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A microplate chemiluminescence enzyme immunoassay (CLEIA) with high sensitivity, selectivity and reproducibility was developed for the determination of free thyroxine (FT4) in human serum. A competitive assay has been utilized with horseradish peroxidase (HRP) labeled thyroxine analog in the chemiluminescence (CL) detection. The CL signal produced...
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... amount of enzyme-T4 analog conjugate binding to the solid phase varies inversely with the concentration of FT4 in the serum, constituting the estimation of FT4 concentration in the serum. The competitive reaction principle is shown in Figure 1. It is based on the HRP-T4 analog conjugate and the chemiluminesence reaction between the enzyme sub- strate of luminol and hydrogen peroxide. ...
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A coating procedure that could provide immobilization of antibodies, with increased binding capacity, that is cost effective, simple, robust, and appropriate for production scale application, is described. This coating approach of T3 antibodies to the polystyrene tubes has been systematically investigated to determine its utility for the developmen...
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... In ELISA, a targeted antigen can be quantified by measuring the activity of the labeling enzyme after the formation of the enzyme-labeled immunocomplex (antigen-antibody). Compared with radioimmunoassay [6], which employs radioisotope labeling, ELISA is easier to handle, safer, and has excellent stability [7]. The signal generated in ELISA by the enzyme could be color development, fluorescence, or chemiluminescence (CL). ...
... The signal generated in ELISA by the enzyme could be color development, fluorescence, or chemiluminescence (CL). A method wherein the enzyme is used to produce a CL signal is called a chemiluminescent enzyme immunoassay (CLEIA) [7,8]. CLEIA is widely used because of CL's highly sensitive detection, simplicity, and wide dynamic range [9][10][11][12]. ...
The most used kind of immunoassay is enzyme-linked immunosorbent assay (ELISA); however, enzymes suffer from steric effects, low stability, and high cost. Our research group has been developing quinone-linked immunosorbent assay (QuLISA) as a new promising approach for stable and cost-efficient immunoassay. However, the developed QuLISA suffered from low water-solubility of synthesized quinone labels and their moderate sensitivity. Herein, we developed a new approach for signal multiplication of QuLISA utilizing the water-soluble quinone anthracycline, doxorubicin, coupled with dextran for signal multiplication. A new compound, Biotin-DexDox, was prepared in which doxorubicin was assembled on oxidized dextran 40, and then it was biotinylated. The redox-cycle-based chemiluminescence and the colorimetric reaction of Biotin-DexDox were optimized and evaluated, and they showed very good sensitivity down to 0.25 and 0.23 nM, respectively. Then, Biotin-DexDox was employed for the detection of biotinylated antibodies utilizing avidin as a binder and a colorimetric assay of the formed complex through its contained doxorubicin redox reaction with NaBH4 and imidazolium salt yielding strong absorbance at 510 nm. The method could detect the plate-fixed antibody down to 0.55 nM. Hence, the application of Biotin-DexDox in QuLISA was successfully demonstrated and showed a significant improvement in its sensitivity and applicability to aqueous assays.
... [8,17,18]. Enzymatic labeling of immunoassay is more sensitive and safer than radioimmunoassay, which widened its use [9,19]. However, enzyme labeling of immunoassays possesses many complications, including the instability of enzymes and their big size that will lead to non-reproducible results and steric inhibition of Ag-Ab reaction, respectively. ...
Enzyme-labeled immunoassays are widely used for trace analysis of clinically significant compounds owing to their high selectivity. However, utilizing an enzyme for labeling the antibody leads to many problems, including low reproducibility and inhibition of antigen-antibody immuno-reaction due to the enzymes’ instability and bulkiness, respectively. Hence, our research group has been using quinones as stable non-enzymatic tags for antibodies utilizing the redox cycle of quinones. Herein, we aimed to use the broadly available and cost-effective quinone, 1,2-naphthoquinone-4-sulfonate (NQS, Folin’s reagent), for signal tagging of immunoassays. NQS could bond with biotin in a relatively short time and without using a catalyst forming biotin-1,2-naphthoquinone (BT-NQ). The synthesized BT-NQ produces strong chemiluminescence or color upon mixing with reductant and luminol or tetrazolium salts, with sensitivity down to 7.7 nM and 49.0 nM, respectively. Next, BT-NQ was used for developing the first quinone-linked immunosorbent assay (QuLISA) through labeling biotinylated-detection antibodies using avidin and BT-NQ, and it was targeted to detect food allergen, β-casein using sandwich-immunoassay. Our method showed good linearity for determination of β-casein with good sensitivity down to 20.2 ng/mL. The results of our method were compared with ELISA kit results, and QuLISA showed good matching and higher sensitivity. Moreover, we applied Folin’s reagent for direct labeling of avidin, and the resulted compound possessed strong chemiluminescence originating from its quinone content. At last, we can conclude that the use of Folin’s reagent offered a simple, stable, sensitive, and cost-effective approach for labeling immunoassays and direct chemiluminescence labeling of proteins.
