A.C. was supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada and the Training Program in Regenerative Medicine (Canadian Institutes of Health Research). F.Y.Y.H. was supported by the Training Program in Regenerative Medicine. This work was funded by a grant from Astellas Pharma Canada. We also would like to thank Dr. Mark Menenghini (University of Toronto) for use of his Axon microarray scanner.

1. 희석 항원 및 생성 항원 마이크로 어레이 <ol><li> 0.2 mg / ml의 최종 농도로 PBS에 희석 항원. 9 핀과 미세 배열 구성과 중복에서 162까지 고유의 항원을 인쇄합니다. 항원 라이브러리로 양성 대조군에서의 IgG 및 IgM의 항원을 포함합니다. 단지 음성 대조군으로 PBS를 포함합니다. </li><li> 384 잘 소스 판에 각 항원의 20 μl를 추가합니다. 프린트 헤드의 설치 미러 그룹의 소스 플레이트에 항원을 추가 (9 핀 인쇄에 사용하는 경우 등, (9)의 그룹이 항원을 준비). </li><li> 배열을 인쇄 할 준비가 될 때까지 -80 ° C에서 박 및 동결와 커버 소스 판. </li><li> 3 × 1 분 동안 탈 이온수와 초음파 처리 조에서이를 배양하여 고체 미세 배열 핀을 청소합니다. 랙에 배치 핀은 건조 후 마이크로 어레이 프린트 헤드의 핀을 배치합니다. 9 핀, 3 × 3 구성을 사용합니다. </li><li> 프린트 헤드 번호 핀을 설정하여 실행 인쇄 프로그램 미세 배열,슬라이드의 수는 각 슬라이드에, 패드의 번호를 인쇄하고, 각 항원에 대한 복제 지점 수 있습니다. 일반적으로 9 핀, 70 슬라이드, 2 패드 / 슬라이드, 각 항원에 대한 2 복제 지점을 사용합니다. 프로그램 미세 배열은 항원의 다른 그룹 사이에 물에 핀을 초음파 처리합니다. </li><li> 해동 소스 플레이트와 100 × g에서 1 분 후 원심 분리 판. 미세 배열의 지정된 자리에 소스 판을 놓습니다. 인쇄 할 항원의 첫 번째 그룹을 덮고 박의 부분을 제거합니다. </li><li> Arrayer에 표면 및 실행 인쇄 프로그램에 인쇄되지 않은 슬라이드를 정렬합니다. 어레이 시스템의 가습기의 빌드 60 % - 습도 RT에서 인쇄 슬라이드 (55)에 Arrayer에에 설정합니다. </li><li> 항원의 각 그룹은 모든 슬라이드 상에 인쇄 된 후, 미세 배열을 일시. 다만 호일로 인쇄 된 표지 항원 (증발을 방지하기 위해) 및 항원의 다음 그룹에 인쇄 될 발견. 인쇄 프로그램을 계속합니다. </li><li> 모든 항원에 인쇄 한 후, 커버 소스 PL-80 ° C에서 박 및 동결의 새로운 조각을 먹었다. 슬라이드 상자와 진공 밀봉에 인쇄 된 슬라이드를 놓습니다. 슬라이드 다음날를 사용하거나 최대 한 달 후에 할 수있다. </li></ol> 2. 희석 혈청과 항원 마이크로 어레이를 프로빙 <ol><li> 배양 챔버를 사용하여 프레임으로 슬라이드를 놓습니다. 각 배열 표면에 차단 버퍼 (2.5 %의 [권 / 권] 태아 혈청 (FCS), PBS에 트윈 20 0.1 % [의 / 권 권])의 700 μl를 추가합니다. </li><li> 젖은 조직의 조각 밀폐 용기에 프레임과 장소를 통해 접착 필름을 배치합니다. 로커에 4 ° C에서 O / N을 품어. </li><li> 혈청 샘플 1을 희석 : (100) 버퍼를 차단에서. 대기음 배열에서 솔루션을 차단 및 각 배열 표면에 희석 한 시료 500 μl를 추가합니다. </li><li> 접착 필름으로 덮고 락으로 4 ° C에서 1 시간 동안 배양한다. </li><li> 버퍼를 차단에서 이차 항체를 희석. 인간 연구의 경우, 0.33 μg의 / ㎖ 및 Cy5에로 Cy3가 표지 된 염소 항 - 인간 IgG 항체가 희석 된 염소 항 - 구호 물자 표지0.25 μg의 / ㎖에 대한 IgM 항체 n은. 마우스 연구를 들면, Cy3에가 ㎖의 0.38 μg의 /에 염소에게 항 - 마우스 IgG 항체를 표지와 Cy5에가 / ㎖ 0.7 μg의에 염소 항 - 마우스 IgM의 항체를 표지 희석. </li><li> 어레이에서 대기음 샘플 및 배열 표면을 세척 완충액 (PBS 중 0.1 % 트윈 20)로 4 회 헹군다. 