Name: a protein microarray assay for serological determination of antigen-specific antibody responses following clostridium difficile infection
Uploaded: 2018-06-15T14:00:00
Description: Watch this Scientific Journal Video about A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection at JoVE.com

이 연구는 헤르메스 친목 올라요 Negm 타 냐 모나 고 노팅엄 소화 질환 센터와 NIHR 노팅엄 소화 질병 생물 의학 연구 센터에서 별도 자금을 통해 지원 했다.

1. 준비 Microarray 플레이트
<ol><li>(이 환자 세라를 실행 하기 전에 미리 정의 된) 최적 농도에서 인쇄 버퍼 [PBS-트윈-트 레 할 로스 (50mm)] C. 남과 어울리지 않는 항 원 희석: 독 소는 (200 µ g/mL) 및 독 소 B (100 µ g/mL), pCDTb (200 µ g/mL), 및 순화 된 네이티브 전체 SLPs ribotypes 001, 002, 027 (200 µ g/mL)에서 파생 된. 참고: 독 소 B 독 소 B 표현 스트레인 CCUG 20309 (90 µ g/mL)에서 얻은 것입니다.<ol>
<li>긍정적?...

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이 프로토콜에서 우리는 microarray C. 남과 어울리지 않 는 단백질 항 원 환자 세라 (그림 3 과 6)와 IVIg (그림 5)의 상업적인 준비에 체액 성 면역 응답을 정의 하기 위한 적합 한 플랫폼입니다 것으로 나타났습니다. 우리는 또한 microarray 기술은 기존의 ELISA (그림 2)에 비교 될 때 잘 수행 하 고 내부 및 남북 assay variabilities 정?...

