Rapid Chemiluminescent Enzyme Immunoassay Research Articles

Presepsin in Human Milk Is Delivery Mode and Gender Dependent.

Breast milk (BM) is a unique food due to its nutritional composition and anti-inflammatory characteristics. Evidence has emerged on the role of Presepsin (PSEP) as a reliable marker of early sepsis diagnosis. In the present study, we aimed to investigate the measurability of PSEP in BM according to different maturation stages (colostrum, C; transition, Tr; and mature milks, Mt) and corrected for delivery mode and gender. We conducted a multicenter prospective case-control study in women who had delivered 22 term (T) and 22 preterm (PT) infants. A total of 44 human milk samples were collected and stored at -80 °C. BM PSEP (pg/mL) levels were measured by using a rapid chemiluminescent enzyme immunoassay. PSEP was detected in all samples analyzed. Higher (p < 0.05) BM PSEP concentrations were observed in the PT compared to the T infants. According to the grade of maturation, higher (p < 0.05) levels of PSEP in C compared to Tr and Mt milks were observed in the whole study population. The BM subtypes' degrees of maturation were delivery mode and gender dependent. We found that PSEP at high concentrations supports its antimicrobial action both in PT and T infants. These results open the door to further studies investigating the role of PSEP.

Utility of a rapid assay for prostaglandin E-major urinary metabolite as a biomarker in pediatric ulcerative colitis

Prostaglandin E-major urinary metabolite (PGE-MUM) is a urinary biomarker reflecting ulcerative colitis (UC) activity. This prospective observational study aimed to evaluate the usefulness of PGE-MUM via rapid chemiluminescent enzyme immunoassay in detecting endoscopic remission (ER) and histologic remission (HR) in pediatric UC (6–16 years) in comparison with fecal calprotectin (FCP). ER and HR were defined as Mayo endoscopic score (MES) of 0 and Matts’ histological grades (Matts) of 1 or 2, respectively. A total of 104 UC and 39 functional gastrointestinal disorder (FGID) were analyzed. PGE-MUM levels were significantly higher in the UC group than in the FGID group (P < 0.001). FCP levels were significantly elevated in the group without ER and HR than in the group with ER and HR (P < 0.001 and P = 0.001), whereas PGE-MUM levels were significantly higher in the group without ER compared to the group with ER (P < 0.001). No significant differences were noted in the AUCs for PGE-MUM and FCP in detecting ER and HR. Although PGE-MUM was inferior to FCP for the detection of HR, it might have the potential for application as a biomarker of endoscopic activity in pediatric UC owing to its noninvasive and rapid method.

Analytical and clinical validation of rapid chemiluminescence enzyme immunoassay for urinary thioredoxin, an oxidative stress-dependent early biomarker of acute kidney injury

BackgroundOxidative stress is now recognized to be an important therapeutic target in kidney diseases. However, there are currently no biomarkers that can be used clinically to diagnose renal oxidative stress. MethodsA rapid assay system for urinary thioredoxin 1, an oxidative stress-dependent biomarker of acute kidney injury (AKI), was developed as a chemiluminescence enzyme immunoassay and validated analytically and clinically. ResultsAnalytic evaluation revealed that hemolytic hemoglobin caused measurements to be abnormally high, above the detectable range. However, urine sediment containing red blood cells did not affect the measurements. Assays using our proposed chemiluminescence enzyme immunoassay were completed within as little as 6 min, whereas a conventional ELISA > 4 h. Aciduria <pH 6.0 led to a significant underestimation of thioredoxin 1 concentrations. However, the effects of aciduria were completely reversible with use of a buffer developed for pH adjustment. Urinary thioredoxin 1 was increased in patients with AKI, but was unaffected by extrarenal oxidative stress diseases, including hypoxemia and myocardial infarction, or by chronic kidney disease in which serum creatinine concentrations were comparable. ConclusionsThese results suggest that the chemiluminescent enzyme immunoassay system for urinary thioredoxin 1 enables rapid and specific diagnosis of AKI associated with oxidative stress.

Sorption of his-tagged Protein G and Protein G onto chitosan/divalent metal ion sorbent used for detection of microcystin-LR.

A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR) by using strategies for oriented immobilization of functionally intact polyclonal antibodies on chitosan surface. Several physicochemical parameters such as metal ion adsorption, hexahistidine-tagged Protein G sorption, the dilution ratio polyclonal antibody concentration, and peroxidase-labeled MC-LR concentration were studied and optimized. The sorption in batch system of G-histidine and G-proteins was studied on a novel sorbent consisting of chitosan/divalent metal ions. Transition metals as Ni++ and Zn++ were immobilized through interaction with -NH2 groups of chitosan in order to supply a material capable to efficiently remove the proteins from aqueous solutions. The maximum uptake of divalent metals onto the chitosan material was found to be 230mgg-1 for Zn++ and 62mgg-1 for Ni++. Experimental data were evaluated using the Langmuir and Freundlich models; the results were well fitted with the Langmuir model; chitosan/Ni++ foam was found to be the best sorbent for G-protein, maximum sorption capacity obtained was 17mgg-1, and chitosan/Zn++ was found to be the best for G-histidine with a maximum sorption capacity of 44mgg-1. Kinetic data was evaluated with pseudo-first- and pseudo-second-order models; the sorption kinetics were in all cases better represented by a pseudo-second-order model. Under optimum conditions, the calibration curve obtained for MC-LR gave detection limits of 0.5 ± 0.06μgL-1, the 50% inhibition concentration (IC50) was 2.75 ± 0.03μgL-1, and the quantitative detection range was 0.5-25μgL-1. The limit of detection (LOD) attained from the calibration curves and the results obtained demonstrate the potential use of CLEIA with chitosan support as a screening tool for the analysis of pollutants in environmental samples.

