Chemiluminescent Enzyme-linked Immunoassay Research Articles

High-sensitivity chemiluminescent immunoassay investigation and application for the detection of T-2 toxin and major metabolite HT-2 toxin.

T-2 toxin is a widely distributed mycotoxin in cereals. HT-2 toxin is the major metabolite, which is also a contaminant in cereals. T-2 toxin and HT-2 toxin have been identified as having carcinogenic, hepatotoxic, teratogenic and immunotoxic properties. To reduce the risk of contamination, a rapid, highly sensitive and inexpensive assay for the detection is required. In this study a high-sensitivity chemiluminescent enzyme-linked immunoassay (CL-ELISA) of T-2 toxin and HT-2 toxin was developed. With the help of the chemiluminescent substrate, this protocol showed a highly sensitive character with an IC50 as low as 33.28 ng mL-1 and 27.27 ng mL-1 for T-2 and HT-2, respectively. In addition, this method had no cross-reaction with other structurally related mycotoxins. These results indicated that the developed CL-ELISA could be applied for the detection of T-2 toxin and HT-2 toxin in actual samples without complicated steps. © 2016 Society of Chemical Industry.

Application of functional microsphere in human hepatitis B virus surface antigen detection.

A novel and simple emulsifier-free emulsion polymerization technique was developed for preparation of mono-dispersed amino functionalized polymer microspheres with well defined diameters (about 400 nm). Various characterization methods demonstrated that the obtained amino microspheres had a uniform size and good dispersity which were confirmed by scanning electron microscope (SEM). Zeta potential and Fourier transform infrared spectrometer (FT-IR) demonstrated that amino groups have been successfully introduced to the microsphere surface. These functionalized microspheres have been shown to be efficient and controllable carriers capable of immobilizing and enriching monoclonal antibodies. Moreover, a newest chemiluminescent enzyme-linked immunoassay (ELISA) approach has been developed for human Hepatitis B virus surface antigen (HBsAg) detection. HBsAg was sandwiched between goat anti-HBsAg polyclonal antibody and mouse anti-HBsAg antibody. Alkaline phosphatase (ALP) conjugated horse anti-mouse immunnogloblin was used to bond with monoclonal antibody. Finally, chemiluminesent (CL) signals were recorded after adding 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) which was used as a chemiluminescent substrate reagent of ALP. This novel chemiluminescent ELISA assay was proved to be of excellent specificity and high sensitivity when using ALP and AMPPD luminescence systems for specific HBsAg detection.

Detection of Ultratrace Chloramphenicol Residues in Milk and Chicken Muscle Samples Using a Chemiluminescent ELISA

A competitive indirect chemiluminescent enzyme-linked immunoassay (CL-ELISA) for chloramphenicol (CAP) residues in milk and chicken muscle has been developed. Due to the unique characteristic of the polyclonal antibody, special reaction system and modified extract method, then after optimization (concentration of Tween-20, concentration of PB and pH, incubation time, and temperature), the method gave a detection limit of 0.92 ng/L and a detection range of 3.16–3035 ng/L, with the IC50 of 17.29 ng/L in optimum condition and real sample matrix. When CAP was spiked in milk and chicken muscle at levels of 5–100 ng/L, recoveries ranged from 104.9%–114.8% and 101.0%–118.8%, with coefficients of variation of 3.0%–14.6% and 9.5%–14.4%, respectively. In an actual chicken muscle residue study, although the extract of samples diluted 10-fold, or even 100-fold, which represents extremely lower concentration of CAP, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The developed method is therefore suitable for screening of ultratrace CAP residues in milk and chicken muscle samples.

Weight reduction can decrease circulating soluble lectin-like oxidized low-density lipoprotein receptor–1 levels in overweight middle-aged men

Circulating soluble lectin-like oxidized low-density lipoprotein receptor–1 (sLOX-1) has been reported to be associated with acute coronary syndrome, but its association with obesity has not been elucidated. In this study, we examined whether weight reduction would reduce the serum levels of sLOX-1 in a 12-week weight reduction intervention. Thirty-eight overweight middle-aged men were enrolled in the study, and 32 completed the intervention. The serum level of sLOX-1 was measured using a chemiluminescent enzyme-linked immunoassay. After the intervention program, body weight and the serum level of sLOX-1 decreased significantly (−7.5% ± 4.8% and −72.1% ± 35.9%, respectively). Changes in serum levels of sLOX-1 were positively correlated with changes in body weight (r = 0.54, P = .003), body mass index (r = 0.57, P = .001), body fat mass (r = 0.57, P = .002), total cholesterol (r = 0.41, P = .03), subcutaneous fat area (r = 0.50, P = .007), high-sensitivity C-reactive protein (r = 0.56, P = .002), leptin (r = 0.47, P = .01), and tumor necrosis factor–α (r = 0.32, P = .09); but no correlations were observed with fasting glycemic-related factors (blood glucose, hemoglobin A1c, and insulin). Changes in body mass index and high-sensitivity C-reactive protein were selected as significant predictors of sLOX-1 changes by multiple regression analyses. These results suggest that LOX-1 induction may be related to adipocyte metabolism, inflammation, and immune response associated with obesity.