이 프로토콜 개발 및 검출을 위한 소설과 맞춤형 단백질 microarray 분석 결과의 검증 및 세균성 긴장 및 C. 남과 어울리지 않는 항 원에 항 체 isotype-특정 응답의 반 정량화에 설명합니다. 우리는 성공적으로 우리의 C. 남과 어울리지 않는 사용-특정 항 체 환자 세라1,2, IVIg3, 준비에에서 콘텐츠 구성 bioanalysis 유망한 새로운 툴으로 서 분석 결과 특정 microarray 및 CDI4가난한 결과 연관 항 체 특이성의 식별. 시연 어떻게 biobanked 혈 청 샘플 및 IVIg의 상업적인 준비 microarray 슬라이드에 분석 될 수 있다 C. 남과 어울리지 않는 병원 체 특정 항 체 반응을이 분석 결과의 높은-품질 프로 파일링를 재현할 수 있도록 합니다.
많은 건강 한 아이 들 및 성인 있고 C. 남과 어울리지 않는 독 소 A에 감지 혈 청 IgG와 IgA 항 체 B5,6. 이러한 초기와 성인에서 C. 남과 어울리지 않 는 노출 다음 중 과도 노출 다음 발생 생각 된다. 이러한 이유로, polyclonal IVIg 되었습니다 재발 및 fulminant CDI7,,89치료에 오프 라벨을 사용. 그러나, 그것의 확실 한 역할 그리고 행동의 모드 불분명 남아 있습니다. 몇 가지 연구는 C. 남과 어울리지 않는 독 소를 체액 면역 반응에서 역할을 질병 프레 젠 테이 션 및 결과 나타났습니다. 특히, 무 증상 환자 증상 질병10을 개발 하는 환자에 비해는 증가 혈 청 항 독 소 A IgG 농도 표시. 명백한 협회 중간 항 독 소 A IgG titers 및 30 일 모든 원인 사망11보고 되었습니다. 여러 보고서 또한 독 소 A, B, 그리고 여러 비 독 소 항 (Cwp66, Cwp84, 플, FliD, 및 표면 층 단백질 (SLPs)) 재발과 항 체 응답에 대 한 보호와 연결을 계시 했다12,13 , 14 , 15. 이러한 관측을가 했다가 개발의 첫 번째 수동 immunotherapy 마약 대상 C. 남과 어울리지 않는 독 소 B (bezlotuxumab)는 최근 미국 식품 의약품 안전 청 및 유럽 의약품에 의해 승인 되었습니다 재발 CDI16의 예방을 위한 기관. 사 독 소 또는 재조합 독 소 조각을 사용 하 여 예방 접종 전략은 또한 현재 개발17,,1819에서. 이러한 새로운 치료 접근 의심할 여 지 없이 큰 샘플 크기에 여러 항 원에 체액 성 면역 반응 평가 대 한 요구를 자극할 것 이다.
오늘날, 세균성 긴장 및 C. 남과 어울리지 않는 항 원에 항 체 isotype-특정 응답을 동시에 평가 가능 상업적으로 사용 가능한 높은 처리량 분석 실험의 주목할 만한 부족이 있다. 미래 연구 및 임상 응용을 촉진 하기 위하여 이러한 분석을 개발 하는 충족 되지 필요가 있다. 단백질 microarrays는 현미경 슬라이드 기반 표면에 반점의 공간적 조직 배열로20 접촉 또는 비 접촉 될 수 있는 로봇 시스템을 사용 하 여 개별적으로 순화 된 단백질의 많은 수를 무력화 하는 방법 인쇄 도구21. 관광 명소는 세포 lysates, 항 체, 조직 homogenates, 내 인 성 또는 재조합 형 단백질 또는 펩 티 드, 체액 또는 약물22,23등 복잡 한 혼합물을 나타낼 수 있습니다.
단백질 microarray 기술 표준 사내 ELISA 뚜렷한 장점이 안티-C. 남과 어울리지 않는 항 체 응답을 평가 하기 위해 전통적으로 사용 된 기술을 제공 합니다. 이들은 다양 한 단백질 목표, 항 원, 예제 및 시 약에 대 한 요구 사항 감소 볼륨의 더 광범위 한 패널에 대 한 멀티-isotype-특정 항 체를 탐지 하기 위한 용량 증가 및 더 큰 통합 향상된 된 능력 기술 복제 뿐만 아니라 여러 내부 품질 관리 (QC)의 수1를 측정합니다. Microarrays는 따라서 더 정확한, 과민 하 고 재현할 수 있고 큰 동적 범위. 이러한 요인 microarrays는 저렴 하 고 잠재적으로 유리한 대안 ELISAs 알려진된 단백질의 대규모 검색에 대 한 확인합니다. 그러나, microarray 기술의 단점 매우 순화 된 항 원의 패널 구축 및 기술 플랫폼을 설정 관련 된 큰 초기 비용에서 주로 유래한 다.
단백질 microarrays 임상 응용 프로그램에 진단 및 기본 연구 공구로 지난 2 년간 광범위 하 게 사용 되었습니다. 단백질 발현 프로 파일링, 효소-기질 관계, 바이오 마커 검사, 호스트-미생물 상호작용의 분석의 연구 및 항 체 특이성23,24, 프로 파일링을 포함 하는 특정 응용 프로그램 25,26,,2728. 말라리아 (변형 체)29, 에이즈-130,31독감, 중증 급성 호흡기 증후군 (SARS)32, 바이러스 성 출혈 성 열33 포함 하 여 많은 새로운 병원 체 단백질/항 microarrays 설립 되었습니다. , herpesviruses34및 결핵35.
현재 프로토콜 쉬운 운영 C. 남과 어울리지 않 는 반응 항 원 microarray 분석 결과, 멀티 isotype 및 c.에 대 한 스트레인 특정 항 체 응답의 정밀, 정확 하 고 특정 정량화를 가능 하 게의 설립에 관한 남과 어울리지 않는 세라와 IVIg polyclonal 항 원. 여기, 우리는 프로필 ELISA 분석 결과 정밀도 및 재현성에 비해 허용 microarray 분석 결과 성능에 관한 대표적인 결과 포함 합니다. 이 분석 결과 다른 임상 샘플 프로 파일링에 더 개발 될 수 있습니다 고 CDI의 분자 기준으로 연구에 대 한 새로운 표준을 설정 합니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:1827px;" width="1826">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BioRobotics MicroGrid II arrayer</td>
			<td style="width:296px;">Digilab, Malborough, MA, USA</td>
			<td style="width:296px;">N/A</td>
			<td style="width:836px;">Contact arrayer used to automated spotting of the antigens onto the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanner InnoScan 710.</td>
			<td>Innopsys</td>
			<td>N/A</td>
			<td>A fluorescent microarray slide scanner with a red (Cy5) laser to read the reaction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAPIX software version 7.2.0</td>
