이 작품은 오스트리아 과학 기금 (FWF) 부여 P18820 - B13에 의해 투자되었다. 윤리 성명 : 연구는 1983 년에 개정하여 참여 모든 환자들이 서면 동의 준 정보로 헬싱키 Declaraction 준수 잘츠부르크 대학에서 인간 및 / 또는 동물에 관련된 실험 윤리위원회 (PLUS_Ethik_090514) 승인되었습니다.

1. 약물 conjugates의 준비 <ol><li> 1.5 ML 반응 관에서 10 MG DF 1 ML DH 2 O의를 해산. 더 이상 진행이 DF 솔루션의 9​​5 μl를 사용합니다. 로 활용하기 위해 PP HSA 500 μl 0.3 M의 수산화 나트륨 (NaOH)의 30.4 MG PP 유도체 (304g / 몰)를 해산. </li><li> (N - morpholino) ethanesulfonic 산성 (MES) 버퍼 산도 해산 DF의 6.5-95 μl - 430.6 μl DH 2 O 100 μl 0.5 M 2를 추가합니다. 33.1 μl DH 2 O와 PP 예제 2 M MES 버퍼 산도 2.9 140 μl를 추가합니다. </li><li> 신선한 N - 에틸 - N &#39;의 57.5 μl를 추가 - (3 - 디메틸 아미노) MG / ML DF 샘플 100의 농도, 또는 10 μl EDC 솔루션과 함께 DH 2 O에 녹아있는 carbodiimide 하이드로 클로라이드 (EDC) 재고 솔루션을 PP 샘플. 소용돌이 1 분 샘플. </li><li> 각 샘플에 118 MG / ML의 농도와 HSA 솔루션의 16.9 μl를 추가합니다. </li><li> 300 라운드 / 분 동안 흔들어 상온에서 2 시간 동안 샘플을 품어. </li><li> 몇 시간마다 인산 버퍼 식염수 (PBS) 산도 7.4 2 리터에 대항하는 DF와 PP 샘플 3 번 Dialyze. 4 ° C.에있는 모든 dialyzing 단계를 수행하여 </li><li> 14,000 XG에서 샘플 10 분 원심하여 뜨는 포함된 DF 또는 HSA에 복합 PP를 수집합니다. </li><li> 12 % 젤을 사용하여 SDS - PAGE를 수행하여 단백질을위한 뜨는을 확인합니다. 한 겔 슬롯에 HSA 공액 12 μg에 대한로드합니다. </li></ol> 2. 약물 conjugates의 결합 비율 (그림 2)를 결정 <ol><li> HPSEC에 의해 DF와 PP conjugates의 결합 률의 분석 150 MM NaCl 및 0.05 % 나트륨 azide를 포함한 100 MM 나트륨 인산염 버퍼 산도 6.5를 사용합니다. 쯧 - 겔 - G2000 SWXL 칼럼 mm 7.8 X 300을 사용합니다. </li><li> 온라인 결합 RI 및 UV 검출기 시스템을 사용하여 신호의 감지를 수행합니다. </li><li> DH 2 O. 1 MG / ML의 농도와 HSA 표준 샘플을 준비 검출기 보정 50 μl HSA 표준 솔루션을 주입. </li><li> RI 상수 K의 RI 및 검출기에 대한 UV 상수 K의 UV를 계산합니다. 수식 영역 RI를 사용 = K의 RI * [(DN / DC) HSA * C (HSA)] 지역 UV = K UV * [(DA / DC) HSA * C (HSA)]와 함께 (DN / DC) HSA = 0.185과 (DA / DC) HSA = 0.518 5. </li><li> DF 두 표준의 5, 10, 30 nmol 금액이 쉽게 주입 수 HPSEC에 대한 PP 파생 표준을 준비합니다. </li><li> 약물과 두 표준 (DA / DC) 약물 (DN / DC)의 값을 의미 계산합니다. DF, (DN / DC) 0.235의 39.000의 ± 0.010과 (DA / DC) ± 0.493 결정한다고 들었습니다. PP 들어, 0.328 ± 0.016의 (DN / DC)와 (DA / DC) 53.155 ± 2.464의 대부분이 결정되었다. </li><li> HPSEC에 약물 conjugates를 주사하고 RI 및 UV 피크 영역을 결정합니다. </li><li> = K의 RI * [(DN / DC) 약물 * C (약) + (DN / DC) HSA * C (HSA)] 지역의 UV 두 방정식 영역 RI를 해결하여 DF, PP 유도체 및 HSA의 농도를 계산 = K UV * [(DA / DC) 약물 * C (약) + (DA / DC) HSA * C (HSA)] 신원 미상하십시오. </li></ol> 3. 