Name: chemo-enzymatic synthesis of n-glycans for array development and hiv antibody profiling
Uploaded: 2018-02-05T13:00:00
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저자는 얇은 영화 기술, 계측 기술 연구 센터 (ITRC)와 국가 적용 연구 실험실, Hsinchu 과학 공원, 대만 감사합니다. 이 작품은 국가 과학 위원회에 의해 지원 되었다 (no를 부여 합니다. 대부분 105-0210-01-13-01) 및 학계 Sinica.

1. 준비의 D1/D2 팔 모듈22
<ol><li>중간 2의 준비
	<ol>
<li>( 그림 2, p-methoxyphenyl-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranoside (100mg, 0.204 mmol)에 표시 된) 15 mL 튜브에 재료 1 을 시작 무게와 트리 스 포함 하는 버퍼 (25 m m, pH 7.5)에 용 해 (MnCl2, 10 m m) 5mm의 최종 glycan 농도 달성 하기에 dichloride를 망간</li>
		<li>Uridine diphos...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+Array+on+Aluminum+Oxide-Coated+Glass+Slides+Through+Phosphonate+Chemistry.">Glycan Array on Aluminum Oxide-Coated Glass Slides Through Phosphonate Chemistry.</a> J. Am. Chem. Soc. 132, 13371-13380 (2010).</li><li>Tseng, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aluminum+Oxide-Coated+Glass+Slides+for+Glycan+Microarrays.">Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays.</a> ACS Omega. 1, 773-783 (2016).</li><li>Shivatare, S. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Hiv-1+gp120+V1/V2+Domain+with+Broadly+Neutralizing+Antibody+PG9.">Structure of Hiv-1 gp120 V1/V2 Domain with Broadly Neutralizing Antibody PG9.</a> Nature. 480, 336-343 (2011).</li><li>Julien, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+Recognition+of+the+HIV-1+Trimer+by+Broadly+Neutralizing+Antibody+PG9.">Asymmetric Recognition of the HIV-1 Trimer by Broadly Neutralizing Antibody PG9.</a> Proc Natl Acad Sci U S A. 110, 4351-4356 (2013).</li><li>Willis, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Antibody+HCDR3s+from+HIV-Native+Donors+Presented+on+a+PG9+Neutralizing+Antibody+Background+Mediate+HIV+Neutralization.">Long Antibody HCDR3s from HIV-Native Donors Presented on a PG9 Neutralizing Antibody Background Mediate HIV Neutralization.</a> Proc Natl Acad Sci U S A. 113, 4446-4451 (2016).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Organic+Chemistry.+Performing+1D+Thin+Layer+Chromatography.">Essentials of Organic Chemistry. Performing 1D Thin Layer Chromatography.</a> JoVE. , (2017).</li><li>de Castro, M. 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에이즈-1 bNAbs PG9, PG16, 및 PGTs 128, 141-145를 포함 한 클래스 중화 HIV-1 분리 순환의 70-80%에서 매우 강력한 보고 했다. 이러한 bNAbs epitopes는 높은 전체 HIV-1 그룹 M의 변종 중 보존, 그러므로 그들은 유도 중화 항 체23,24,25 에이즈 백신에 대 한 효과적인 immunogen 디자인을 안내할 수 있습니다. . 에이즈-1 광범위 하 게 항 체를 중화의 glyca...