			<td>Innopsys</td>
			<td>N/A</td>
			<td>Measure signal intensities of the spots.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon contact pin</td>
			<td>Parallel Synthesis Technologies</td>
			<td>SMT-P75</td>
			<td>Print the samples onto the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific Nalgene Desiccator</td>
			<td>Thermo Scientific</td>
			<td>41102426</td>
			<td>To store the new and printed slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384-well plate</td>
			<td>Genetix</td>
			<td>X7022UN</td>
			<td>To prepare the antigens.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate cover</td>
			<td>Sigma Aldrich, UK</td>
			<td>CLS6570-100EA</td>
			<td>To reduce evaporation of the samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminosilane slides</td>
			<td>Schott,&#160; Germany</td>
			<td>1064875</td>
			<td>The slide of choice for printing the antigens.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide holders</td>
			<td>GraceBio Labs, USA</td>
			<td>204862</td>
			<td>Divide the slides into identical 16 subarrays. These holders are re-usable, removable, leak-proof wells .&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Candida albicans lysate</td>
			<td>NIBSC</td>
			<td>PR-BA117-S</td>
			<td>Positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetanus Toxoid&#160;</td>
			<td>Athens Research and Technology</td>
			<td>04/150</td>
			<td>Positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G (IgG), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707</td>
			<td>Purified Native Human Immunoglobulin G IgG, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G1 (IgG1), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-1</td>
			<td>Purified Native Human Immunoglobulin G1 IgG1, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G2 (IgG2), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-2</td>
			<td>Purified Native Human Immunoglobulin G2 IgG2, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G3 (IgG3), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-3</td>
			<td>Purified Native Human Immunoglobulin G3 IgG3, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin G4 (IgG4), Normal Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090707-4</td>
			<td>Purified Native Human Immunoglobulin G4 IgG4, Human Plasma.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A (IgA), Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701</td>
			<td>Purified Native Human Immunoglobulin A (IgA), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A1 (IgA1), Human Myeloma Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701-1M</td>
			<td>Purified Native Human Immunoglobulin A1 (IgA1), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin A2 (IgA2), Human Myeloma Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090701-2M</td>
			<td>Purified Native Human Immunoglobulin A2 (IgA2), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunoglobulin M (IgM), Human Plasma</td>
			<td>&#160;Athens research and&#160; technology&#160;&#160;</td>
			<td>16-16-090713</td>
			<td>Purified Native Human Immunoglobulin M (IgM), Human Plasma.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated Goat Anti-Human IgG Antibody, gamma chain specific</td>
			<td>Vector Labs</td>
			<td>BA-3080</td>
