Freeze-drying of few microliters of antibody formulations to implement 384-wells homogeneous instant assays.

Enzyme-linked immunosorbent assay protocols have traditionally complex workflows with several intensive wash steps. Analytical tools with both shorter time-to-result and hands-on-time using smaller sample and assays reagents volumes are now investigated. In this context, fluorescence resonance energy transfer (FRET)-based assays are emerging as one of the most promising analytical tools in high-throughput screening (HTS). These immunoassays allow fast quantification of antigens at the nano-gram level in a final assay volume of only a few µL. We used a homogeneous time-resolved FRET (called HTRF) assay to develop a freeze-dried screening and ready-to-use format with only one rehydration step called "instant assay". To assure optimal performance of the developed homogeneous instant assay, we investigated the critical quality attributes by studying the functionality and stability of the critical reagents and fluorophores. The cyclic adenosine 3'-5'-monophosphate (cAMP) was selected as the antigen target. We tested various formulations (with different buffers, sugars, bulking reagents, surfactants and co-solvants) combined with a slow freezing and the use of an aluminium plate holder during the freeze-drying of few microliter of bioreagents. The optimized freeze-drying procedure permits to preserve more than 70% of Ab recognition properties. The developed off-the-shelf homogeneous FRET immunoassay allows direct and fast quantification of cAMP at a nanogram level.

A case series and literature review of multiple sclerosis and COVID-19: Clinical characteristics, outcomes and a brief review of immunotherapies.

BACKGROUND: In view of the emerging coronavirus pandemic, the demand for knowledge about the impact of SARS-CoV-2 on people with Multiple Sclerosis (MS) continues to grow. Patients receiving disease modifying therapy (DMT) for MS have a higher background risk of infection-related health care utilization when compared to the general population. Therefore, there is a need of evidence-based recommendations to reduce the risk of infection and also managing MS patients with SARS-CoV-2. CASE DESCRIPTION: We present three patients with history of Multiple Sclerosis (MS) on DMTs presenting with worsening MS symptoms likely pseudo exacerbation who were diagnosed with COVID-19. DISCUSSION: An extensive review of 7 articles was performed, in addition to a brief review on DMTs use in MS patients with COVID-19. In our cases, all patients were on DMT and severe course of disease was noted in 2 cases. No fatality was observed. CONCLUSIONS: This review provides a base on the clinical characteristics, outcomes and the roles of DMTs in MS patients suffering from n-cov-2. Physicians need to be vigilant about considering COVID-19 infection related relapse in the MS patients, especially in this COVID-19 pandemic era and look for pseudo-exacerbation. As most cases are found to have mild course and full recovery on DMTs, further research is needed to formulate evidence-based guidelines. This review will particularly be helpful for the researchers and registries to collect future data on MS and COVID-19.

Profiles of influenza A/H1N1 vaccine response using hemagglutination-inhibition titers.

To identify distinct antibody profiles among adults 50-to-74 years old using influenza A/H1N1 HI titers up to 75 days after vaccination. Healthy subjects 50 to 74 years old received the 2010-2011 trivalent inactivated influenza vaccine. We measured venous samples from Days 0, 28, and 75 for HI and VNA and B-cell ELISPOTs. Of 106 subjects, HI titers demonstrated a ceiling effect for 11 or 10% for those with a pre-vaccination HI titer of 1:640 where no subject post-vaccination had an increase in titer. Of the remaining 95 subjects, only 37 or 35% overall had at least a 4-fold increase by Day 28. Of these 37, 3 waned at least 4-fold, and 13 others 2-fold. Thus 15% of the subjects showed waning antibody titers by Day 75. More than half failed to respond at all. The profiles populated by these subjects as defined by HI did not vary with age or gender. The VNA results mimicked the HI profiles, but the profiles for B-cell ELISPOT did not. HI titers at Days 0, 28, and 75 populate 4 biologically plausible profiles. Limitations include lack of consensus for operationally defining waning as well as for the apparent ceiling. Furthermore, though well accepted as a marker for vaccine response, assigning thresholds with HI has limitations. However, VNA closely matches HI in populating these profiles. Thus, we hold that these profiles, having face- and content-validity, may provide a basis for understanding variation in genomic and transcriptomic response to influenza vaccination in this age group.