N-glycans glycoproteins에 covalently에 연결 되는 아스파라긴 (Asn) 잔류물 합의 Asn-Xxx-Ser/Thr sequon의 단백질 구조, antigenicity, 가용성, 및 lectin 인식 등 여러 생물 학적 과정에 영향을 1 , 2. 화학 합성 N-연결 된 oligosaccharides 그들의 거 대 한 구조 마이크로 고 높은 분기 건축 때문에 중요 한 합성 챌린지를 나타냅니다. 보호의 신중한 선택 그룹 빌딩 블록, anomeric 센터, 및 발기인의 적절 한 사용에서 선택 달성의 반응성을 조정/activator(s) 복잡 한 oligosaccharides의 합성에서 핵심 요소입니다. 복잡 한이 문제를 해결 하려면 사전 N-glycan 합성 작품의 좋은 금액으로 알려졌다 최근3,4. 이러한 강력한 접근에도 불구 하 고 찾는 다양 한 N-glycans의 준비에 대 한 효과적인 방법 주요 도전 (~ 20000) 남아 있다.
에이즈-1 광범위 한 유전적 다양성 및 중화 항 체 응답에서 탈출 하는 능력의 급속 한 돌연변이 율 에이즈-15,6 에 대 한 안전 하 고 예방 백신을 개발 하는 가장 큰 과제 중 하나입니다. , 7. 에이즈를 사용 하 여 호스트 면역 반응을 피하기 위해 하나의 효과적인 전술 이다 봉투 당단백질 gp120 다양 한 N의 닢 glycosylation-호스트 glycosylation 기계8에서 파생 된 glycans 연결 9. 인간 미 발달 신장 (HEK) 293T 세포에서 재조합 단위체 HIV-1 gp120 glycosylation의 정확한 분석에 대 한 최근 보고서 제안 특성 셀 전용 패턴10 구조 microheterogeneity의 발생 , 11 , 12. 따라서 이해의 HIV-1 bNAbs glycan 특이성 필요 잘 특징이 gp120 N-glycan 구조 분석에 대 한 충분 한 수량에 관련.
Glycan microarray 기술 발견 제공 다양 한 탄수화물 묶는 단백질, 바이러스/박테리아 adhesins, 독 소, 항 체, 및 lectines13,14의 특이성의 높은 처리량 기반 탐사 . 기준점과 칩 기반 형태로 체계적인 glycans 배열 문제가 낮은 선호도 단백질 glycan 상호 작용 multivalent 프레 젠 테이 션15,16,,1718을 통해 결정 합니다. 이 칩 기반 glycan 배열 셀 인터페이스를 효과적으로 모방을 편리 하 게 나타납니다. 기술을 풍부 관련 된 기존의 배열 형식 고르지 못한 문제를 극복 하 고, 우리의 그룹 최근 개발 phosphonic 산-엔드 glycans를 사용 하 여 신호 강도 향상 하는 알루미늄 산화물 코팅 유리 (ACG) 슬라이드에 glycan 배열 동질성, 그리고 감도19,20.
Glycan epitopes 광범위 중화 항 체 (bNAbs) 새로 격리 된 에이즈-1의에 대 한 현재 이해를 개선 하기 위해, 우리는 N의 광범위의 준비에 대 한 매우 효율적인 모듈형 전략 개발-glycans21 연결 22 는 ACG에 인쇄할 슬라이드 ( 그림 1참조). 매우 강력한 bNAb PG9 HIV 분리 되었던의 이종 glycan 바인딩 동작의 특이 한 검출 감염 개인23,,2425를 제공 하는 프로 파일링 연구 에이즈-1 bNAbs ACG 배열에의 한 특이성

