Competitive Immunoassay Technique Research Articles

A novel competitive color-tone change fluorescence immunochromatographic assay for the ultrasensitive detection of pesticide and veterinary drug residues

One important challenge faced by competitive immunoassay techniques is the inability to visually distinguish weak color changes in the T-line during the detection of trace analyte concentrations. In this study, we introduced multi-color fluorescence into the immunoassay system and developed a novel competitive dual-channel color-tone change fluorescent immunochromatographic assay (CFICA) for the visual and quantitative detection of kanamycin (KAN) and carbendazim (CBZ). At the same time, we have developed matching, portable fluorescence-reading equipment, which can analyse the spectral data of the RGB channel individually and can effectively solve the problems of the traditional single-fluorescence reader – large reading error and poor detection sensitivity. After optimization, our CFICA, combined with the corresponding instrument, achieved low limits of detection (LOD) for KAN and CBZ, as low as 1.98 and 12.82 pg/mL, respectively. We believe that this CFICA detection system, with its high accuracy, low error, and multi-color detection capabilities, holds tremendous potential for visual and multi-channel competitive point-of-care testing.

Effect of Gender, Physical Activity and Stress-Related Hormones on Adolescent's Academic Achievements.

Background: Physical activity (PA) has been shown to develop better fitness and body function in children. Various studies have shown that as the age of students increases, its correlation with school achievement decreases. Different hormonal changes during adolescence make it difficult to adjust in his/her environment, causing stress. To the best of our knowledge, no study has studied the correlation between stress-related hormones and school performance among adolescents. This study was conducted to evaluate physical activity and stress-related hormones, cortisol and serotonin, among school adolescents aged 12–18 years old and find their association with academic achievements. Methods: A total of 300 students were invited to participate in this study. Physical activity of the participants was assessed in relation to the time spent performing various physical activities. End of the academic year grades were obtained from the school as a collective measure of academic achievement and executive function. The levels of cortisol and serotonin were measured using the competitive immunoassay techniques. Results: There was a significant correlation between age, gender, BMI, cortisol, serotonin, physical activity score; and academic achievement, and executive functioning among participants. Academic achievement and executive functioning scores correlated positively with gender, serotonin, physical activity score, but negatively with age, BMI and salivary cortisol. Stepwise regression analysis showed that physical activity and demographic parameters and stress-related hormones, cortisol and serotonin, explained around 61.9–77.9% of academic performance and executive functioning variation in school adolescents, especially females. Conclusions: Optimal physical activity and release of stress-related hormones could be the determining factor for performance in school and other activities. These results should be taken into consideration while planning the school curriculum.

SERS-based competitive immunoassay of troponin I and CK-MB markers for early diagnosis of acute myocardial infarction

We report a SERS-based competitive immunoassay technique for the early diagnosis of acute myocardial infarction (AMI). Simultaneous quantification of the dual cardiac markers, CK-MB and troponin I, was achieved by single wavelength excitation.

Assessment of Seminal Estradiol and Testosterone Levels as Predictors of Human Spermatogenesis

The proposed hypothesis for this study was that seminal testosterone/estradiol levels and/or their ratios may be a good indicator for predicting normal spermatogenesis. The concentrations of estradiol and testosterone in seminal fluid were measured using competitive immunoassay techniques in specimens collected from 192 infertile patients and 103 normospermic men. Infertile patients were subdivided into three groups according to their semen analysis results and testicular biopsy: oligozoospermia, obstructive azoospermia (OA), and nonobstructive azoospermia (NOA). Results showed that seminal testosterone levels in the infertile groups were lower than in the normospermic individuals (P < .01), whereas seminal estradiol levels in the OA group were significantly higher than those in normospermic and NOA groups (P < .01). Testosterone/estradiol ratios in the seminal plasma from the infertile groups were significantly lower than that in the normospermic group (P < .01). However, seminal estradiol levels among normospermic and NOA groups showed no significant differences. These results suggest that the local balance between androgen and estrogen, or their ratios, may play an important role in maintaining normal spermatogenesis. Also, decreased seminal testosterone/estradiol ratio may be a good indicator for identifying the absence of sperm production in NOA patients.

