RESUMO
Oral fluid (OF) is a highly effective substrate for population-based HIV screening efforts, as it is noninfectious and significantly easier to collect than blood. However, anti-HIV antibodies are found at far lower concentrations in OF compared with blood, leading to poor sensitivity and a longer period of time from infection to detection threshold. Thus, despite its inherent advantages in sample collection, OF is not widely used for population screening. Here we report the development of an HIV OF assay based on Antibody Detection by Agglutination-PCR (ADAP) technology. This assay is 1,000-10,000 times more analytically sensitive than clinical enzyme-linked immunoassays (EIAs), displaying both 100% clinical sensitivity and 100% specificity for detecting HIV antibodies within OF samples. We show that the enhanced analytical sensitivity enables this assay to correctly identify HIV-infected individuals otherwise missed by current OF assays. We envision that the attributes of this improved HIV OF assay can increase testing rates of at-risk individuals while enabling diagnosis and treatment at an earlier time point.
Assuntos
Anticorpos Anti-HIV/genética , Infecções por HIV/diagnóstico , Reação em Cadeia da Polimerase/métodos , Saliva/virologia , Aglutinação , DNA/química , Diagnóstico Precoce , Anticorpos Anti-HIV/análise , Proteína do Núcleo p24 do HIV/genética , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/genética , Infecções por HIV/prevenção & controle , Humanos , Programas de Rastreamento/métodos , Sensibilidade e Especificidade , Fluxo de TrabalhoRESUMO
Screening for islet autoantibody markers to identify individuals who are at high risk for developing type 1 diabetes (T1D), often years in advance of clinical symptoms, is both a challenge and a necessity. Identifying high-risk individuals not only reduces hospitalization and rates of life-threatening diabetes ketoacidosis (DKA), but also directs enrollment into prevention trials that require patients who are in the early stages of disease. Here we describe an automated high-throughput multiplex islet autoantibody assay that integrates antibody detection by agglutination-PCR (ADAP) chemistry on the Hamilton Microlab STAR liquid handling platform. The automated system features on-deck thermal cycling and plate sealing to minimize the level of human intervention. The automated multiplex ADAP T1D assay performed similarly to that of manual methods using two distinct cohorts of clinical specimens obtained from the Lucile Packard Children's Hospital at Stanford University and the 2018 Islet Autoantibody Standardization Program (IASP). Notably, the automated assay requires only 4 µL of serum sample for the simultaneous analysis of GAD, IA-2 and insulin autoantibodies. Up to 96 samples may be processed in as little as 3 hours, and the only user intervention required is to transfer a final sealed 96-well plate containing PCR amplicons onto a quantitative PCR (RT-qPCR) instrument for quantification. The automated system is particularly well suited for large-scale analysis of islet autoantibodies in a reproducible, timely, and cost-effective manner.
Assuntos
Diabetes Mellitus Tipo 1 , Aglutinação , Autoanticorpos , Automação , Criança , Diabetes Mellitus Tipo 1/diagnóstico , Humanos , Reação em Cadeia da Polimerase MultiplexRESUMO
Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. However, the reliance on radioactive components, large sample volumes and limited throughput renders radioassay testing costly and challenging. We developed a multiplex analysis platform based on antibody detection by agglutination-PCR (ADAP) for the sample-sparing measurement of GAD, IA-2 and insulin autoantibodies/antibodies in 1 µL serum. The assay was developed and validated in 7 distinct cohorts (n = 858) with the majority of the cohorts blinded prior to analysis. Measurements from the ADAP assay were compared to radioassay to determine correlation, concordance, agreement, clinical sensitivity and specificity. The average overall agreement between ADAP and radioassay was above 91%. The average clinical sensitivity and specificity were 96% and 97%. In the IASP 2018 workshop, ADAP achieved the highest sensitivity of all assays tested at 95% specificity (AS95) rating for GAD and IA-2 autoantibodies and top-tier performance for insulin autoantibodies. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1µL serum with high sensitivity. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies.
Assuntos
Aglutinação/imunologia , Autoanticorpos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , Adolescente , Adulto , Feminino , Glutamato Descarboxilase/imunologia , Humanos , Anticorpos Anti-Insulina/imunologia , Masculino , Programas de Rastreamento , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Adulto JovemRESUMO
A Ga(III)-catalyzed cycloisomerization reaction provides expedient access to a benzannulated cycloheptadiene bearing a cyano group, which has been applied to the syntheses of several icetexane diterpenoids including icetexone and epi-icetexone. Key to the synthesis is a novel in situ generated diazene rearrangement.