Competitive Enzyme Immunoassay Method Research Articles

Effects of thyroxine immersion on Sterlet sturgeon (Acipenser ruthenus) embryos and larvae: Variations in thyroid hormone levels during development

This study was planned to investigate the effects of immersion of Sterlet sturgeon (Acipenser ruthenus) eggs by thyroxine hormone (T4) on the embryonic survival rate (ESR), hatching rate (HR), larval survival rate (LSR), and thyroid hormone levels. Hence, oocytes of six females were pooled, fertilized with pooled semen of three males and 45 min after fertilization process, the eggs were placed 120 min in four batches that were immersed in T4 solutions as follows: D1 (the control treatment: T4-free, hatchery water), D2 (0.01 mg L−1 of T4), D3 (0.05 mg L−1 of T4), D4 (0.1 mg L−1 of T4), and D5 (1 mg L−1 of T4). Next, the eggs were transferred to the incubator. The ESR was recorded 6 h after fertilization while HR, and LSR were measured at 1 day post hatching. In addition, the levels of thyroid hormones were measured using the competitive enzyme immunoassay method three hours and three days after fertilization and at the five-day pre-larval stage. The highest ESR was obtained following D5 treatment (P < .05). Besides, the highest and lowest HR were observed in D5 and D1 treatments, respectively (P < .05). The D5 treatment resulted in the highest pre-larval SR. During all sampling times, the D5 treatment resulted in the highest level of thyroid hormones among the other treatments (P < .05). However, the T3:T4 ratio was significant only within the three days after fertilization in D1 treatment (P < .05). In general, it can be concluded that the immersion of Sterlet sturgeon eggs in T4 improved the ESR, hatching, and LSR of this species, resulting in a promising future for the aquaculture of Sterlet sturgeon and other sturgeon species.

Direct competitive enzyme immunoassay method for the determination of ochratoxin a in feeds and foods

A reagent kit EIA-OCHRATOXIN A for the determination of mycotoxin ochratoxin A in feeds and foods by a direct competitive enzyme immunoassay using microtitration plate has been developed and tested. The conjugate of ochratoxin A with horseradish peroxidase was synthesized using the mycotoxin aminoderivative joined to the carbohydrate chain of the enzyme. A monoclonal antibody to the mycotoxin was immobilized on the surface of plate wells through sheep anti-mouse antibodies. The evaluated parameters of the kit and metrological characteristics of the technique of measurements correspond to the modern level of immunoassay development and provide the determination of ochratoxin A content of agricultural products in a range of 5 to 375 mg/kg with proper accuracy and precision. The limit of quantitative determination of ochratoxin A in grain and cereal foods does not exceed 5 mg/kg.

Malachite green residue in farmed fish in north-west part of Iran

ABSTRACTThe occurrence of malachite green (MG) residue was determined in 177 farmed fish samples (i.e., 85 carp, Cyprinus carpio, and 92 rainbow trout, Oncorhynchus mykiss) purchased from north-west part of Iran from 2014 to 2015. Competitive enzyme immunoassay method validated according to the criteria of Commission Decision 2002/657/EC was used to determine the MG residue. The mean recoveries were in the range of 79.1–86.2% and the limit of detection of the method was 0.30 µg/kg. The value of detection capability for MG in fish muscle was less than 1 µg/kg, which was below the minimum required performance limit of 2 µg/kg. MG was detected in 108 fish samples, corresponding to 61.0% of the total examined samples, ranging between 0.35 and 7.12 µg/kg. Most of the contaminated samples (28.8%) contained MG at the level of 0.3–1 µg/kg. Altogether, 18 out of 177 samples contained residue of MG higher than 2 µg/kg. The results indicated that the usage of MG in Iranian fish farms is frequent. It is alarming and could be a potential hazard for public health.

Aflatoxin M1 contamination in white and Lighvan cheese marketed in Rafsanjan, Iran

This study was a screening survey to determine the occurrence of aflatoxin M1 (AFM1) in 82 cheese samples composed of white cheese (45 samples) and Lighvan cheese (37 samples) obtained from supermarkets and retail outlets in Rafsanjan city of Iran. The competitive enzyme immunoassay method was used for determination of the toxin in the samples. Aflatoxin M1 was detected in 39 (47.6%) samples, consisting of 29 (64.4%) white cheese (mean: 135 ng/kg; range: 93.3–309 ng/kg) and 10 (27%) Lighvan cheese samples (mean: 90.8 ng/kg; range: 70.5–203 ng/kg). According to Iranian national standard limit for AFM1 in cheese (200 ng/kg), 9 samples (20%) of white cheese and 1 sample (2.70%) of Lighvan cheese had levels above the limit. It was concluded that the contamination of the samples with AFM1 in such a level could be considered as a serious public health problem.