슬라이드에 버퍼 빠르게 떨어져 가볍게 붓는다. </li><li> 락과 RT에서 각 배열 표면을 씻어 10 분 부화 버퍼를 차단의 700 μl를 추가합니다. 반복 세척 단계를 두 번 더. </li><li> 각 슬라이드 표면에 희석 차 항체의 500 μl를 추가하고 접착 필름 커버. 락과 4 ° C에서 45 분을 품어. </li><li> 대기음 차 항체 슬라이드에서 혼합하고 위의 4 번 헹군다. 워시는 상기 차단 / 희석액 700 ㎕를 3 번​​ 슬라이드. </li><li> PBS에 침지 금속 슬라이드 랙 프레임과 장소에서 슬라이드를 제거합니다. 궤도 흔들림와 실온에서 20 분을 품어. PBS의 새로운 용기에 놓고 다른 20m를 품다진탕한다. </li><li> 탈 이온수 15 초 용기에 넣어 슬라이드 랙. 물이 새 컨테이너에 선반을 놓고 다른 15 초를 품어. </li><li> 건조 슬라이드, RT에서 5 분 220 XG에 원심 분리기와 스핀에서 ELISA 플레이트 어댑터에 장소 슬라이드 랙. </li><li> 스캔 할 준비가 될 때까지 빛을 꽉 상자에 슬라이드를 놓습니다. </li></ol> 3. 스캔 항원 마이크로 어레이 및 데이터 내보내기 <ol><li> 를 Cy3 및 Cy5의 형광 신호를 검출 할 수있는 마이크로 어레이 스캐너를 사용하여 스캔 슬라이드. 스캐너의 광전자 증 배관 (PMT) 레벨을 조정하기 위해, 단지 이차 항체로 프로빙 하였다 슬라이드 사전 스캔. 참고 :이 슬라이드 전체 양 (IgG에와 IgM의)를 포함하여 그것에 인쇄 항원 라이브러리뿐만 아니라 음 (PBS) 컨트롤이 있어야합니다. 프리 스캔 최적 PMT 설정을 찾기 위해 신속하게 사용자를 허용하는 낮은 해상도로 스캔한다. </li><li> PMT를 값을 설정되도록합니다 (Cy3에 채널에서) 인간 IgG 기능과 잡음합니다 (Cy5에 채널)을 IgM의 기능은 비슷한 평균 형광 강도를 뺀 배경 (MFI-B) (일반적으로 40000)가 있습니다. PMT를 레벨은 &quot;하드웨어&quot;버튼을 누름으로써 설정된다. 일단 설정되면, 일정이 PMT 설정을 유지. </li><li> 은 &quot;스캔&quot;버튼을 누름으로써 두 개의 채널에 대한 실험 슬라이드 스캔. 각 스캔 한 후 슬라이드 이미지 (두 채널 모두)를 저장합니다. </li><li> 슬라이드를로드하는 &quot;파일&quot;버튼을 사용하여 마이크로 어레이 소프트웨어로 분석된다. </li><li> &quot;파일&quot;버튼을 이용하여 유전자 배열 목록 (GAL) 파일을로드합니다. 는 GAL 파일 어레이 피처들의 신원을 가진 어레이의 레이아웃을 가진다. </li><li> 배열 기능을 최대한 가깝게 템플릿을 일치하도록 스캔 한 이미지를 통해 배열 템플릿을 놓습니다. </li><li> 은 &quot;정렬&quot;버튼을 눌러 템플릿으로 모든 블록의 기능을 맞 춥니 다. 프로그램은 일반적으로 원형의 기능의 개요를 확인할 수 있지만, 일부 수동 조정이 필요할 수있다. 이러한 조정 기능 모드를 입력 할 수있다. 소프트웨어는 각 항원에 대해 MFI-B를 계산한다. </li><li> 텍스트 파일로 결과를 내보내려면 &quot;파일&quot;버튼을 사용합니다. </li></ol> 마이크로 어레이의 의미 분석 4. 분석 배열 데이터 (SAM) <ol><li> 엑셀로 개별 텍스트 파일을로드하고를 Cy3와 Cy5에 채널 MFI-B가 열을 식별합니다. </li><li> 중복 기능의 평균을 계산합니다. </li><li> 어떤 부정적인 MFI-B의 경우, (10)와 음수 로그베이스 (2)를 계산하여 다음 100 원 MFI-B를 나누고하여 데이터를 변환 대체합니다. </li><li> 이전에 7 설명한 바와 같이 마이크로 어레이 (SAM)의 의미 분석과 로그 변환 된 데이터를 분석 할 수 있습니다. </li><li> 두 그룹 (예를 들어, 건강한 대조군 대 명), 하나의 그룹 라벨 「1」및 그룹을 이용하여 연구를위한 &quot;2&quot; 의있는 항원의 반응성을 식별하기 위해 SAM의 두 클래스 짝이 분석을 사용하여두 군간 ignificantly 다른 (Q 값 &lt;0.05). </li><li> 프리젠 테이션 8 이미지를 만들기 위해 클러스터링 및 열 맵 생성 소프트웨어를 사용합니다. </li></ol>

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The authors have nothing to disclose.