			<td>Goat anti- human IgG (&#947;-chain specific)-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG1 Hinge-BIOT</td>
			<td>Southern Biotec</td>
			<td>9052-08&#160;</td>
			<td>Goat anti- human IgG1 biotin antibody reacts specifically with human IgG1 but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG2 Fc-BIOT</td>
			<td>Southern Biotec</td>
			<td>9060-08&#160;&#160;</td>
			<td>Goat anti- human IgG2 -biotin antibody reacts specifically with human IgG 2but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG3 Hinge-BIOT</td>
			<td>Southern Biotec</td>
			<td>9210-08&#160;&#160;&#160;</td>
			<td>Goat anti- human IgG3-biotin antibody reacts specifically with human IgG3 but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgG4 pFc&#39;-BIOT</td>
			<td>Southern Biotec</td>
			<td>9190-08&#160;&#160;</td>
			<td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Human IgA, alpha chain specific, made in goat - Biotinylated</td>
			<td>Vector Labs</td>
			<td>BA-3030</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgA1-BIOT</td>
			<td>Southern Biotec</td>
			<td>9130-08</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgA2-BIOT</td>
			<td>Southern Biotec</td>
			<td>9140-08&#160;&#160;</td>
			<td>Goat anti- human IgG -biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human IgM-BIOT</td>
			<td>Southern Biotec</td>
			<td>9020-08&#160;</td>
			<td>Goat anti- human IgG-biotin antibody reacts specifically with human IgG but not with other immunoglobulins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 mm syringe filter</td>
			<td>Thermo scientific</td>
			<td>723-2520</td>
			<td>Filter the 5% BSA.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich, UK</td>
			<td>A7284</td>
			<td>Use 5% BSA for blocking the slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody diluent</td>
			<td>Dako, UK</td>
			<td>S3022</td>
			<td>To dilute the serum and the secondary antibody.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Cy5</td>
			<td>eBioscience</td>
			<td>SA1011</td>
			<td>Detection of the immune reaction.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087)</td>
			<td>Toxins Group, Public Health England</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified whole C. difficile toxin B (CCUG 20309 toxin B only expressing strain)</td>
			<td>Toxins Group, Public Health England</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precursor form of B fragment of binary toxin, pCDTb</td>
			<td>University of Bath&#160;</td>
			<td>NA</td>
			<td>Produced in E. Coli from wholly synthetic recombinant gene construct. Amino acid sequence based on published sequence from 027 ribotype (http:www.uniprot.org/uniprot/A8DS70)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified native whole ribotype-specific (001, 002, 027) surface layer proteins</td>
			<td>Dublin City University&#160;</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vigam (IVIg preparation 1)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Privigen (IVIg preparation 2)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Intratect (IVIg preparation 3)</td>
			<td>Nottingham University Hospitals NHS Trust</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protein microarray assay for serological determination of antigen-specific antibody responses following clostridium difficile infection

