Childhood screening for type 1 diabetes comparing automated multiplex Antibody Detection by Agglutination-PCR (ADAP) with single plex islet autoantibody radiobinding assays.

BACKGROUND: Two or more autoantibodies against either insulin (IAA), glutamic acid decarboxylase (GADA), islet antigen-2 (IA-2A) or zinc transporter 8 (ZnT8A) denote stage 1 (normoglycemia) or stage 2 (dysglycemia) type 1 diabetes prior to stage 3 type 1 diabetes. Automated multiplex Antibody Detection by Agglutination-PCR (ADAP) assays in two laboratories were compared to single plex radiobinding assays (RBA) to define threshold levels for diagnostic specificity and sensitivity. METHODS: IAA, GADA, IA-2A and ZnT8A were analysed in 1504 (54% females) population based controls (PBC), 456 (55% females) doctor's office controls (DOC) and 535 (41% females) blood donor controls (BDC) as well as in 2300 (48% females) patients newly diagnosed (1-10 years of age) with stage 3 type 1 diabetes. The thresholds for autoantibody positivity were computed in 100 10-fold cross-validations to separate patients from controls either by maximizing the χ2-statistics (chisq) or using the 98th percentile of specificity (Spec98). Mean and 95% CI for threshold, sensitivity and specificity are presented. FINDINGS: The ADAP ROC curves of the four autoantibodies showed comparable AUC in the two ADAP laboratories and were higher than RBA. Detection of two or more autoantibodies using chisq showed 0.97 (0.95, 0.99) sensitivity and 0.94 (0.91, 0.97) specificity in ADAP compared to 0.90 (0.88, 0.95) sensitivity and 0.97 (0.94, 0.98) specificity in RBA. Using Spec98, ADAP showed 0.92 (0.89, 0.95) sensitivity and 0.99 (0.98, 1.00) specificity compared to 0.89 (0.77, 0.86) sensitivity and 1.00 (0.99, 1.00) specificity in the RBA. The diagnostic sensitivity and specificity were higher in PBC compared to DOC and BDC. INTERPRETATION: ADAP was comparable in two laboratories, both comparable to or better than RBA, to define threshold levels for two or more autoantibodies to stage type 1 diabetes. FUNDING: Supported by The Leona M. and Harry B. Helmsley Charitable Trust (grant number 2009-04078), the Swedish Foundation for Strategic Research (Dnr IRC15-0067) and the Swedish Research Council, Strategic Research Area (Dnr 2009-1039). AL was supported by the DiaUnion collaborative study, co-financed by EU Interreg ÖKS, Capital Region of Denmark, Region Skåne and the Novo Nordisk Foundation.

CalScope: Monitoring Severe Acute Respiratory Syndrome Coronavirus 2 Seroprevalence From Vaccination and Prior Infection in Adults and Children in California May 2021-July 2021.

Background: Understanding the distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies from vaccination and/or prior infection is critical to the public health response to the pandemic. CalScope is a population-based serosurvey in 7 counties in California. Methods: We invited 200 000 randomly sampled households to enroll up to 1 adult and 1 child between April 20, 2021 and June 16, 2021. We tested all specimens for antibodies against SARS-CoV-2 nucleocapsid and spike proteins, and each participant completed an online survey. We classified participants into categories: seronegative, antibodies from infection only, antibodies from infection and vaccination, and antibodies from vaccination only. Results: A total of 11 161 households enrolled (5.6%), with 7483 adults and 1375 children completing antibody testing. As of June 2021, 33% (95% confidence interval [CI], 28%-37%) of adults and 57% (95% CI, 48%-66%) of children were seronegative; 18% (95% CI, 14%-22%) of adults and 26% (95% CI, 19%-32%) of children had antibodies from infection alone; 9% (95% CI, 6%-11%) of adults and 5% (95% CI, 1%-8%) of children had antibodies from infection and vaccination; and 41% (95% CI, 37%-45%) of adults and 13% (95% CI, 7%-18%) of children had antibodies from vaccination alone. Conclusions: As of June 2021, one third of adults and most children in California were seronegative. Serostatus varied regionally and by demographic group.

Detection of neutralizing antibodies against multiple SARS-CoV-2 strains in dried blood spots using cell-free PCR.