A disposable electrochemical immunofiltration test strip for rapid detection of α-fetoprotein

A disposable electrochemical immunofiltration test strip for the rapid detection of α-fetoprotein (AFP) was developed. The test strip was constructed by assembly of screen-printed carbon electrodes, absorption-water pad, nitrocellulose membrane modified by anti-AFP antibody and glass fiber membrane conjugated with ferrocene monocarboxylic acid (FC) labeling AFP. The analytical system utilizes flow-through immunofiltration and competitive immunoassay techniques in combination with an amperometric sensor. The parameters affecting the immunoassay such as selection of filter membrane, membrane pore-size, and antibody binding capacity were investigated and optimized. The immunofiltration system allows us to specifically and directly detect AFP in serum with a low detection limit of 6 ng/mL. The working range is from 6 to 500 ng/mL with an overall analysis time of 5 min for one sample. This electrochemical immunoassay system enabled us to construct a novel point-of-care testing device for the monitoring of biomarker including AFP.

Piezoelectric immunosensor with gold nanoparticles enhanced competitive immunoreaction technique for quantification of aflatoxin B1

A simple, rapid and highly sensitive piezoelectric immunosensor has been proposed and applied to detect aflatoxin B1 (AFB1). It is unlikely that direct binding of small molecules such like AFB1 to the piezoelectric sensor surface could result in a satisfactory detection limit and sensitivity. Thus, indirect competitive immunoassay technique had been used for the detection of the target and gold nanoparticles (GNP) been employed as a ‘weight label’ to the secondary antibody for amplifying the response. This method is proven in its ability to detect AFB1 down to a level of 0.01ngmL−1 in artificially contaminated milk, which is comparable to or even exceeding the sensitivity of microtitre plate ELISA. Furthermore, the frequency responses of the immunoassay are linearly correlated to the logarithm of AFB1 concentration in the range of 0.1–100ngmL−1. The sensor could be regenerated under very mild conditions simply by immersing the sensor into glycine buffer solution to desorb the combined antibody. It is found that the as-renewed sensor could be reused at least 9 runs without obvious loss of sensing sensitivity.

Detection of Deoxynivalenol in Foods and Indoor Air Using an Array Biosensor

Deoxynivalenol (DON), a mycotoxin produced by several Fusaruim species, is a worldwide contaminant of foods and feeds. Because of the potential dangers due to accidental or intentional contamination of foods with DON, there is a need to develop a rapid and highly sensitive method for easy identification and quantification of DON. In this study, we have developed and utilized a competitive immunoassay technique to detect DON in various food matrixes and indoor air samples using an array biosensor. A DON-biotin conjugate, immobilized on a NeutrAvidin-coated optical waveguide, competed with the DON in the sample for binding to fluorescently labeled DON monoclonal antibodies. To demonstrate a simple procedure amenable for on-site use, DON-spiked cornmeal, cornflakes, wheat, barley, and oats were extracted with methanol-water (3:1) and assayed without cleanup or preconcentration. The limits of detection ranged from 0.2 ng/mL in buffer to 50 ng/g in oats. The detection limit of DON spiked into an aqueous effluent from an air sampler was 4 ng/mL.

Influence of Circadian Time Structure on Acute Hormonal Responses to a Single Bout of Heavy-Resistance Exercise in Weight-Trained Men

Both testosterone (T) and cortisol (C) exhibit circadian rhythmicity being highest in the morning and lowest in the evening. T is a potent stimulator of protein synthesis and may possess anti-catabolic properties within skeletal muscle, and C affects protein turnover, thereby altering the balance between hormone-mediated anabolic and catabolic activity. Physiological reactions of these hormones and training adaptations may influence the post-exercise recovery phase by modulating anabolic and catabolic processes, therefore affecting metabolic equilibrium, and may lead to intensification of catabolic processes. We investigated the effect of the circadian system on the T and C response of weight-trained men to heavy resistance exercise. Thirteen young (21.8±2.2 yr) weight-trained men (12 months training experience) performed an eight-station heavy-resistance exercise protocol on two separate occasions (AM: 06:00 h and PM: 18:00 h), completing 3 sets of 8–10 repetitions at 75% of each subject's one-repetition maximum (1-RM). Blood samples were obtained prior to, during, and following the exercise bout, and serum total T and C concentrations were determined by competitive immunoassay technique. Performing the single bout of heavy-resistance exercise in the PM as compared to the AM positively altered the C and T/C ratio hormonal response. Pre-exercise C concentrations were significantly lower (p < 0.05) in the PM session, which resulted in a lower peak value, and the accompanying increased T/C ratio suggested a reduced catabolic environment. These data demonstrate that the exercise-induced hormonal profile can be influenced by the circadian time structure toward a profile more favorable for anabolism, therefore optimizing skeletal muscle hypertrophic adaptations associated with resistance exercise.

