Reliable Enzyme‐Linked Immunosorbent Assay Systems for Pathogenic Factors of Pseudomonas aeruginosa Alkaline Proteinase, Elastase, and Exotoxin A: A Comparison of Methods for Labeling Detection Antibodies with Horseradish Peroxidase

Abstract

Pseudomonas aeruginosa is an important opportunistic pathogen that sometimes causes fatal infections in vulnerable hosts. Several components related to its virulence have been identified and characterized. Among these, alkaline proteinase (aeruginolysin), elastase (pseudolysin), and exotoxin A are considered to play important roles in the pathogenesis of P. aeruginosa infections (25). Therefore, in order to study the virulence of P. aeruginosa, it is essential to precisely evaluate the amounts of these pathogenic factors. Alkaline proteinase is a 49-kDa enzyme with optimal enzymatic activity at alkaline pH (24). Elastase is a 33-kDa enzyme (24). These enzymes are metalloproteinases that hydrolyze many proteinaceous substrates. Elastase hydrolyzes a wide range of substrates, including elastin and IgG (6), and inactivates complement components (31). In contrast, alkaline proteinase is unable to hydrolyze elastin or IgG. Exotoxin A is a 66-kDa enzyme that transfers the ADP-ribose moiety of NAD to 1149 Microbiol. Immunol., 51(12), [1149][1150][1151][1152][1153][1154][1155][1156][1157][1158][1159] 2007 Abstract: Sensitive sandwich enzyme-linked immunosorbent assay (ELISA) systems for the quantification of 3 pathogenic factors of Pseudomonas aeruginosa-alkaline proteinase (aeruginolysin), elastase (pseudolysin), and exotoxin A-were developed. The maleimide-pyridyl disulfide method was applied for the labeling of rabbit anti-each antigen IgG with horseradish peroxidase (HRP) and the conjugates were used as secondary antibodies (detection antibodies) in the ELISA systems. The EDTA, a chelating agent, was added to the buffers for sample and detection antibody, which inhibited the degradation of IgG by elastase derived from P. aeruginosa for improving the assay precision. The ELISA systems using the HRPlabeled detection antibodies produced by the maleimide-pyridyl disulfide method exhibited higher sensitivity than previously reported methods. The detection limits for alkaline proteinase, elastase, and exotoxin A were 18 pg/ml, 34 pg/ml, and 22 pg/ml, respectively. The intra-assay coefficients of variation for alkaline proteinase, elastase, and exotoxin A were 3.4%-5.0%, 1.9%-3.5%, and 1.3%-5.4%, respectively. These ELISA systems exhibited good inter-assay precision, non-cross-reactivity, dilution linearity, and recovery. Employing these ELISA systems, we revealed that pathogenic factor concentrations were different among the P. aeruginosa strains tested, which may relate to the different pathogenicity of each strain.

Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression ofPseudomonas aeruginosaIn Vitro

“…Exotoxin A was measured according to the method of Shigematsu et al [22] and was determined using a commercially available Human Pseudomonas Exotoxin A (PEA) ELISA Kit (Cusabio Biotech Co., Ltd., Hubei, PR China, product code: CSB-E11252 h), according to the manufacturer's instructions. The data were recorded as ng/mL.…”

Effects of Allicin on the Formation of Pseudomonas aeruginosa Biofilm and the Production of Quorum-Sensing Controlled Virulence Factors

“…Exotoxin A assay (Kozak and Saelinger, 1988;Shigematsu et al, 2007). Bacteria were grown in a dialysate of trypticase soy broth (TSBD), consisting of TSB with 1% glycerol and 50 mM monosodium glutamate, at 32°C with shaking for 18 h. e culture was then divided into two groups: TSBD medium alone (control) and allicin treatment at 128 µg/ml (experimental).…”

Negative-Pressure Wound Therapy in aPseudomonas aeruginosaInfection Model

“…Exotoxin A was measured according to the method of Shigematsu et al [ 26 ] and was determined using a commercially available human Pseudomonas exotoxin A enzyme-linked immunosorbent assay kit (Cusabio Biotech Co., Ltd., Hubei, China), according to the manufacturer's instructions. The data were recorded as ng/mL.…”