약물 conjugates를 사용하여 BAT <ol><li> 일곱 유동세포계측법의 튜브를 준비하고 자신의 콘텐츠에 따라 그들을 분류 : 흠없는 제어, 음성 제어, 긍정적인 제어 및 약물 conjugates 있습니다. </li><li> 피펫 흠없는과 부정적인 제어를위한 소유 유리병에 B - CCR - STB의 자극 버퍼 (Flow2 CAST, Bühlmann 연구소, Schönenbuch, CH) 50 μl. 긍정적인 컨트롤로 Flow2 CAST 키트에서 제공하는 안티 FcεRI 솔루션의 50 μl를 사용합니다. 0.02 -20 μg / ML DF 활용 및 PP 활용 (200 μl의 분석 볼륨의 최종 농도)에서 1시 10분 희석 계열을 만들어 마약 conjugates을 위해 예약된 유리병에 활용 솔루션의 적절한 금액을 추가합니다. 이러한 적정 실험은 각 환자에 대한 최적의 샘플 basophil 활성화의 농도를 결정하기 위해 수행되어야합니다. </li><li> 각 튜브 50 μl 환자의 EDTA (에틸렌 다이아 민 테트라 초산) 전체 혈액 100 μl 자극 버퍼를 추가합니다. 신중하게 샘플을 섞는다. </li><li> 피펫 20 μl Flow2 캐스트 얼룩의 phycoerythrin (PE)와 각 튜브에 플루오레신 isothiocyanate (FITC)로 표시 방지 CD63 항체로 분류 안티 CCR3 항체를 포함하는 시약 다시 섞는다. 흠없는 샘플에 얼룩 시약을 피펫하지 마! </li><li> 37 ° C의 물을 욕조에 45 분 동안 샘플을 품어. </li><li> 5 분 얼음에 반응을 중지합니다. </li><li> 부드럽게 각 유리병과 와동 2.0 ML Flow2 주조 lysing 시약을 추가하여 적혈구를 Lyse. 상온에서 10 분 동안 어둠 속에 샘플을 품어. </li><li> 5 500 XG 분만에 신중하게 가만히 따르다 뜨는에 대한 샘플을 원심 분리기. </li><li> 유동세포계측법까지 얼음에 500 μl Flow2 CAST 세척 버퍼와 저장소에 세포를 Resuspend. </li><li> 흐름 cytometer의 전압 s을 (를) 조정앞으로 측면 산란 (FSC, SSC)에 ettings 흠없는 샘플을 인수하면서. </li><li> 게이트 세포는 SSC - PE의 음모에 SSC - FSC의 플롯에서 lymphocytes로 예측했다. Basophils는 CCR3 안녕 SSC ro와 게이트들을 FITC - PE의 음모로로 PE - 라벨 안티 CCR3 항체에 의해 식별됩니다. 활성 basophils을 감지 안티 CD63 항체는 FITC와 함께 표시됩니다. </li><li> 부정적인 컨트롤에 CD63 하이로 basophils의 최대 절단 2.5 %를 설정합니다. &gt; 5 % basophils는 CD63 안녕과 대조군의 MFI 나눈 샘플의 평균 형광 지수 (MFI) 2을 초과하는 (그림 3)하는 경우 샘플 basophil 활성화를위한 긍정적인 간주됩니다. </li></ol> 4. 대표 결과 : 이 실험은 IGE - 의존 약물 과민성을 감지하는 유익한 도구로 방망이를 평가합니다. NSAIDs의 단백질 conjugates은 basophils의 활성화를 위해 사용됩니다. HPSEC함으로써 RI 및 UV 감지 집합 상태, 결합도, 그리고 conjugates의 효과적인 수율로 결정됩니다. 그림 4 보여주는 바와 같이, DF의 conjugates의 43.5 %가 5.0 DF / HSA의 결합 학위 monomeric 있었다. 집계를위한 9.5의 결합 정도가 결정되었습니다. propyphenazone 활용은 21.2의 결합 학위 68.5 %의 단량체로 구성되어에 표시되었다. 집계의 결합 정도는 27.9했다. 