Antisense Oligonucleotide Directed to Human Apolipoprotein B-100 Reduces Lipoprotein(a) Levels and Oxidized Phospholipids on Human Apolipoprotein B-100 Particles in Lipoprotein(a) Transgenic Mice

Lipoprotein (a) [Lp(a)] is a genetic cardiovascular risk factor that preferentially binds oxidized phospholipids (OxPL) in plasma. There is a lack of therapeutic agents that reduce plasma Lp(a) levels. Transgenic mice overexpressing human apolipoprotein B-100 (h-apoB-100 [h-apoB mice]) or h-apoB-100 plus human apo(a) to generate genuine Lp(a) particles [Lp(a) mice] were treated with the antisense oligonucleotide mipomersen directed to h-apoB-100 mRNA or control antisense oligonucleotide for 11 weeks by intraperitoneal injection. Mice were then followed up for an additional 10 weeks off therapy. Lp(a) levels [apo(a) bound to apoB-100] and apo(a) levels ["free" apo(a) plus apo(a) bound to apoB-100] were measured by chemiluminescent enzyme-linked immunoassay and commercial assays, respectively. The content of OxPL on h-apoB-100 particles (OxPL/h-apoB) was measured by capturing h-apoB-100 in microtiter wells and detecting OxPL by antibody E06. As expected, mipomersen significantly reduced plasma h-apoB-100 levels in both groups of mice. In Lp(a) mice, mipomersen significantly reduced Lp(a) levels by approximately 75% compared with baseline (P<0.0001) but had no effect on apo(a) levels or hepatic apo(a) mRNA expression. OxPL/h-apoB levels were much higher at baseline in Lp(a) mice compared with h-ApoB mice (P<0.0001) but decreased in a time-dependent fashion with mipomersen. There was no effect of the control antisense oligonucleotide on lipoprotein levels or oxidative parameters. Mipomersen significantly reduced Lp(a) and OxPL/apoB levels in Lp(a) mice. The present study demonstrates that h-apoB-100 is a limiting factor in Lp(a) particle synthesis in this Lp(a) transgenic model. If applicable to humans, mipomersen may represent a novel therapeutic approach to reducing Lp(a) levels and their associated OxPL.

Elevated Soluble Lectin‐like Oxidized LDL Receptor‐1 (sLOX‐1) Levels in Obese Postmenopausal Women

We investigated the association between soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) levels and obesity in older women. Fifty-one postmenopausal women (10 lean, 22 overweight, and 19 obese) were included in this small retrospective analysis. Plasma sLOX-1 levels were measured using a chemiluminescent enzyme-linked immunoassay. Plasma levels of sLOX-1 were significantly higher in obese women (55.33 +/- 4.49 pg/ml) compared to lean (30.91 +/- 6.19 pg/ml, P = 0.002) and overweight women (38.31 +/- 4.18 pg/ml, P = 0.017). Plasma sLOX-1 levels were positively associated with body weight, BMI, total body fat, and trunk fat. The relationship between sLOX-1 and BMI was attenuated after adjustment for age, hormone replacement therapy, and body fat. In conclusion, obese women have higher sLOX-1 levels, which may reflect increased LOX-1 expression in adipose tissue.

Intraindividual Variation in Total and Percent Free Prostate-Specific Antigen Levels in Prostate Cancer Suspects

Objectives: To investigate intraindividual total and percent free serum prostate-specific antigen (PSA) in prostate cancer suspects and to understand the clinical implications. Patients and Methods: Total and percent free PSA were measured using Tandem-R or chemiluminescent enzyme-linked immunoassay for a median of three times in 126 men. Prostate biopsies were performed in all patients; benign prostatic hyperplasia was diagnosed in 81 patients and prostate cancer in 45 patients. Results: The overall mean coefficients of variation for total and percent free PSA were 16.10 ± 11.94% and 15.45 ± 15.91%, respectively. A significant correlation (p = 0.0056) was observed between the two variations. The variations in total and percent free PSA were related to none of such stratifications as baseline total PSA level, histology, age, or measurement interval, but for measurement interval on that for total PSA. Conclusion: Intraindividual variation in serum PSA should be considered in decision-making about performing prostate biopsies. Also, care should be taken in interpreting repeated percent free PSA measurements in order to enhance the specificity of total PSA, because it had a similar variation to total PSA variation.

Quantitation of thymidylate synthase in human tumors using an ultrasensitive enzyme-linked immunoassay

Thymidylate synthase (TS; EC 2.1.1.45) is an important therapeutic target for fluoro-pyrimidine cytotoxic drugs that are widely used for the treatment of solid tumors. Using the monoclonal antibody TS 106, we have developed an ultrasensitive enzyme-linked immunoassay (ELISA) for the detection and quantitation of TS. Using a chemiluminescent ELISA technique, TS was detectable in serially diluted lysates from NCI H630 and HCT 116 human colon carcinoma cell lines. The ELISA assay was reliably able to detect activity down to a level of 30 attamol of TS protein above background (P 2 = 0.016). The usable range of detection was from 0.03 to 500 fmol of enzyme. There was a close correlation between the optical density signal and the total TS enzyme between both cell lines ( r 2 = 0.96). The ELISA was used to measure TS in cytosolic extracts from human tumor samples, and it was able to quantitate TS levels using as little as 1-mg tumor biopsy samples. The mean total TS measured by ELISA in seven tumor samples from patients with breast cancer and sarcomas was 131 fmol/mg cytosolic protein (range 60–240) compared with a mean TS of 85 fmol/mg cytosolic protein (range 35–163) using the fluorodeoxyuridine monophosphate binding assay. While the TS levels were uniformly higher when measured by ELISA, there was close proportional agreement between both assays ( r 2 = 0.84). Thus, the chemiluminescent TS ELISA would appear to be an extremely sensitive and specific assay that may be used to quantitate TS in tumor tissue specimens.