<table width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetic acid</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">64197</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetonitrile</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">75058</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetic anhydride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">108247</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Anhydrous magnesium sulfate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7487889</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Boron trifluoride ethyl etherate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">109637</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Bovine serum albumin</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">9048468</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Bio-Gel P2 polyacrylamide</td>
			<td style="width:123px;">Bio-Rad</td>
			<td style="width:119px;">1504118</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Bis(cyclopentadienyl)hafnium(IV) dichloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">12116664</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">&#946;-1, 4 Galactosyl transferases from bovine milk</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">48279</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="width:168px;height:84px;">BioDot Cartesion technology with robotic pin SMP3 (Stealth Micro Spotting Pins)</td>
			<td style="width:123px;">Arrayit</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Cerium ammonium molybdate</td>
			<td style="width:123px;">TCI</td>
			<td style="width:119px;">C1794</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Cerium ammonium nitrate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">16774213</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Clean glass slide&#160;</td>
			<td style="width:123px;">Schott&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Cytidine-5&#8242;-monophospho-N-acetylneuraminic acid</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">3063716</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Deuterated chloroform</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">865496</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Donkey Anti-Human IgG (Alexa Fluor647 conjugated</td>
			<td style="width:123px;">Jackson Immuno Research, USA</td>
			<td style="width:119px;">709605098</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Dichloromethane</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">75092</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Diethylaminosulfur trifluoride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">38078090</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Dimethylformamide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">68122</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Ethyl acetate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">141786</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Ethylene glycol</td>
			<td style="width:123px;">Acros Organic</td>
			<td style="width:119px;">107211</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">FAST frame slide incubation chambers</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Guanosine 5&#39;-diphospho-b-L-fucose disodium salt&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">15839700</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab tracer 2.0 software&#160;</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:164px;">Section 4 of the Protocol</td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">GenePix Pro 4300A reader (microarray image analysis)</td>
			<td style="width:123px;">moleculardevices</td>
			<td style="width:119px;"></td>
			<td style="width:164px;">www.moleculardevices.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">GraphPad Prism Software (Image processing )</td>
			<td style="width:123px;">GraphPad Software, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:164px;">http://www.graphpad.com/guides/prism/6/user-guide/</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Lithium hydroxide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">1310652</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Manganese chloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7773015</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Methanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">67561</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">N-butanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">71363</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Oxalic acid</td>
			<td style="width:123px;">Acros Organic</td>
			<td style="width:119px;">144627</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Palladium hydroxide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">12135227</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Phosphate Buffered Saline</td>
			<td style="width:123px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">10010023</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Pyridine</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">110861</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">P-Toluene sulfonic acid monohydrate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">773476</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Silver triflate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">2923286</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium bicarbonate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">144558</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium chloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7647145</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Sodium hydrogen carbonate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">144558</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium methoxide&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">124414</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium sulfate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7757826</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Toluene&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">108883</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Tris buffer&#160;</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:119px;">N/A</td>
			<td style="width:164px;">Ultra-pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Tween-20</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:119px;">9005645</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Uridine diphosphate galactose (UDP-galactose)</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">137868521</td>
			<td style="width:164px;"></td>
		</tr>
	</tbody>
</table>

chemo-enzymatic synthesis of n-glycans for array development and hiv antibody profiling

우리 N의 광범위의 빠른 준비를 위한 매우 효율적인 방법 제시-oligosaccharides (초과 20000 구조 추정) 인간 glycoproteins에 일반적으로 발견 되는 연결. 원하는 구조 다양성을 달성 하기 위해 전략 뒤에 3-O 및 6-O 에 그들의 stepwise α 선택적 glycosylations oligosaccharyl 불 소 단위의 세 종류의 화학 효소 합성 시작의 위치는 중요 한 β-mannoside 결합 하는 데 일반적인 코어 trisaccharide의만 노 오 스 잔류물. 우리는 더 HIV 항 체와 이종 ligand 상호 작용의 분석에 대 한 공유 혼합된 배열을 만드는 알루미늄 산화물 코팅 유리 (ACG) 슬라이드의 표면에 N-glycans를 첨부. 특히, 남자 (5)는 새로 고립 된 HIV-1 광범위 중화 항 체 (bNAb), PG9, 밀접 하 게 간격 둔된 남자5GlcNAc2 의 혼합물에의 바인딩 동작 및 2, 6-디-sialylated bi antennary 복잡 한 입력 N-glycan (SCT ) ACG 배열, 에이즈 백신 개발을 위한 효과적인 immunogen 디자인 가이드에 새로운 길을 엽니다. 또한, 우리의 ACG 배열 hetero ligand 바인딩 동작에 대 한 다른 HIV 항 체를 연구 하는 강력한 도구를 구현 합니다.