총, DF의 conjugates의 결정 커플링 학위 7.6 DF와 PP의 conjugates의가 23.2이었습니다. BAT는 NSAID 과민성에 IGE 종속 반응 조사를 허용 최적의 조건 (conjugates의 농도, 자극 시간)에 따라 conjugates와 함께 수행했습니다. basophils의 20.6 %가 활성화되었다 형광 강도 로그 이동 특징 : 그림 5에서와 같이 단지 PP 활용은 basophil 활성화 CD63 표면 표현의 upregulation에 의해 시각 트리거 수있었습니다. MFI는 1893-386 (대조군)에서 4.9의 요소에 의해 증가했습니다. 대조적으로, DF 공액 사용 basophils만이 3.1 %가 5% 차단 아래어진 활성화되었습니다. 또한 MFI는 210-197 (대조군)에서 요소 1.1 (한도 2.0)에 의해서만 증가했습니다. 검정 유효 기간에 의해 주어집니다은 (i) &lt;부정적인 컨트롤의 5 % basophil 활성화, basophil subpopulation의 형광 강도 (2) 로그인 변화, 그리고 (iii)&gt; 50 % 활성 basophils (긍정적인 제어). <img alt="그림 1" src="/files/ftp_upload/3263/3263fig1.jpg" /> 그림 1. HSA에 DF와 PP 파생의 커플링. 해소 후 약품, 물, MES 버퍼 및 EDC (커플링 시약)가 추가됩니다. HSA는 마약 솔루션 pipetted되기 전에 샘플은 1 분 vortexed 있습니다. 상온에서 흔들어 2 시간 이내에 약물 covalently HSA에 바인딩. 단량체뿐만 아니라 집계가 발생할 수 있습니다. <img alt="그림 2" src="/files/ftp_upload/3263/3263fig2.jpg" /> 그림 2. 커플링 도의 계산. 첫째, RI 및 K의 UV K 상수가 결정됩니다. 따라서, 알려진 농도와 HSA 표준 HPSEC 시스템에 주입합니다. (DN / DC) 및 HSA의 (DA / DC)은 각각 5, 0.185과 0.518의 가치를 부여하고 있습니다. 정상 지역은 상수를 계산하는 데 사용됩니다. 둘째, 각 약물 세 기준은 약물과 집중 특정 RI 및 UV 영역을 결정하기 위해 주입하고 있습니다. K의 RI 및 K의 UV 상수가 위의 단계에서 알려져 있으므로, 약물 관련 (DN / DC) 및 (DA / DC) 파생 상품을 계산하실 수 있습니다. 셋째, 약물의 HSA conjugates는 HPSEC로 주입됩니다. 그 결과 피크 영역은 두 개의 알 수없는 변수로 두 개의 방정식을 해결하여 최대마다 약물 및 HSA의 농도를 계산하는 데 사용됩니다. <img alt="그림 3" src="/files/ftp_upload/3263/3263fig3.jpg" /> 그림 3. Basophil의 게이팅. lymphocytes 앞으로 뿌리 대 게이트 사이드 분산형 (SSC - FSC 줄거리)입니다 같은 세포는 예측했다. basophils는 문이 lymphocytes (SSC - CCR3 - PE의 줄거리)에서 CCR3 안녕 SSC 이오으로 식별됩니다. Basophils는 CD63 - FITC - CCR3 - PE의 음모에 활성화 (CD63 하이)에 대한 분석입니다. 대조군은 CD63 안녕을 남아있는 최대 2.5 %의 basophils의 차단을 설정하는 데 사용됩니다. <img alt="그림 4" src="/files/ftp_upload/3263/3263fig4.jpg" /> 그림 4. 약물 conjugates의 결정 커플링도. RI 및 UV 신호는 약물의 HSA conjugates의 집계 (낮은 유지 볼륨에서 eluting)와 단량체를 (높은 유지 음량 eluting) 대표이 봉우리를 보여줍니다. 단량체 및 집합체의 비율 (RI 신호에서 결정)과 커플링 학위는 (N = 1)을 묘사하고 있습니다. <img alt="그림 5" src="/files/ftp_upload/3263/3263fig5.jpg" /> 그림 5. Basophil activati시험. 약물 활용 - 활성화된 샘플, 부정적인 통제, 그리고 긍정적인 컨트롤의 결과는 CD63 - FITC - CCR3 - PE의 플롯 (N = 1)에 표시됩니다.