우리Clostridium 남과 어울리지 않는 항 체 수준의 polyclonal 정 맥 면역 글로불린 (IVIg)의 별도 준비와 인간의 세라에서의 안티-결정에 대 한 소설 높은 처리량 단백질 microarray 분석 결과 대 한 자세한 개요를 제공합니다. 프로토콜 샘플 준비, 배열, 시험 절차, 및 데이터 분석의 인쇄에 관련 된 방법론 단계를 설명합니다. 또한,이 프로토콜 플라즈마를 포함 한 다양 한 임상 샘플을 통합 하 고 문화 supernatants 셀을 더 개발 될 수 있습니다. 우리는 단백질 microarray를 사용 하 여 isotype (IgG, IgA, IgM), 서브 클래스 (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2)의 조합을 결정 하는 방법을 보여 및 스트레인-특정 항 체를 매우 전체 C. 남과 어울리지 않 는 정화 독 소 A와 B (toxinotype 0, 스트레인 VPI 10463, ribotype 087), C. 남과 어울리지 않는 독 소 B만 표현 스트레인 (CCUG 20309)에서 독 소 B, 이진 독 소, pCDTb, ribotype 특정 전체 표면 층 단백질 B 조각의 선구자 형태 (SLPs; 001, 002, 027), 단백질 (파상풍을 제어 사망 toxoid 그리고 Candida albicans)입니다. 실험 기간 동안 microarrays 세라 C. 감염과 (CDI), 낭 성 섬유 증 (CF) 설사, 건강 한 컨트롤 (HC), 없이 개인으로 개인 및 개인 사전 및 포스트 IVIg 치료와 함께 조사 됩니다는 CDI, 결합 된 면역 결핍 장애 및 만성 염증 demyelinating polyradiculopathy의 치료. 우리 환자 단체, IVIg, 그리고 전에 세라의 상업적인 준비와 다음 IVIg 관리 사이 독 소 중립화 신진대사 및 멀티 isotype 특정 항 체 수준에 큰 차이가 발생합니다. 또한, 있다 microarray 및 효소 연결 된 immunosorbent 분석 결과 (ELISA) 사이의 중요 한 상관 관계 항 독 소 IgG 수준에 대 한 혈 청 샘플에서. 이러한 결과 microarray 예방 접종 또는 감염 된 인간 C.difficile 항 원에 항 체 응답을 프로 파일링 하기 위해 유망한 도구가 될 수 것이 좋습니다. 항 원 패널과 생산 비용에 있는 감소의 더 수정, 우리는 microarray 기술을 최적화 하 고 환자 별로 C. 감염과 대 한 가장 임상적으로 유용한 immunotherapies를 선택 도움이 될 수 있습니다 예상.

