Combined Enzyme-linked Immunosorbent Assay Research Articles

Method Validation of a Bridging Enzyme-Linked Immunosorbent Assay in Combination with Acid-Dissociation and Bead Treatment for Detection of Anti-Drug Antibody

Method Validation of a Bridging Enzyme-Linked Immunosorbent Assay in Combination with Acid-Dissociation and Bead Treatment for Detection of Anti-Drug Antibody

Multicenter Validation Study of a Diagnostic Algorithm to Detect NASH and Fibrosis in NAFLD Patients With Low NAFLD Fibrosis Score or Liver Stiffness

OBJECTIVES:Nonalcoholic steatohepatitis (NASH) and fibrosis play critical roles for the prognosis of patients with nonalcoholic fatty liver disease (NAFLD). Identification of patients at risk of NASH and fibrosis is therefore critical for disease management. NAFLD Fibrosis Score (NFS) and transient elastography (TE) have been suggested to exclude advanced fibrosis. However, there is increasing evidence that also patients with NASH and early fibrosis are at risk of disease progression and complications, emphasizing the need for improved noninvasive risk stratification in NAFLD.METHODS:Because hepatocyte apoptosis plays an early role in NASH pathogenesis, we evaluated whether the apoptosis biomarker M30 might identify NAFLD patients who are at risk of NASH and fibrosis despite low NFS or TE values. Serum M30 levels were assessed by enzyme-linked immunosorbent assay in combination with NFS and/or TE in an exploration (n = 103) and validation (n = 100) cohort of patients with biopsy-proven NAFLD.RESULTS:Most patients with low NFS (cutoff value < −1.455) revealed increased M30 levels (>200 U/L) in the exploration (62%) and validation (67%) cohort, and more than 70% of them had NASH, mostly with histological fibrosis. Vice versa, most patients with NFS < −1.455 but nonelevated M30 levels showed no NASH. NASH was also detected in most patients with indeterminate NFS (−1.455 to 0.676) but elevated M30 levels, from which ∼90% showed fibrosis. Similar results were obtained when using TE instead of NFS.DISCUSSION:The combination of the M30 biomarker with NFS or TE enables a more reliable identification of patients with an increased risk of progressed NAFLD and improves patient stratification.

A Nuclear-encoded Subunit of the Photosystem II Reaction Center

A nuclear-encoded polypeptide of 6.1 kDa was identified in isolated photosystem II (PSII) reaction center from Spinacia oleracea. The hydrophobic membrane protein easily escapes staining procedures such as Coomassie R-250 or silver staining, but it is clearly detected by immunodecoration with peptide-directed IgG. This additional subunit was found to be present in PSII reaction centers previously known to contain only the D1/D2/cytb559 proteins and the psbI gene product. Furthermore, cross-linking experiments using 1-(3-dimethylaminopropyl-) 3-ethylcarbodiimide showed that the nearest neighbors were the D1 and D2 proteins and the cytb559. The 6.1-kDa protein was purified by immune affinity chromatography. N-terminal sequence analysis of the isolated protein confirmed the identity of the 6.1-kDa protein and enabled finding of strong similarities with a randomly obtained cDNA from Arabidopsis thaliana. Using enzyme-linked immunosorbent assay in combination with thylakoid membrane preparations of different orientation, the N terminus of the protein, predicted to span the membrane once, is suggested to be exposed at the lumen side of the membrane. Consequently the 6.1-kDa protein seems to be the only subunit in the PSII reaction center that is nuclear encoded and has its N terminus on the lumen side of the membrane. These findings open for new interesting suggestions concerning the properties of photosystem II reaction center with respect to the photosynthetic activity, regulation and assembly in higher plants.

Identification, cellular localization and in vitro release of a novel teleost melanin-concentrating hormone gene-related peptide.

The melanin-concentrating hormone (MCH) precursor encodes MCH and a second peptide named neuropeptide EI (NEI) in mammals, neuropeptide EV (NEV) in salmonids and MCH gene-related peptide (Mgrp) in other fish. The primary structure of the putative Mgrp of the cichlid fish tilapia (Oreochromis mossambicus) appears to be very different from mammalian NEI and salmonid NEV. To investigate the processing and release of tilapia Mgrp (tMgrp), in the present study an antiserum was raised against synthetic tMgrp. By immunocytochemistry, tMgrp immunoreactivity was colocated with MCH immunoreactivity in the tilapia hypothalamus and pituitary. In addition, a tMgrp enzyme-linked immunosorbent assay in combination with reversed phase HPLC was used to demonstrate the presence of processed tMgrp in tilapia hypothalamus and pituitary. The release of tMgrp from neuro-intermediate lobes (NILs) of tilapia pituitaries was demonstrated after in vivo incubation of chopped NILs. Depolarizing concentrations of potassium significantly stimulated tMgrp release. Six weeks of adaptation of tilapia to white or black backgrounds had no effect on in vitro tMgrp release or on the tMgrp content of NIL and hypothalamus. Tilapia Mgrp, unlike MCH, had no effect on tilapia scale melanophores, nor did it modulate the melanin-concentrating effect of MCH. We conclude that tMgrp is processed from the MCH preprohormone, that it is released in vitro, and that the peptide has no direct role in the melanin concentration of fish scale melanophores. Therefore a neuroendocrine or neuromodulatory function is proposed for tMgrp.

Detection of rubella virus-specific polymeric immunoglobulin A by enzyme-linked immunosorbent assay in combination with streptococcal pretreatment of serum

An enzyme-linked immunosorbent assay combined with streptococcal treatment of serum was assessed for its ability to detect serum polymeric immunoglobulin A. This technique detects rubella virus-specific polymeric immunoglobulin A antibody, which appears for only a short time after infection, and it is useful for serodiagnosis of recent rubella virus infection.