<ol>
	<li>Himly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgE-mediated+immediate-type+hypersensitivity+to+the+pyrazolone+drug+propyphenazone.">IgE-mediated immediate-type hypersensitivity to the pyrazolone drug propyphenazone.</a> J Allergy Clin Immunol. 111, 882-888 (2003).</li><li>Demoly, P., Pichler, W., Pirmohamed, M., Romano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+questions+in+Allergy:+1--drug+allergy/hypersensitivity.">Important questions in Allergy: 1--drug allergy/hypersensitivity.</a> Allergy. 63, 616-619 (2008).</li><li>Ebo, D. G., Hagendorens, M. M., Bridts, C. H., Schuerwegh, A. J., Clerck, L. S., Stevens, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+in+vitro+activated+basophils,+specific+IgE+and+skin+tests+in+the+diagnosis+of+pollen-associated+food+allergy.">Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy.</a> Cytometry Part B (Clinical Cytometry). 64B, (2005).</li><li>Harrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diclofenac+hypersensitivity:+antibody+responses+to+the+parent+drug+and+relevant+metabolites.">Diclofenac hypersensitivity: antibody responses to the parent drug and relevant metabolites.</a> PLoS One. 5, e13707-e13707 (2010).</li><li>Wen, J., Arakawa, T., Philo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-exclusion+chromatography+with+on-line+light-scattering,+absorbance,+and+refractive+index+detectors+for+studying+proteins+and+their+interactions.">Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.</a> Anal Biochem. 240, 155-166 (1996).</li><li>De Weck, A. L., Sanz, M. L., Gamboa, P. M., Aberer, W., Bienvenu, J., Blanca, M., Demoly, P., Ebo, D. G., Mayorga, L., Monneret, G., Sainte Laudy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tests+based+on+human+basophils:+more+potentials+and+perspectives+than+pitfalls.+II.+Technical+issues.">Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. Technical issues.</a> J Investig Allergol Clin Immunol. 18, 143-155 (2008).</li><li>Ebo, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-assisted+allergy+diagnosis:+current+applications+and+future+perspectives.">Flow-assisted allergy diagnosis: current applications and future perspectives.</a> Allergy. 61, 1028-1039 (2006).</li><li>Poulsen, L. K., Quan, S., Kragh, C., Platzer, M. H., Skov, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basophil+granulocyte+in+allergic+reactions:+experimental+models+and+their+use+for+the+identification+of+drugs+with+effects+or+side+effects+on+basophils.">The basophil granulocyte in allergic reactions: experimental models and their use for the identification of drugs with effects or side effects on basophils.</a> Curr Med Chem - Anti-inflammatory &#38; Anti-Allergy Agents. 3, 167-180 (2004).</li><li>Sanz, M. L., Gamboa, P., de Weck, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+combined+test+with+flowcytometric+basophil+activation+and+determination+of+sulfidoleukotrienes+is+useful+for+in+vitro+diagnosis+of+hypersensitivity+to+aspirin+and+other+nonsteroidal+anti-inflammatory+drugs.">A new combined test with flowcytometric basophil activation and determination of sulfidoleukotrienes is useful for in vitro diagnosis of hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs.</a> Int Arch Allergy Immunol. 136, 58-72 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