An easily implementable serological assay to accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies is urgently needed to better track herd immunity, vaccine efficacy and vaccination rates. Herein, we report the Split-Oligonucleotide Neighboring Inhibition Assay (SONIA) which uses real-time qPCR to measure the ability of neutralizing antibodies to block binding between DNA-barcoded viral spike protein subunit 1 and the human angiotensin-converting enzyme 2 receptor protein. The SONIA neutralizing antibody assay using finger-prick dried blood spots displays 91-97% sensitivity and 100% specificity in comparison to the live-virus neutralization assays using matched serum specimens for multiple SARS-CoV-2 variants-of-concern. The multiplex version of this neutralizing antibody assay, using easily collectable finger-prick dried blood spots, can be a valuable tool to help reveal the impact of age, pre-existing health conditions, waning immunity, different vaccination schemes and the emergence of new variants-of-concern.

Multiplex agglutination-PCR (ADAP) autoantibody assays compared to radiobinding autoantibodies in type 1 diabetes and celiac disease.

Multiplex Antibody-Detection by Agglutination-PCR (ADAP) assay was compared to singleplex standard radiobinding assays (RBA) to detect autoantibodies against insulin (IAA), GAD65 (GADA), islet antigen-2 (IA-2A), ZnT8 (ZnT8A) and tissue transglutaminase (TGA). Serum samples from 273 (114F/158M), 15-73 years of age healthy controls and 227 (109F/118M) newly diagnosed type 1 diabetes children, 1-11 years of age, were analyzed in both assay systems.The original WHO standard 97/550 and in-house reference standards for RBA were compared to ADAP. The ADAP and RBA generated parallel reference standards in all assays except TGA. Lower detection limits were observed in the ADAP assay for GADA,IAA and ZnT8A, markedly for TGA, but not for IA-2A. The Receiver Operating Characteristics (ROC) curve AUC analyses for pairwise comparison of ADAP with RBA showed no difference for GADA (n.s.), ADAP greater AUC for IAA (p = 0.005), RBA greater AUC for IA-2A (p = 0.0004) and ZnT8A (p < 0.0001) while ADAP TGA had a greater AUC compared to both RBA TGA-IgG (p < 0.0001) and TGA-IgA (p < 0.0001). These data suggest that the ADAP and RBA assays are comparable with equal performance for GADA, better ADAP performance for IAA while the RBA showed better performance in both IA-2A and ZnT8A associated with greater heterogeneity in autoantibody levels. The simultaneous analysis of 5 different autoantibodies by ADAP in sample volume reduced to only 4 µL and at an increased lower detection limit in all assays except IA-2A makes the ADAP automated autoantibody assay a distinct advantage for high throughput screening.

Automation of a multiplex agglutination-PCR (ADAP) type 1 diabetes (T1D) assay for the rapid analysis of islet autoantibodies.

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.

Sensitive detection of multiple islet autoantibodies in type 1 diabetes using small sample volumes by agglutination-PCR.

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.

The evolution of vascular tissue engineering and current state of the art.

Dacron® (polyethylene terephthalate) and Goretex® (expanded polytetrafluoroethylene) vascular grafts have been very successful in replacing obstructed blood vessels of large and medium diameters. However, as diameters decrease below 6 mm, these grafts are clearly outperformed by transposed autologous veins and, particularly, arteries. With approximately 8 million individuals with peripheral arterial disease, over 500,000 patients diagnosed with end-stage renal disease, and over 250,000 patients per year undergoing coronary bypass in the USA alone, there is a critical clinical need for a functional small-diameter conduit [Lloyd-Jones et al., Circulation 2010;121:e46-e215]. Over the last decade, we have witnessed a dramatic paradigm shift in cardiovascular tissue engineering that has driven the field away from biomaterial-focused approaches and towards more biology-driven strategies. In this article, we review the preclinical and clinical efforts in the quest for a tissue-engineered blood vessel that is free of permanent synthetic scaffolds but has the mechanical strength to become a successful arterial graft. Special emphasis is given to the tissue engineering by self-assembly (TESA) approach, which has been the only one to reach clinical trials for applications under arterial pressure.

syn,syn-15,17-Di-2-naphthyl-hexa-cyclo-[10.2.1.1.1.0.0]hepta-decane deuterochloro-form monosolvate.

The main molecule of the title compound, C(37)H(36)·CDCl(3), is a hydro-carbon with two naphthalene segments attached to opposite ends of a rigid norbornylogous spacer with an overall structure that is approximately C-shaped. The dihedral angle between the naphthalene ring planes is 9.27 (7)°. The cleft that exists between the naphthalene rings is large enough that the compound crystallizes with a solvent mol-ecule (CDCl(3)) in the cleft. The CDCl(3) solvent mol-ecule is present in two disordered orientations in a 3:2 ratio, each involving C-D⋯π to C(6) ring centers.