다양 한 N-glycans의 합성에 대 한 모듈형 화학 효소 전략은 그림 1에 표시 됩니다. 전략은 다양성 α-특정 mannosylation는 3-O 및 6-O 에 다음 세 가지 중요 한 모듈의 화학 효소 합성 하 여 처음에 만들 수 있습니다는 사실에 근거한 일반적인만 노 오 스 잔류물의 위치 trisaccharide Nglycans의 핵심. Bi-트라이-, tetra antennary 복합 형식 N<...

Watch this Scientific Journal Video about Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling at JoVE.com

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Glycan microarray를 개발 하 고 광범위 하 게 항 체를 중화 하는 HIV의 프로 파일링에 대 한 사용 설명 되는 산화 알루미늄 코팅 유리에 첨부 파일에 대 한 N-glycans의 합성에 모듈 접근 (ACG 슬라이드) 슬라이드.

N-glycans 배열 개발 및 HIV 항 체의 항 암 치료 효소 합성 프로 파일링

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

The overall goal of this methodology, is to develop a modular chemo-enzymatic approach to the synthesis of N-glycans by constructing a new glycan microarray on a aluminum oxide coated glass slide for detecting heteroglycan binding of HIV antibodies. This methods can help answer key questions in glycobiology for identification of cell virus specific glycans for much development. The major advantage of this method is to detect weak binding and identify a mixture of sugars that bind to a protein such as, antibody from HIV patient.The implications of this technique extend to a diagnosis of influenza infections because sialic catabolist express on human airways epithelial cells and issues are common while the symptoms. Therefore, glycoarray will be an ideal tool to determine specificity of wild subtypes. Though this method can provide inside into the glycan specificities of HIV antibodies it can also be applied to other systems such as, diagnosis of various cancers based on the binding pattern of human serum antibodies toward human specific serum antigens printed on the surface.To begin this procedure, add 2-5 micromoles of previously prepared glycan and a five milliliter round bottom flask. Add a magnetic stir bar to the flask and cap it with a rubber septum. Add 400 microliters of freshly dried DMF and 10-25 micromoles of the phosphonic acid linker to the flask.Then, stir the reaction mixture at 800 rpm for five hours. Once the reaction is complete, evaporate the solvent using a rotary evaporator at five to 10 millibar vacuum pressure and 40 degrees Celsius. Following this, dissolve the dried reaction mixture and 0.5 milliliter of water and load on top of polyacrylamide gel bed.Elute the products using water and collect one to two milliliter fractions. To check the collected fractions by TLC, spot each fraction on a TLC plate and dip the plate in 0.25 molars Cerium Ammonium Molybdate. Heat the stained plate with a hot plate to visualize the product.After combining the product containing fractions and freezing the solution biofolize to obtain the desired product as a white powder. Next, dissolve the dried product in two milliliters of water and add Palladium Hydroxide. Attach a hydrogen containing balloon to the flask and stir the reaction mixture at 800 rpm for 15 hours under hydrogen atmosphere.Once the reaction is complete, filter the mixture through a Celite Bed and wash with two milliliters of methanol and two milliliters of distilled deionized water. Then, evaporate the solvent using a rotary evaporator at 300 millibar vacuum pressure and 40 degree Celsius. Following this, dissolve the crud mixture in approximately one milliliter of water and load it on top of a polyacrylamide gel bed.Elute the product with water and collect one to two milliliter fractions. After combining the product containing fractions and freezing the solution biofolize to obtain the desired product as a white powder. Once the product is dry, characterize it by enimar and mass spectroscopy using Deuterium Oxide as the solvent.For surface anodization, set a temperature controlled incubator to four degree Celsius. Then transfer a previously prepared 0.3 molar aqueous oxalic acid solution to a 500 milliliter beaker containing a magnetic stir bar. Place a 10 centimeter long platinum rod as a cathode into the solution.Keep stirring the oxalic acid at 300 rpm throughout the anodization process. In the lab tracer software, click on the setup button and then click on function choice Sweep Voltage. Set the start and stop voltage to 25.8 volts.The number of points to 100, the compliance to one, and the sweep delay to 1200 milliseconds. Then, click okay. Clamp an aluminum