Immunoassay Biotechnology

Lateral flow immunoassay strip is a simple, reliable and rapid qualitative tool for primary screening of transgenic proteins. The extraction of proteins from sample products for performing lateral flow immunoassay (LFIA) strip test is an important step. In this study, the protein extraction efficiency of various buffers prepared in-lab and some extraction buffers from commercial kits were tested. In addition, the effects of blocking buffers along with hybridization timings, for blocking of unoccupied sites, on prepared nitrocellulose membranes were examined during assembly of LFIA strips. It was estimated that 1XPBS of pH 7 is best protein extraction buffer according to our sample, Bt-Cry 1Ac from Bt-Cotton seeds. Furthermore, during manual assembly of LFIA strips, we found that the sealing tapes used as covering for one-unit assay has
no effect on efficiency of LFIA strip tests. In our experiments, we optimized hybridization timing for blocking of unoccupied sites on nitrocellulose membranes is 30 minutes. We also optimized a simple composition for blocking buffer

As the COVID-19 pandemic has profoundly impacted human life, prompt diagnostic tests are becoming an essential part of the social activities. However, the expensive and time-consuming laboratory-based traditional methods do not suffice the enormous needs for massive number of tests, especially in resource-limited settings. Therefore, more affordable, rapid, sensitive and specific field-practical diagnostic devices play an important role in the fight against the disease. In this review, we present the current status and advances in the biosensing technologies for diagnosing COVID-19, ranging from commercial achievements to research developments. Starting from a brief introduction to the disease biomarkers, this review summarizes the working principles of the biosensing technologies , followed by a review of the commercial products and research advances in academia. We recapitulate the lit-eratures with a wide scope of bio/marker detections, embracing nucleic acids, viral proteins, human immune responses, and other potential bio/markers. Further, the challenges and perspectives for their employment in future point-of-care applications are discussed, with an extended appraisal on the practical strategies to enlarge the testing capability without high cost. This critical review provides a comprehensive insight into the diagnostic tools for COVID-19 and will encourage the industry and academia in the field of diagnostic biosensing for future evolvement to large-scale point-of-care screening of COVID-19.

BACKGROUND:
The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC(-)ssaV(-)) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.
OBJECTIVES:
We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.
METHODS:
Subjects were randomly assigned to receive either a nominal dose of 5x10(9) CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.
PRINCIPAL FINDINGS:
One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18-5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12-36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI] = 1.23 [0.550-2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC(-)ssaV(-)) ZH9 was detected in 51 (51%; 95% CI, 41-61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92-99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7-29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88-100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.
CONCLUSIONS:
This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.
TRIAL REGISTRATION:
Controlled-trials.com ISRCTN91111837.