BAT는 잘 설립된 아직 정기적으로 IGE - 중재 알레르기 질환 6,7의 진단 방법을 사용하지만. 낮은 분자량 화합물이 crosslink IGE 수용체, basophil 활성화 8 필수 수 없습니다와 같은 약물에 대한 과민성, 그러나, 그 적용 손실이 크다. 따라서, 조사중인 의약품 covalently 적합한 캐리어 단백질 (예 : HSA)에 결합해야합니다. 중요한 것은, 커플링 학위 (캐리어 단백질마다 약물 분자 즉, 숫자) conjugates의 (예 : IGE 수용체 상호 연결) 면역 활동을 보장하기 위해 제어해야합니다. 이론적으로, 캐리어 분자 당 두 haptens는 IGE 수용체에 대한 충분해야 교차 연결 및 HSA 당 다섯 DF 분자처럼 몇 가지는 세포 기반 분석 4 중재자 릴리스를 실행하는 표시되었습니다. 두 conjugates, PP - HSA 및 DF - HSA는 충분히 높은 커플링 학위 (HPSEC)을 표시하고 BAT에서 사용하기에 적합 시약 만들기 immunologically 적극적인 것으로 결정되었습니다. 또 다른 문제는 대신 부모 약물의 대사로 과민성 반응을 일으킬 수, 마약 metabolites는 역할을 수 있습니다. DF의 경우,이 가능성은 이전에 5 개의 주요 단계 전 metabolites 하나의 결합의 변형 4를 사용하여 상세하게 평가되었습니다. 마스터 설명한 기법을위한 중요한 요소는 혈액 샘플 (&lt;혈액 수집, 검색 basophils의 수를 이후 12시간&gt; 500)와 자극 조건 최적화 (시간, 투약)의 품질을 포함합니다. 또한,도 긍정적인 컨트롤 (항체는 IGE 수용체에 대한 감독) 식별해야 할과 반응하지 않은 조치를 소위. 따라서, 검증 기준은 긍정적인 제어로 안티 FcεRI 항체를 포함해야합니다. 약물 conjugates를 사용하는 중요한 이점은 그들은 마치 BAT 4 반 FcεRI 항체와 공동 자극에 의해 DF - HSA 활용에 표시되는 순수한 약물과는 달리 무독성 표시는 사실이다. 순수 DF는 반면, BAT 9 interpretability 문제를 일으키는 1.25 MG / ML의 농도에서 세포 독성 효과를 표시합니다. 일반적으로, 잠재적인 세포 독성에 대한 테스트를 강력하게 모든 새로 생산된 마약을 활용하는 것이 좋습니다. 여기, 우리는 IGE - 중재 과민성을 나타내기 위해 BAT에 사용되는 PP - HSA 활용의 잠재력을 보여주었다. 대조적으로, DF 과민성는 DF - HSA 활용에 대한 응답으로 basophil 활성화의 부족에 의해 입증 IGE와 관련되지 않습니다. 중요한 것은 IGE - 중재 메커니즘에 관한 불확실성의 경우, conjugates는 DF 4 보여왔다로 체외 세포 기반 분석 시스템에 의해 면역 활동을 평가해야합니다. 적당한 단백질 회사에 covalently 낮은 분자량의 약물을 결합의 설명 설정을 사용하면 항생제를 포함한 약물, 다른 NSAIDs, radiocontrast 미디어, 근육 relaxants, anesthetics에 대한 과민성 반응의 숫자를 분자 등 IGE - 중재의 참여에 대한 조사 수 있습니다 메커니즘. 따라서, BAT는 진단 (피부 반응 검사, 구강 도발 테스트)에 대한 기존 방법에 추가 될 수있다.