Extension of the molecular analysis to the promoter region of the iduronate 2-sulfatase gene reveals genomic alterations in mucopolysaccharidosis type II patients with normal coding sequence.

Hunter disease or mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by the deficit of the enzyme iduronate-2-sulfatase (IDS), involved in the catabolism of the glycosaminoglycans heparan and dermatan sulfate. Our aim was to search for molecular defects in the promoter region of the IDS gene in patients with previous biochemical diagnosis of MPS II and after we sequenced the whole IDS coding region and the exon/intron boundaries without detecting any pathogenic mutations. Screening of the promoter region of four patients detected in two of them a 178 bp deletion and in the other two a single nucleotide substitution 818 bp upstream of the coding region. The latter had never been described before in MPS II patients and it turned out to be a polymorphism. Our experience suggests that MPS II patients with no mutations detected in the IDS coding region should be screened in the promoter region of the gene. Findings will hopefully help to clarify the relationship between genotype and phenotype and will be useful for the correct molecular diagnosis of Hunter patients and the identification of female carriers, the latter particularly important for genetic counseling.

Novel polymorphisms in UTR and coding region of inducible heat shock protein 70.1 gene in tropically adapted Indian zebu cattle (Bos indicus) and riverine buffalo (Bubalus bubalis).

Due to evolutionary divergence, cattle (taurine, and indicine) and buffalo are speculated to have different responses to heat stress condition. Variation in candidate genes associated with a heat-shock response may provide an insight into the dissimilarity and suggest targets for intervention. The present work was undertaken to characterize one of the inducible heat shock protein genes promoter and coding regions in diverse breeds of Indian zebu cattle and buffaloes. The genomic DNA from a panel of 117 unrelated animals representing 14 diversified native cattle breeds and 6 buffalo breeds were utilized to determine the complete sequence and gene diversity of HSP70.1 gene. The coding region of HSP70.1 gene in Indian zebu cattle, Bos taurus and buffalo was similar in length (1,926 bp) encoding a HSP70 protein of 641 amino acids with a calculated molecular weight (Mw) of 70.26 kDa. However buffalo had a longer 5' and 3' untranslated region (UTR) of 204 and 293 nucleotides respectively, in comparison to Indian zebu cattle and Bos taurus wherein length of 5' and 3'-UTR was 172 and 286 nucleotides, respectively. The increased length of buffalo HSP70.1 gene compared to indicine and taurine gene was due to two insertions each in 5' and 3'-UTR. Comparative sequence analysis of cattle (taurine and indicine) and buffalo HSP70.1 gene revealed a total of 54 gene variations (50 SNPs and 4 INDELs) among the three species in the HSP70.1 gene. The minor allele frequencies of these nucleotide variations varied from 0.03 to 0.5 with an average of 0.26. Among the 14 B. indicus cattle breeds studied, a total of 19 polymorphic sites were identified: 4 in the 5'-UTR and 15 in the coding region (of these 2 were non-synonymous). Analysis among buffalo breeds revealed 15 SNPs throughout the gene: 6 at the 5' flanking region and 9 in the coding region. In bubaline 5'-UTR, 2 additional putative transcription factor binding sites (Elk-1 and C-Re1) were identified, other than three common sites (CP2, HSE and Pax-4) observed across all the analyzed animals. No polymorphism was found within the 3'-UTR of Indian cattle or buffalo as it was found to be monomorphic. The promoter sequences generated in 117 individuals showed a rich array of sequence elements known to be involved in transcription regulation. A total of 11 nucleotide changes were observed in the promoter sequence across the analyzed species, 3 of these changes were located within the potential transcription factor binding domains. We also identified 4 microsatellite markers within the buffalo HSP70.1 gene and 3 microsatellites within bovine HSP70.1. The present study identified several distinct changes across indicine, taurine and bubaline HSP70.1 genes that could further be evaluated as molecular markers for thermotolerance.