여기에 설명 된 프로토콜 항원 마이크로 어레이 기술을 이용하여자가 항체의 정량을 허용한다. 항원 마이크로 어레이는자가 항체 선별에 기존의 ELISA에 비해 여러 가지 장점을 제공한다. 우선, 핵산, 단백질, 펩티드, 및 세포 용 해물을 포함하는 다양한 항원에 따라서 다중화자가 항체 스크리닝을 허용 니트로 셀룰로스 - 코팅 된 슬라이드 상에 배치 될 수있다. 항원 나노 리터 각 슬라이드에 발견?...

Autoantibodies are present in many disease states and can often have direct pathogenic activity1. Identification of autoantibodies is important for diagnosis of certain diseases, for prognosis of disease outcome, and for the classification of patients who may benefit from specific therapies2. Autoantibodies are typically identified in patient serum using the ELISA technique; however, screening for multiple antigens with this technique is laborious and consumes a large quantity of patient sample. New technologies are therefore needed to profile autoantibodies on a larger scale.
The antigen microarray technique is a proteomic technology that allows autoantibodies to be profiled in a multiplex fashion3. In the first step of this process, an antigen library is arrayed onto a slide surface using a robotic microarrayer. The slides are probed with diluted serum and then fluorescent-labeled secondary antibodies are added. Antibody reactivities are visualized by scanning the slides with a microarray scanner and quantified by fluorescent intensities. Antigen microarrays offer multiple advantages over the ELISA technique in screening for autoantibodies: 1) they require only microliters of serum to profile autoantibodies to multiple antigens simultaneously, 2) they use antigen sparingly, as only nanoliters of antigen are spotted onto the arrays, 3) they have enhanced sensitivity3 compared to ELISA and 4) they allow for the simultaneous yet, separate detection of more than one antibody isotype. Antigen microarrays have been used to profile autoantibodies in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus4-6. In all three of these diseases, new insight into disease pathogenesis was obtained from profiling autoantibodies on a large scale with the arrays. 
Here we describe a protocol to generate antigen microarrays using nitrocellulose-coated slides. A variety of antigens including proteins, peptides, and cell lysates can be arrayed onto the slides using this technique. The arrays can be easily customized by including antigens of interest in the source plate that holds the antigen library. In addition, we show how a pair of secondary antibodies can be used to separate IgG and IgM reactivities on the same slide surface. We have now optimized this technique to measure autoantibodies in both humans and mice.

<table><tbody><tr><td>Ribosomal P0</td><td>Diarect</td><td>14100</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>human IgG</td><td>Jackson Immuno</td><td>009-000-003</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>human IgM</td><td>Jackson Immuno</td><td>009-000-012</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>mouse IgG</td><td>Sigma-Aldrich</td><td>I5381</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>mouse IgM</td><td>Biolegend</td><td>401601</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>double-stranded DNA</td><td>Sigma-Aldrich</td><td>D1626</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>single-stranded DNA</td><td>Sigma-Aldrich</td><td>D8899</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>microarrayer</td><td>Virtek</td><td>VersArray Chipwriter Pro</td><td>many types of arrayers are suitable</td></tr><tr><td>solid printing pins</td><td>Arrayit Corporation</td><td>SSP015</td><td></td></tr><tr><td>software for robotic microarrayer</td><td>Virtek</td><td>Chipwriter Pro&amp;nbsp;</td><td></td></tr><tr><td>FAST slides (2 Pad)</td><td>GVS Northa America</td><td>10485317</td><td></td></tr><tr><td>FAST frame</td><td>GVS Northa America</td><td>10486001</td><td></td></tr><tr><td>FAST incubation chambers (2 Pad)</td><td>GVS Northa America</td><td>10486242</td><td></td></tr><tr><td>384 well plates</td><td>Whatman</td><td>7701-5101</td><td></td></tr><tr><td>plate sealers</td><td>VWR</td><td>60941-062</td><td></td></tr><tr><td>foil plate covers</td><td>VWR</td><td>60941-124</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Fetal calf serum</td><td>Invitrogen</td><td>12483020</td><td></td></tr><tr><td>Cy3 goat anti-human IgG</td><td>Jackson Immuno</td><td>109-165-096</td><td>use working stock in 50% glyercol</td></tr><tr><td>Cy5 goat anti-human IgM</td><td>Jackson Immuno</td><td>109-175-129</td><td>use working stock in 50% glyercol</td></tr><tr><td>Cy3 goat anti-mouse IgG</td><td>Jackson Immuno</td><td>115-165-071</td><td>use working stock in 50% glyercol</td></tr><tr><td>Cy5 goat anti-mouse IgM</td><td>Jackson Immuno</td><td>115-175-075</td><td>use working stock in 50% glyercol</td></tr><tr><td>Microarray Scanner</td><td>Molecular Devices</td><td>Axon 4200A</td><td></td></tr><tr><td>Microarray software</td><td>Molecular Devices</td><td>Genepix 6.1</td><td></td></tr><tr><td>Clustering software</td><td>eisenlab.org</td><td>Cluster 3.0</td><td></td></tr><tr><td>Heatmap software</td><td>eisenlab.org</td><td>Treeview 1.60</td><td></td></tr><tr><td>Microarray statistical software</td><td>Stanford University</td><td>SAM 4.0 (Significance Analysis of Microarrays)</td><td></td></tr></tbody></table>