... Due to hypothyroidism caused by the removal of part or all of the gland due to the dysfunction, patients receive a salt form of the hormone substitutelevothyroxine-Na pentahydrate throughout life. Side effects of the drug, such as weight gain, hair loss, impaired renal function, tachycardia, arrhythmias, sleep disorders, unexplained anxiety and etc., are observed due to increased appetite during long-term use [14][15][16]. ...
Immobilization of a thyroid hormone substitute of L-thyroxine on chitosan-based smart hydrogel structure was performed to reduce the side effects of the required daily dose. The hydrogel was obtained by the Schiff reaction of chitosan with acetaldehyde and quaternization with methyl iodide then by ion exchange with NaCl salt. Finally, a hydrogel was obtained from the treatment of chitosan-based salt at -20 °C. The amount of levothyroxine immobilized by physical sorption on the hydrogel was determined by molecular electron spectroscopy on basis especially absorption peak at 227 nm. Results of IR- and UV-Vis spectroscopic analyses revealed that the interaction between levothyroxine and hydrogel occurs mainly due to hydrogen bonding and orientation forces of intermolecular interaction.
... It revealed that an increase in temperature of 30-37 °C leads to an increase in the capacity of the levothyroxine which proves that the process is endothermic in nature. Also, an increase in the mobility of the macromolecule up to 37 °C leads to the opening of active sites, and this process is accelerated by increasing the mobility of the levothyroxine molecule [14]. This proves that levothyroxine is immobilized to DEMX-based gel by chemical sorption between 30 and 40 °C. ...
In order to reduce the side effects of thyroid hormone substitute Na-levothyroxine pentahydrate, its sorption with a quaternized salt of a new alkyl derivative of chitosan was studied. The amount of the drug in the salt (gel) is in micrograms, and the gel-levothyroxine is in the form of a complex that can show biological activity. For this purpose, a sorption process of levothyroxine-Na from an aqueous solution to the inside and surface of the hydrogel was carried out under static conditions. The capacity of the hydrogel was studied depending on pH medium, the ionic strength, the dose of hydrogel, the concentration of the drug and the temperature. It was shown that the effective sorption of levothyroxine by chitosan-based hydrogel was optimal at a pH of 6-8.5, in the presence of 10-50 mg of hydrogel dose and at a 50 mg/L concentration of levothyroxine but the sorption degree begins to decreases after T=40 °C. The isotherm results of sorption processes have been found to be subordinate mainly to Langmuir and to some extent Freundlich equations. It was shown that gel degradation in the oxidizing medium is about 70% within 2 weeks, and in the elastase and PBS medium is about 17-20%.
... It revealed that an increase in temperature of 30-37 °C leads to an increase in the capacity of the levothyroxine which proves that the process is endothermic in nature. Also, an increase in the mobility of the macromolecule up to 37 °C leads to the opening of active sites, and this process is accelerated by increasing the mobility of the levothyroxine molecule [14]. This proves that levothyroxine is immobilized to DEMX-based gel by chemical sorption between 30 and 40 °C. ...
In order to reduce the side effects of thyroid hormone substitute levothyroxine sodium pentahydrate, its sorption with a quaternized salt of a new alkyl derivative of chitosan was studied. The drug amount in the salt (gel) is in micrograms, and the gel-levothyroxine is in the form of a complex that can show biological activity. With that end in view, a sorption process of levothyroxine sodium from an aqueous solution to the inside and surface of the hydrogel was carried out under static conditions. The capacity of the hydrogel depending upon the pH medium, the ionic strength, the hydrogel dose, the concentration of the drug and the temperature was studied. It was shown that the effective sorption of levothyroxine by chitosan-based hydrogel was optimal at pH of 6-8.5, at 50 mg/L concentration of levothyroxine in the presence of 10-50 mg of hydrogel dose but the sorption degree begins to decrease after T=40 °C. The isotherm results of sorption processes have been found to be subordinate mainly to Langmuir and to some extent Freundlich equations. It revealed that gel degradation in the oxidizing medium is about 70% within 2 weeks, and in the elastase and PBS medium is about 17-20%.