Presepsyna (rozpuszczalny podtyp CD14-ST) jako diagnostyczny biomarker posocznicy u noworodków

BackgroundDetermination of sCD14-ST (presepsin) concentration as a marker for infections in newborns was not performed till now. Aim1. Assessment of blood presepsin concentration in newborns, including their sex, fetal maturity, birth weight, early-onset sepsis. 2. Determination of correlation between presepsin value and CRP, procalcitonin concentrations and some hematological parameters. Material and methodsThe study comprised 45 newborns, among them 27 septic and 18 without infection, but with perinatal risk factors or symptoms suggesting infection, in which by rapid chemiluminescent enzyme immunoassay, using Pathfast TM analyser from Mitsubishi Kagaku Iatron, sCD14-ST concentration in whole blood was measured. ResultsThe mean presepsin concentration in septic newborns (1772±1009pg/ml) was significantly higher than in group without infection (556±158). Positive correlations between sCD14-ST and CRP, procalcitonin concentrations and negative correlations between the value of presepsin and Ht, Hb, platelet count, birth weight and fetal maturity were noted. Conclusions1. In newborns with early-onset sepsis, independently of their sex, birth weight, fetal maturity, significant increase presepsin concentration, correlating with increase of CRP and procalcitonin values and with decrease of platelet count, Hb and Ht. 2. Measurement of presepsin concentration in neonatal whole blood may be usefulness in diagnosis of early-onset sepsis.

Sensitive and rapid chemiluminescence enzyme immunoassay for microcystin-LR in water samples

A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR). Several physicochemical parameters such as the chemiluminescent assay mediums, the dilution ratio of MC-LR–OVA conjugate, monoclonal antibody concentration, and peroxidase labeled antibody concentration were studied and optimized. Under optimum conditions, calibration curve obtained for MC-LR had detection limits of 0.032 ± 0.003 μg L −1, the 50% inhibition concentration (IC 50) was 0.20 ± 0.02 μg L −1 and the quantitative detection range was 0.062–0.65 μg L −1. The proposed methods was successfully applied to the monitoring of MC-LR in spiked water samples without significant effect of the matrix, and the recovery of MC-LR added to water samples at different concentrations ranged from 80% to 115% with the coefficients of variation (CVs) less than 9%. The LOD attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of CLEIA as a screening tool for the analysis of MC-LR in environmental samples.

Micro-plate Chemiluminescence Enzyme Immunoassay for Clinical Determination of Progesterone in Human Serum

A high-throughput, simple and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the clinical determination of progesterone in human serum, using luminol-hydrogen peroxide as chemiluminescence system catalyzed by horseradish peroxidase (HRP). The solid phase of anti-progesterone antibody was prepared by immunoreaction between anti-progesterone polyclonal antibody and donkey anti-rabbit IgG, i.e. second antibody, which had been physically absorbed on the wells of polystyrene microplate and was used as a universal solid phase. The effect of various factors, such as the dilution of immunoreagents, chemiluminescence substrate, chemiluminescence reaction time and incubation condition were examined and optimized. The optimal dilutions of anti-progesterone antibody and HRP-progesterone conjugate were 1:10000 and 1:15000, respectively. The II substrate was chosen and the luminescence was determined after 10 min incubation. The immunoreacted sample was incubated in water bath at 37°C for 1 h. The assay was evaluated with sensitivity as low as 0.08 μg l −1. The relative standard deviation (RSD) was less than 15% for both intra- and inter-assay precision. The recoveries of three different spiked concentration samples were 101%, 101% and 94.4%, respectively. This proposed method has been successfully applied to the clinical evaluation of progesterone in 36 human sera. The results showed a good correlation with the accredited radioimmunoassay (RIA) with a correlative coefficient of 0.9502.

Chemiluminescence Enzyme Immunoassay Using Bacterial Magnetic Particles

A novel chemiluminescence enzyme immunoassay using bacterial magnetic particles (BMPs) has been developed for highly sensitive and rapid detection of immunoglobulin G. Antibody was immobilized onto BMPs using the heterobifunctional reagents sulfosuccinimidyl 6-[3'-(2-pyridyldithio) propionamido]hexanoate (sulfo-LC-SPDP) and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). For the highly sensitive immunoassay method using these BMPs, a good relationship was obtained between the luminescence intensity and mouse IgG concentration in the range of 1-10(5) fg/mL. Furthermore, in order to reduce assay time and to simplify operations, a rapid chemiluminescence enzyme immunoassay method has been developed. The rapid method was completed in 10 min. A linear relationship was obtained between the luminescence and mouse IgG concentration in the range of 10-1000 ng/mL.