generation of two-color antigen microarrays for the simultaneous detection of igg and igm autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

도 1에 나타낸 바와 같이 항원 로봇 미세 배열로 슬라이드 상에 384 웰 플레이트에 배치되어 인쇄된다. 2는 배양 챔버와 처리 후의 스캔 슬라이드 프레임에 배치 된 슬라이드를 나타낸다. (3) 양성 및 음성 대조군 슬라이드를 나타낸다. 네가티브 슬라이드은 이차 항체로 프로빙하고, 양성 대조군 슬라이드 전신?...

Watch this Scientific Journal Video about Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies at JoVE.com

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

의 IgG 및 IgM의자가 항체의 동시 검출을위한 2 색 항원 마이크로 어레이의 생성

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

generation-two-color-antigen-microarrays-for-simultaneous-detection

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.7K 조회수.  University Health Network. We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest. 

비디오: Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

교차 reactivity 무료 및 정찰 병 무료 스냅 칩 샌드위치 Immunoassays 다중화

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

연구

생체공학

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Monospecific 및 소설 형 간염-2 엘리트/DFS70 녹아웃 기판으로 아나 심사 중 혼합된 DFS70 패턴의 동시 구별

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...

면역학 및 감염병학

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

인간의 섬 신경에 대 한 Electrochemiluminescence 분석 실험

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

바이러스 특수형에 걸쳐 혈청 항체의 폭을 측정하기 위해 인플루엔자 항원 마이크로어레이의 사용

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

혈청 내 Borrelia 특이적 IgG 및 IgM에 대한 다중 면역분석

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

저자 스포트라이트: Extending the Scope of Multiplex Immunoassay for Lyme Borreliosis Diagnostics and Pathogen Research (라임 보렐리오증 진단 및 병원체 연구를 위한 멀티플렉스 면역분석의 범위 확장) 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

다중 혈청학적 검사에서 다양한 항체 등급의 동시 검출

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

고처리량 전기화학발광 7-Plex 분석으로 제1형 당뇨병 및 다발성 자가면역질환 동시 스크리닝

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

의학

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Source: Olivera-Ardid, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57662">Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates printed glycan array technology for analyzing circulating anti-glycan antibodies in mouse serum. The antibodies in the serum interact with specific glycans on the chip, which is confirmed by immunostaining followed by fluorescence scanning.

의 IgG 및 IgM의자가 항체의 동시 검출을위한 2 색 항원 마이크로 어레이의 생성

요약

더 많은 비디오 탐색

이 비디오의 챕터

교차 reactivity 무료 및 정찰 병 무료 스냅 칩 샌드위치 Immunoassays 다중화

Monospecific 및 소설 형 간염-2 엘리트/DFS70 녹아웃 기판으로 아나 심사 중 혼합된 DFS70 패턴의 동시 구별

인간의 섬 신경에 대 한 Electrochemiluminescence 분석 실험

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

바이러스 특수형에 걸쳐 혈청 항체의 폭을 측정하기 위해 인플루엔자 항원 마이크로어레이의 사용

다중 혈청학적 검사에서 다양한 항체 등급의 동시 검출

다중 혈청학적 검사에서 다양한 항체 등급의 동시 검출

다중 혈청학적 검사에서 다양한 항체 등급의 동시 검출

고처리량 전기화학발광 7-Plex 분석으로 제1형 당뇨병 및 다발성 자가면역질환 동시 스크리닝

Printed Glycan Array를 사용한 순환 항글라이칸 항체 검출