... The targeted antigen can be quantified by measuring the enzyme activity of the antigen-antibody immune-conjugate (Gan and Patel, 2013). This method is widely used at present as it is superior in sensitivity, safety, and applicability as compared with radioimmunoassay that uses radioactive isotope label (Banga-Mboko et al., 2003;Wang et al., 2007). Among enzyme immunoassays, a method utilizing an enzyme to catalyze CL reaction is called CL-enzyme immunoassays (Marquette and Blum, 2009;Nishizono et al., 1991). ...
Chemiluminescence-enzyme immunoassays make it possible to measure trace components with high sensitivity and selectivity due to the high specificity of the antigen-antibody reaction and high sensitivity of chemiluminescence assays. However, using an enzyme-labeled antibody suffers from many problems such as low reproducibility due to the instability of the enzyme and inhibition of antigen-antibody reaction due to its steric effect. Therefore, herein we report an innovative non-enzymatic chemiluminescence immunoassays labeling reagent through using quinone as a signal-generating tag coupled with biotin as a binder, to overcome enzymatic labeling problems. Biotinylated-1,4-naphthoquinone (biotin-NQ) was synthesized and characterized and it could produce long-lasting chemiluminescence upon mixing with dithiothreitol and luminol based on the redox cycle of quinone. Biotin-NQ showed exceptional stability towards different stress factors that may be encountered during performing the immunoassay such as high temperatures, highly acidic and alkaline conditions, and repeated freeze-thaw cycles. On the other hand, all these conditions lead to decreased labeling enzyme reactivity due to possible denaturation of its protein structure. Finally, the measurement of the biotin-labeled antibody was successfully performed using biotin-NQ and avidin. As a result, the antibody could be detected down to 25.7 nM which is 2.5 times sensitive than biotin-HRP chemiluminescence-enzyme immunoassays. Moreover, our method was applied successfully for determination of avidin using immobilized biotinylated antibody and biotin-NQ, which simulates immunoassays. Avidin could be detected down to 23.4 nM with excellent linearity (r = 0.996). Accordingly, our developed reagent, biotin-NQ, could be used as a universal highly stable, cost-effective, and steric free non-enzymatic label for immunoassays.
... thyroid gland causes hyperthyroidism, which involves symptoms such as heat intolerance, weight loss, diarrhea, and an enlarged thyroid gland (Fig. S1B). On the contrary, lack of thyroid hormones (hypothyroidism) can lead to sluggishness, depression, constipation, and weight gain [10][11][12]. These disorders are diagnosed by measuring TSH and T4 levels, and measurement of T3 is useful for more accurate diagnosis [13][14][15]. ...
Thyroid-stimulating hormone (TSH) secretion plays a critical role in regulating thyroid gland function and circulating thyroid hormones (i.e., thyroxine (T4) and triiodothyronine (T3)). A novel multi-immunoreaction-based dual-color capillary electrophoresis (CE) technique was investigated in this study to assess its reliability in diagnosing thyroid gland disease via simultaneous detection of TSH, T3, and T4 in a single run of CE. Compared to the conventional immunoreaction technique, multi-immunoreaction of biotinylated streptavidin antibodies increased the selectivity and sensitivity for individual hormones in human blood samples. Dual-color laser-induced fluorescence (LIF) detection-based CE performed in a running buffer of 25 mM Na2B4O7-NaOH (pH 9.3) allowed for fast, simultaneous quantitative analysis of three target thyroid hormones using different excited wavelengths within 3.2 min. This process had excellent sensitivity and detection limits of 0.05-5.32 fM. The results showed 1,000-100,000 times higher detection sensitivity than previous methods. Method validation with enzyme linked immunosorbent assay for application with human blood samples showed that the CE method was not significantly different at the 98% confidence level. Therefore, the developed CE-LIF method has the advantages of high detection sensitivity, faster analysis time, and smaller sample amount compared to the conventional methods The combined multi-immunoreaction and dual-color CE-LIF method should have increased diagnostic relia bility for thyroid gland disease compared to conventional methods based on its highly sensitive detection of thyroid hormones using a single injection and high-throughput screening.