그림 1 설명된 프로토콜의 주요 단계를 설명 하는 순서도를 보여 줍니다. 그림 2 는 상당한 사이 계약 microarray ELISA IgG와 IgA 항 독 소 A와 B 환자 테스트 세라 수준을 보여주는 Spearman 상관 관계 테스트. 그림 3 설사 없이 CF 환자, 설사, CDI 환자와 HC에서 독 소 A, B, 독 소 및 이진 독 (pCDTb)에 대 한 특정 항 체 응?...

Watch this Scientific Journal Video about A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection at JoVE.com

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

이 문서 Clostridium 남과 어울리지 않는 항 원에 인간 세라 정화 7-플렉스 패널의 체액 면역 반응 높은 프로 파일링에 대 한 간단한 단백질 microarray 메서드를 설명 합니다. Polyclonal 정 맥 면역 글로불린의 준비에 특정 항 체 반응의 결정에 대 한 프로토콜을 확장할 수 있습니다.

항 원 특정 항 체 응답 다음 Clostridium 남과 어울리지 않 는 감염의 혈 청 학적인 결정에 대 한 단백질 Microarray 분석 결과

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

This method may help with optimizing and selecting clinically useful immunotherapies for Clostridium difficile infection in a patient-specific manner, and elucidating human immune responses to Clostridium difficile. The main advantage of this technique is that it enables accurate and simultaneous quantification of multi isotype and strain-specific antibody responses to C.difficile antigens. Generally, individuals that are new to this method will struggle because expertise is required in establishing and optimizing a panel of highly purified antigens, and setting up the technological platform.Visual demonstration of this method is critical, as methodological list types involved in preparing the microwave plate, arrays printing, a safe procedure, and the data analysis require attention to the details. Demonstrating some of the procedure will be by Sam Greenwood, a technician from my laboratory. To begin, dilute Clostridium difficile antigens and printing buffer to optimum concentration.Next, transfer 10 microliters of antigen solution to a 384 well plate. Cover the plate with a plate seal, and centrifuge at 300 times gravity for five minutes at room temperature. For microarray printing, use a hand-held gas burner to heat the silicon PIN three times for two seconds each.Then, rinse the silicon PIN in clean water three times for three seconds each. Place the microarray plate in the loading cartridge of an arrayer. Eject the tray, and then place the amino silane slides in the slide tray and press the OK button to turn the vacuum on.Insure that all of the slides are secured, and then click OK.Return the tray to the origin, and close the lid. Next, launch the appropriate program and go to File, then Microarraying Parameters, then Open. Select Clostridium difficile file for printing all antigens in quadruplicate.Under this window, open the Source tab to indicate sample storage, and Target tab to input microarray details. Then, open the Options tab to select the washing tool. Under the General tab of Run Preferences, on the TAS applications suite toolbar, select Wash at start of run, and Wash at end of run.Open the Climate tab, and set the humidity level between 55 to 60%Now start the run, and periodically check the arrayer to insure proper functioning. After printing, carefully place the printed slides in a slide holder with 16 multi-well chambers. Then add 100 microliters of 5%BSAT to each block.Cover the slides and incubate for one hour at room temperature with shaking. Use 120 microliters of PBST to wash each block five times for one minute each with shaking. Next, thaw out the serum samples obtained from C difficile infected patients and healthy controls on ice for 20 minutes.Add commercially available antibody diluent to the master block, and then add samples to the master block and mix. Discard the washing buffer and add 100 microliters of each diluted sample to the experimental blocks. For the negative control block, add 100 microliters of only the antibody diluent.Then, incubate the slides for one hour at room temperature with gentle shaking. Next, use 120 microliters of PBST to wash each block five times for three minutes each with shaking. Then add 100 microliters of biotinylated goat anti-Human antibody diluted in antibody diluent to each block.Cover the slides and incubate for one hour at room temperature with gentle shaking. Use 120 microliters of PBST to wash each block five times for three minutes each with shaking. Next, add 100 microliters of streptavidin SY5 conjugate diluted in 5%BSAT, in a ratio of one to two thousand to each block.Cover the slides with foil and incubate for 15 minutes at room temperature with gentle shaking. Wash the slides first with 120 microliters of PBST five times for one minute each, and then wash them two more times for one minute each with PBS. To dry the slides, spin them for five minutes at 300 times gravity.Turn the laser scanner on 30 minutes prior to scanning, to allow it to warm up. Then place the slide in the scanner with the printed surface facing up until the plate light turns solid green. Next, press the Settings button.On the first tab, insure that the Automatic scrolling and Barcode are unchecked. Then, set the Pixel size to 10, Scan Mode to Medium, Acquisition to 635 only, Acquisition Mode to Manual, gain to 20, and power to low. On the second tab, insure that the slide type is unlabeled.On the third tab, set the Focus setting to Auto focus. Press the import grid button to select the appropriate array list associated with the scanned image. Then, press the autofind button to align the grid to the spots on the slide.Finally, press the Quantification Process button to measure the fluorescence intensity of each spot. Then, save the results in an appropriate format for further analysis. In this protocol, a protein microarray is compared with ELISA to assess the reproducibility between these two techniques.The Spearman correlation coefficient analysis shows significant agreement between the microarray and ELISA for detecting IgG Anti-toxin A, and Anti-toxin B levels in serum samples. Intro assay and inter assay reproducibility of the protein micro array are calculated using the serum samples of seven patients. The samples analyzed on two different array slides against Toxin A, Toxin B, SLP001, SLP002, SLP027, Toxin B from Toxin B only producing strain CCUG, and pCDTb antigens reveal good reproducibility.A protein microarray is performed to detect an isotype-specific antibody response against Clostridium difficile toxins. The serum samples of patients and healthy individuals are used to detect the levels of IgG and IgA against Toxin A, Toxin B, Toxin B from a Clostridium difficile Toxin B only expressing strain, and precursor form of a B fragment of binary toxin. Once mastered, this technique can be done in seven hours.The quality control experiment, different parameters such as selection of appropriate surface chemistry, printing buffers, blocking buffers, and dilutions of serum samples and secondary antibodies should be evaluated before running samples. Printing a high-quality array requires a good understanding of the array or software to adjust the printing of parameters according to the experimental protocol. It is crucial to maintain optimum performance of the arrayer, and a dust-free environment throughout the experiment.This technique will pave the way for researchers in the field of immunology and molecular biology, in terms of characterizing humoral immune responses to infection, and discovering serodiagnostic antigens. Don't forget that working with infected blood samples and bacterial toxins can be hazardous. In general, in laboratory, safety and standard precautions should always be taken into consideration while performing this procedure.

Research

JoVE Journal

Immunology and Infection

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

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