coated glass slide facing toward the cathode.Click on the run test button and observe the current measurement. After surface anodization, wash the glass slide thoroughly with double distilled water and purge dry with nitrogen gas. Store the glass slide in a 30%relative humidity chamber until further use.For fabrication, prepare 100 microliters solutions of all monovalent glycans in ethanol glycol at 10 millimolar concentration. Dilute the glycan solutions with printing buffer to make 100 micromolar solutions. For the Heteroligand Study, add five microliters of MAN-5 Glycan to five microliters of each glycan solution.Transfer the prepared glycans into the 384 well microplate for microarray printing step. After inserting a microplate on the printer array tech, print microarrays by robotic pin by depositing 0.6 nanoliters of the glycan solutions onto each aluminum coated glass slide. Next prepare 70 microliters of primary antibody PG-9 and PTSB buffer containing 3%BSA.Then prepare 120 microliters of secondary fluorescent tag antibody Donkey and Anti-human Immunoglobulin G and PBST buffer containing 3%BSA in the dark. Mix PG9 and the secondary fluorescent tag antibody in a 1:1 ratio. Then incubate the premixed antibodies for 30 minutes at four degrees Celsius.Following incubation, load an aluminum coated glass slide into the slide incubation chamber, which is divided into 16 wells. Transfer 100 microliters of the premixed antibodies to the glycan array. After incubating at four degrees Celsius for 16 hours remove the premixed antibodies from the glycan array with a pipette and then remove the slide incubation chamber.Wash the glass slide in PBST buffer and deionized water. Then spin dry the glass slide at 2000 times gravity. Next, open the microarray image analysis software.Insert the glass dried slide into the array scanner with the arrayed features facing down. Click on the settings menu. Set the image resolution to five micrometers per pixel.And set the wavelength at 635 nanometers with PMT 450 and power to 100%Click on the scan button to start imaging the slide. Finally click the file icon to save the scan image in TIF format for image analysis. In this study, a wide array of N-Glycans were synthesized using a modular chemo enzymatic strategy.To demonstrate the effectiveness of the strategy a biantennary isometric structure was synthesized by chemoenzymatic methods. Enzymatic galactosylation for disaccharide 1 followed by fucosylation of trisaccharide 2 afforded the desired tetrasaccharide 3. Per satylation and reducing end modifications of building block two and three, afforded to desire molecule four and five.Stereoselective three O-glycosalyation of module four of trisaccharide six provided hexasaccharide seven. De-protection and reaction before module five yielded dedecasaccharide nine. Global detection afforded glycan 10 which was characterized by enimar and mass spectroscopy.Glycans containing a Pentylamine tail at the reducing end were modified with phosphonic acid linkers and attached to the aluminum glass coated surface through phospinide chemistry. HIV-1 broadly neutralizing antibody PG9 was screened for it's glycan specificity using homo and heteroglycan arrays to demonstarte for the first time it's ineteraction with adjacent heteroglycans in the V1/V2 loop of HIV1 PG120 surface. Once mastered, this technique can be done in 24 hours from glycan immobilization to beta analysis if it was performed properly.While attempting this procedure, it is important to remember to use freshly anodized aluminum coated glass slide for Glycan 10. For laying this procedure are the med techs slides. Determination of the body constant can be performed in order to quantify the binding interactions between carbohydrate and protein of interest.After the development of this technique, it paved the way for researchers to better understand the role of carbohydrate in infectious disease in Kenya and help develop new therapies against diseases. Don't forget that working with toxic agents can be extremely hazardess and precautions such as wearing of the clothes, glasses and masks should always be taken when preforming experiments.

Research

JoVE Journal

Chemistry

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

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