Aflatoxin M 1 in raw milk in Croatia

In this study the levels of aflatoxin M 1 (AFM 1) of 61 milk samples delivered from small milking farms were determined in January, February, March and April (winter–spring season), and June, July and September (summer–autumn season) of 2009. The AFM 1 concentration was determined by competitive enzyme-immunoassay method. The maximum mean concentrations of AFM 1 recorded in winter–spring season were in the range of 35.8–58.6 ng/l and in summer–autumn season in the range of 11.6–14.9 ng/l. The AFM 1 levels determined in January, February, March and April were significantly higher in accordance with concentration of AFM 1 in June, July and September ( P < 0.05 to P < 0.0001, respectively). Also, there was significant difference ( P < 0.0001) between the mean concentrations of AFM 1 in samples taken all together in winter–spring and summer–autumn season. Only in one sample delivered in February the level of AFM 1 was higher than the maximum tolerance limit (50 ng/l). Therefore, it was concluded that in 98.4% of milk samples in Croatia the levels of AFM 1 were below maximum tolerance level accepted by the European Union.

Effects of Sexually Activated Rams or Ewes on Pulsatile LH Secretion in Anoestrous Sheep

home Acta Veterinaria Brno Journal of the University of Veterinary Sciences Brno, Czech Republic Search:

Production of prostaglandins E 1 and E 2 by adult human red blood cells

We showed that human adult red blood cells (RBCs) produce prostaglandin E 1 (PGE 1) and E 2 (PGE 2). RBCs that were mechanically stressed in the presence of extracellular Ca 2+ by being injected rapidly through a fine needle produced PGE 1 and PGE 2 within 30 min after this mechanical stress. The amounts of PGE 1 and PGE 2 produced by 1 × 10 9 mechanically stressed RBCs were approximately 50 pg and 100 pg, respectively, which were determined in the cytosolic fraction from sonicated RBCs using a competitive enzyme immunoassay method. A Western blot analysis using anti-cyclooxygenase-2 antibody revealed a band at the 70-kDa position in the samples from RBCs producing PGE 1 and PGE 2. Treatment with 10 μg/mL indomethacin completely inhibited the productions of PGE 1 and PGE 2. The present results may indicate a new role of RBCs in microcirculation.

A novel enzyme immunoassay for total thyroxine using immobilized antibodies and hydrophobic chromatography purified thyroxine-peroxidase conjugate

A new and simple competitive enzyme immunoassay method for the measurement of total thyroxine in serum is described. Anti-thyroxine antibodies, raised in sheep with a bovine serum albumin-thyroxine conjugate prepared with carbodiimide as coupling initiator, were physically adsorbed onto a large surface area polypropylene support. Competition occurred between thyroxine in the sample and a thyroxine-peroxidase conjugate prepared with a glutaraldehyde spacer and further purified by octyl Sepharose hydrophobic chromatography. The entire assay was performed in 2 h with a useful range of 10–300 μg/l. The coefficients of variation ranged from 6.9 to 12% and good agreement ( r = 0.93–0.97) was found between this method and 2 radioimmunoassays on 71 serum samples having well distributed thyroxine values.

Immunoassay of melatonin with enzyme-labeled antibodies.

This paper reports a non equilibrium competitive enzyme immunoassay method using enzyme-labeled antibodies, for the quantitation of melatonin in chloroform-extracted samples. Its principle is as follows: methoxytryptamine hemisuccinate-human serum albumin conjugate physically absorbed onto a polystyrene sphere and melatonin to be measured, compete for a limited and fixed amount of peroxidase-labeled anti melatonin IgG. After incubation and washings the enzymatic activity bound to the sphere was measured with a chromogenic substrate. This simple method can detect as low as 22 fm of melatonin and is fairly precise. We also present its application to the determination of melatonin in serum and pineal gland.

Production of antibodies to calmodulin in rabbits and enzyme immunoassays for calmodulin and anti-calmodulin.

The production of anti-calmodulin antibodies in rabbit was examined with the use of performic acid-oxidized calmodulin from bovine brain or an emulsion of native calmodulin and methylated bovine serum albumin as the antigen. The antibodies in rabbit sera were determined serially by means of a sensitive enzyme immunoassay method that uses a mini-column of goat (anti-rabbit IgG) IgG-coupled Sepharose 4B for separation. The antibodies could be produced in rabbits with either antigen. Although the native calmodulin with methylated bovine serum albumin seemed to be a better antigen, the titer of antiserum was not raised to levels detectable in the double immunodiffusion test. Monospecific antibodies were purified from the antiserum, and a competitive enzyme immunoassay method for the assay of calmodulin was developed with the use of the column-separation technique employed in the assay of anti-calmodulin. The method could determine from 10 ng to 10 micrograms (0.6 pmol to 0.6 nmol) of rat calmodulin and gave no cross-reaction with S-100 protein. Calmodulin contents in rat brain determined by the present method were consistent with those measured by the bioassay using calmodulin-deficient phosphodiesterase.