Detection of benzo[a]pyrene-DNA adducts in human placenta and umbilical cord blood.

Placenta constitutes a vital organ of exchange between mother and foetus. In addition to this favourable effect for foetal development, placenta indirectly may allow transfer of several maternal blood xenobiotics. Human placenta and umbilical cord blood are interesting models for investigating maternal environment and the metabolism, the bioactivation and the transfer of carcinogens such as polycyclic aromatic hydrocarbons. We used them to assess the effect of a woman's smoking on the foetus. Few studies cover this subject. In pregnant women who have continued to smoke, benzo[a]pyrene compound of cigarette smoke is metabolically activated to diol-epoxide derivative: benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide, ultimate carcinogen (BPDE-I). This derivative is covalently fixed on DNA and gives BPDE-I-DNA adducts. By a competitive immunoassay technique, we determined BDPE-I-DNA adducts in 20 samples of placenta and umbilical cord blood from women who smoked (n = 15) and who did not (n = 10). Tobacco consumption was checked by urinary cotinine determination. In the group of smokers levels of adducts were found in 13 specimens of placenta (from 10 to 60 fmol/50 micrograms of DNA) and 12 umbilical cord blood (from 10 to 22.15 fmol/50 micrograms of DNA) samples. These results indicate that a mother's tobacco consumption is linked to the accumulation of BPDE-I-DNA adducts in the placenta, which are seen in smaller quantities in the umbilical cord blood, probably because of the metabolic capacity of the placenta and the transfer of B[a]P from the mother to the foetus.

New immunoassay techniques using Nafion-modified electrodes and cationic redox labels or enzyme labels

Two new competitive immunoassay techniques with sensitive electrochemical detection at Nafion film modified electrodes are described and compared. Method A implies the use of an antigen labeled with a cationic redox group in homogeneous immunoassay. Method B involves an enzyme-labeled antigen, in connection with an anionic substrate + cationic electroactive product of the enzymatic reaction in heterogeneous assay. A 5 min accumulation step precedes the electrochemical detection in both methods. Method B can be applied to the determination of any kind of antigen, whereas method A is restricted to haptens. Phenytoin, an anti-epileptic drug, was chosen as a model antigen and labeled by cobaltocenium and alkaline phosphatase to illustrate methods A and B, respectively. The enzymatic reaction was performed with N-ferrocenoyl)-6-amino-2,4-dimethylphosphate as substrate in method B. The dianionic substrate was repulsed from the Nafion film, whereas the corresponding enzymatically generated alcohol was selectively entrapped within the film as a ferricinium salt by applying a potential of 0.6 V vs. solAg AgCl during the accumulation step performed at pH 7.4. Both methods were tested for the determination of phenytoin in clinical serum samples.

Homogeneous electrochemical immunoassay using a perfluorosulfonated ionomer-modified electrode as detector for a cationic-labeled hapten

A Nafion film-modified electrode has been used as an electroanalytical sensor to detect a drug containing a cationic redox label by square wave voltammetry. A tracer, composed of the model drug amphetamine covalently attached to a cobaltocenium redox label, was synthesized and used in a competitive immunoassay technique. In this assay, only the positively charged tracer not vound to the antibody preconcentrated in the anionic film. Consequently, it was not necessary to separate free from antibody-bound labeled amphetamine, thereby allowing the assay to be carried out in homogeneous media at the electrode surface