스부르크, 독일 </li><li> 폴리에틸렌 글리콜 6000, Rotipuran 박사 유로., 칼 로스 GmbH를, 카를 스루에, 독일 </li><li> Premixed PBS - 버퍼 10X, 로체 진단 GmbH를, 만하임, 독일 </li><li> 롤러 믹서 SRT2, 스튜어트, 스태포드셔, 영국 </li><li> Vibrofix VF2, 장케 &amp; 쿤켈, 이카 - Labortechnik, Staufen i.Br., 독일 </li><li> 난황 스푼, Fackelmann (houshold 효과), 독일 </li></ol>

igy technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (peg) precipitation

암탉은 메신저 예방 접종 (Musculus pectoralis, 왼쪽 및 오른쪽, 사출 볼륨 0.5-1.0 ML)를 통해 또는 유전자 건 플라스미드 - 예방 접종에 의해 예방 접종을하실 수 있습니다. 항원의 immunogenicity에 따라, 높은 항체 - titres은 (최대 1:100,000 - 1:1,000,000) 하나 또는 3 이후 얻을 수 - 4 부스트 예방 접종을. 일반적으로, 암탉은 이후 누워 용량 감소에 대한 72주 끊임없이 알을 낳는. 이 프로토콜은 침전 절차 수단 (PEG. 폴손 외. 1980)로 달걀 노른자에서 총 IgY의 추출을 설명합니다. 방법은 두 가지 중요한 단계를 포함한다. 첫번째는 lipids의 제거이고 두 번째 단계 중 하나 뜨는에서 총 IgY의 강수량입니다. 버퍼 (일반적으로 PBS)에 대한 투석 후 IgY - 추출물은 년 이상 -20 ° C에 저장할 수 있습니다. 추출물의 순도는 80 % 근처이고, 계란 당 총 IgY가 누워있는 암탉의 연령에 따라, 40-80 MG에서 다릅니다. 최대 80 MG / 계란 (PEG 침전에 관한)에 40 MG / 계란에서 암탉의 나이가 총 IgY 콘텐츠 증가합니다. 연간 암탉의 누워있는 용량은 325 계란 주위에있다. 60 MG 총 IgY / 계란 (표 1 참조)의 의미 IgY의 콘텐츠를 기반으로 20g 총 IgY / 올해의 총 잠재 수확을 의미합니다.

Watch this Scientific Journal Video about IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation at JoVE.com

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

이 프로토콜은 폴리에틸렌 글리콜 석출에 의해 난황에서 총 IgY의 특정 추출에 대해 설명하고 IgY 기술에 대한 일반적인 정보를 제공합니다.

IgY 기술 : 폴리에틸렌 글리콜의 노른자위에서 치킨 항체의 추출 (PEG) 강수량

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

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JoVE Journal

Immunology and Infection

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

64.1K 조회수.  Robert Koch-Institute. 이 프로토콜은 폴리에틸렌 글리콜 석출에 의해 난황에서 총 IgY의 특정 추출에 대해 설명하고 IgY 기술에 대한 일반적인 정보를 제공합니다.

비디오: IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

의 드문 경우에 이가와 조직 Transglutaminase이 항체에 의해 표피 Transglutaminase의 인식 붉은 털원숭이 피부염

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

연구

면역학 및 감염병학

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

에서 DNA 추출을위한 간단한 Chelex 프로토콜<em&gt; 아노펠레스</em&gt; 종.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

측면 흐름 기술을 사용하여 Haematological 강한 악의 환자에서 침윤성 폐 아스 페르 길 루스 증의 탐지

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

핫 수성 - 페놀 추출에 의한 그람 음성 박테리아에서 정화와 Lipopolysaccharide의 시각화

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

인간 백신 닭 계란에 인플루엔자 바이러스 전파

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

정렬 된 불멸화 된 B 세포로부터 재조합 인간 IgG 단일 클론 항체의 생성

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

하이 브리 도마 기술에 의해 쥐 단일 클론 항체의 생성

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

독감 조류 특정 항 체에 의해 유도 된 Fc 중재 Effector 기능을 평가 하는 방법

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

희귀 한 항 원 특정 B 세포 혈액 순환에서 구별 인간 단일 클론 항 체의 생성

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

미생물 감염 및 분석에 대 한 초파리 melanogaster 애벌레에서 Hemocytes의 추출

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...