<table border="1"><tbody><tr><th> 시약 / 기기의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 2 - (N - Morpholino) ethanesulfonic 산성 </td><td> 시그마 </td><td> M3671 </td><td> - </td></tr><tr><td> 7.8 X 300mm가 젤 쯧 - G2000SWXL 칼럼 </td><td> Bioscience Tosoh </td><td> 08,540 </td><td> - </td></tr><tr><td> BD FACSCanto II </td><td> Becton 디킨슨 </td><td> 338962 </td><td> - </td></tr><tr><td> 벤치 최고 원심 분리기 </td><td> 시그마 </td><td> - </td><td> - </td></tr><tr><td> Diclofenac 나트륨 소금 </td><td> 시그마 </td><td> D6899 </td><td> - </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) - Vacutainer </td><td> Becton 디킨슨 </td><td> 368589 </td><td> - </td></tr><tr><td> Flow2 CAST 키트 </td><td> Bühlmann 연구소 </td><td> FK - CCR </td><td> 효과적인 2011년 6월 14일 Flow2 CAST 키트는 현재 흐름 CAST입니다 </td></tr><tr><td> HP1100 HPLC 시스템 </td><td> 휴렛 팩커드 </td><td> - </td><td> - </td></tr><tr><td> 인간 혈청 알부민 </td><td> 시그마 </td><td> A9511 </td><td> - </td></tr><tr><td> 실험실 원심 분리기 </td><td> Eppendorf </td><td> - </td><td> - </td></tr><tr><td> N - 에틸 - N&#39; - (3 - 디메틸 아미노) carbodiimide 하이드로 클로라이드 (EDC) </td><td> 시그마 </td><td> E6383 </td><td> - </td></tr><tr><td> PBS의 정제 </td><td> AppliChem </td><td> A9199 </td><td> - </td></tr><tr><td> Propyphenazone 유도체 </td><td> 분자 생물학의학과, 잘츠부르크 대학, 오스트리아 </td><td> - </td><td> - </td></tr><tr><td> 셰이커 </td><td> Eppendorf </td><td> - </td><td> - </td></tr><tr><td> 나트륨 azide </td><td> 시그마 </td><td> S8032 </td><td> - </td></tr><tr><td> 나트륨 염화물 (NaCl) </td><td> 시그마 </td><td> S1679 </td><td> - </td></tr><tr><td> 나트륨 탄산수 소염 </td><td> 시그마 </td><td> 90421C </td><td> - </td></tr><tr><td> 나트륨은 hydroxid </td><td> 시그마 </td><td> S5881 </td><td> - </td></tr><tr><td> 인산 나트륨 </td><td> 시그마 </td><td> 342483 </td><td> - </td></tr><tr><td> 트리플 검출기 어레이 </td><td> Viscotek </td><td> TDA302 </td><td> - </td></tr><tr><td> 개인 BioVortex V - 1 플러스 </td><td> Peqlab </td><td> 90 V - 1 </td><td> - </td></tr><tr><td> 물 목욕 1,012 </td><td> GFL </td><td> 1012 </td><td> - </td></tr></tbody></table>

basophil activation test for investigation of ige-mediated mechanisms in drug hypersensitivity

propyphenazone (PP)와 diclofenac (DF)와 같은 비 steroidal 항염증성 약물 (NSAIDs)에 대한 과민성 반응은 알레르기 반응 하나 I - 같은 유형 매니 페스트 수 있습니다. 피부 테스트는 신뢰성이되지 않고 구두 도발 시험은 환자 2 생명을 위협하는 위험을 저승으로 임상 연습에서 약물 과민성의 진단은 주로 환자의 역사에 의해 수행됩니다. 따라서, 기본 IGE - 중재 pathomechanism에 대한 증거는 얻을 수 어렵습니다. 여기, 우리는 생체내에 알레르기 반응을 모방 이펙터 약물 과민 환자에서 파생된 인간 basophils의 사용에 따라 체외 방식에 제시. 약물 알레르기 환자의 basophils는 범인의 약물에 대한 IGE 분자가 특정 휴대으로, 그들은 IGE 수용체 crosslinking에 활성화되고 알레르기 이펙터 분자를 릴리스된다. basophils의 활성화는 유동세포계측법 3을 사용 CD63 표면 표현의 upregulation의 결정에 의해 모니터할 수 있습니다. 낮은 분자량의 약물의 경우, conjugates은 basophils에서 IGE 수용체 crosslinking을 사용하도록 설계되었습니다. 그림 1, NSAIDs의 두 대표에 묘사된, PP와 DF는 covalently 리신 잔류물의 기본 아미노 그룹과 반응 카르 복실 그룹을 통해 (HSA) 인간 혈청 알부민에 바인딩됩니다. 카르 복실 그룹이 포함된 PP의 파생은 organochemically 사전 연구 1 합성이 분명해 반면, DF는, 따라서 직접 사 사용할 수있는 고유 카르 복실 그룹과를 수행합니다. 단백질 캐리어 분자에서 낮은 분자량 화합물과 그 공간적 분포의 결합 정도는 두 IGE 수용체 분자의 crosslinking을 보장하는 것이 중요합니다. 여기에서 설명한 프로토콜은 순차적 굴절률 (RI)와 울트라 바이올렛 (UV) 커플링 학위 결정에 대한 감지 시스템이 장착된 고성능 크기 제외 크로마 토그래피 (HPSEC)을 적용합니다. 설명 방법론이 다른 약물에 대해 적용될 수 있습니다으로서, basophil 인증 시험 (BAT)는 약물 과민성의 IGE - 중재 메커니즘의 결정에 사용될 수있는 잠재력을 맺는다. 여기, 우리는 IGE - 중재와 DF의 과민성 BAT에 의해 비 IGE - 중재만큼 PP 과민성을 결정합니다.

Watch this Scientific Journal Video about Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity at JoVE.com

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Basophil 인증 테스트 IGE에 의존 알레르기의 탐지를위한 강력한 도구입니다<em&gt; 체외에서</em&gt;. 여기, basophil 활성화 시험에 대한 최적화된 프로토콜이 약물 과민성을 조사하는 데 사용됩니다. 공유 결합 약물 단백질 conjugates 및 물리 특성의 효율적인 생산 방법을 설명합니다.

약물 과민성의 IGE - 중재 메커니즘을 조사 Basophil 인증 시험

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

basophil-activation-test-for-investigation-ige-mediated-mechanisms

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33.3K 조회수.  University of Salzburg. Basophil 인증 테스트 IGE에 의존 알레르기의 탐지를위한 강력한 도구입니다 체외에서. 여기, basophil 활성화 시험에 대한 최적화된 프로토콜이 약물 과민성을 조사하는 데 사용됩니다. 공유 결합 약물 단백질 conjugates 및 물리 특성의 효율적인 생산 방법을 설명합니다.

비디오: Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B 세포의 CD40 - 정품 인증 Murine 모델

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

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Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

촉진 약물 디스커버리 : Zebrafish의 자동화된 하이 컨텐츠 염증 분석

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

항원 특정 규제 트랜스 생체 지연 형 과민 분석

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

활성화 및 인간 단핵구 유래 수지상 세포에서 IL-1β를 사용하여 NLRP3 inflammasome을 활동의 측정

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T 림프구에있는 인테그린 활성화의 연구에 대한 정적 접착 분석

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

높은 처리량 정량 실시간 RT-PCR 분석 유형 I과 III 인터페론 하위 유형의 발현 양상을 결정하는

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

다제 내성 항 감염 블루 라이트 치료의 생체 조사에서 아시네 토 박터 바우 마니는 생물 발광 영상을 사용하여 감염을 굽기

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

병원성 메커니즘 연구를위한 BALB / c 마우스에서 약물 유도,자가 면역 간염의 유도

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

광견병 백신 효능을 평가하기 위한 시험관 내 ELISA 테스트

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.