... Lumimol and horseradish peroxidase (HRP) are most widely used as the CL label and catalyst, respectively [8,9]. In the majority of sandwich-type methods, a microplate is always used as an immobilization support [10]. Based on these, many of the classical biomarkers related to diseases in human blood have been detected accurately [11][12][13], which have greatly promoted the development of clinical diagnostic studies. ...
... The effect of labeling time on the RLU and concentration of components in each elute tube. (A-D) correspond to a series of durations required for the labeling reaction(10,20,30, and 60 min, respectively). ...
An ultrasensitive and rapid sandwich-type chemiluminescence immunoassay (CLIA) was developed for the clinical determination of human epididymis protein 4 (HE4) in human serum, using GoldMag nanoparticles as solid phase and acridinium ester (AE) as chemiluminescence system (GMP-CLIA). The process of AE labeling antibodies was systematically studied and evaluated. The effect of varies factors such as molar ratio of AE to antibodies, labeling time, and the components of elution buffer and trigger solution were optimized. Under the selected conditions, AE labeling experiments were successfully performed with the average labeling efficiency of 1.92 ± 0.08, and antibody utilization rate of 69.77 ± 1.19%. Antibody activity remained unchanged after labeling. The established GMP-CLIA method can detect HE4 in the range of 0.25-50 ngmL⁻¹ (10-2000 pM) with a detection limit of 0.084 ngmL⁻¹ (3.36 pM). The sensitivity has reached a high level, comparable with the current commercial detection kits. This proposed method has been successfully applied to the clinical determination of HE4 in 65 human sera. The results showed a good correlation with a clinical method, microplate-based chemiluminescence enzyme immunoassay (CLEIA), with the correlation coefficient of 0.9594.
... It has been applied broadly in clinical and biomedical research due to such advantages as relatively simple and inexpensive instrumentation, low detection limit, and broad dynamic range. [23][24][25][26] Chemiluminescence detection methods commonly employ horseradish peroxidase (HRP) and alkaline phosphatase (ALP) for enzymatic signal amplication. ...
Multiplexed detection of pathogen-specific DNA sequences in a simple and reliable way is in great demand for clinical and biomedical applications. However, there is still a lack of available DNA detection methods that are simple and pathogen-selective for point-of-care (POC) testing. Here, we report a novel zinc finger protein (ZFP)-based chemiluminescence method for direct detection of pathogenic double-stranded DNA (dsDNA) in a multiplexed platform. ZFPs are custom-designed to identify unique pathogenic DNA sequences. ZFP-based chemiluminescence detection of dsDNA provides sufficient sensitivity (≤50 fmol) and high specificity without target DNA amplification. Our study addresses the potential of developing a simple and selective pathogen detection method in a multiplexed fashion needed for POC applications.
... Immunoassays are based on the highly specific molecular recognition of antibodies and antigens, which facilitates the sensitive detection of biomolecules such as proteins (Dipti et al., 2012;Kim et al., 2012;Lin et al., 2012;Maple et al., 2012), hormones (Borque et al., 2011;Islam et al., 2011;Troutt et al., 2011;Wang et al., 2007), and drugs, as well as microorganisms (Sandhu et al., 2012). Immunoassays have developed significantly in recent years due to improvements in their specificity, analytical time, and practicability, which can be attributed to the elimination of complex separation and extraction steps. ...
The high sensitivity of optical detection techniques and the highly specific reactions between antibodies and antigens mean that optical immunoassays have attracted much interest in the fields of protein, hormone, drug, and microorganism detection, without the need for complex separation and extraction steps. The immobilization of an antibody on a solid support is a crucial step for optical immunoassays. This review surveys the latest advances in current antibody immobilization techniques in detail, including physical adsorption, covalent attachment, bioaffinity immobilization, and some recently developed methods. Furthermore, specific consideration is given to oriented immobilization, which may improve the homogeneous surface covering the accessibility of the active site and surface coverage, and the analytical performance of immunoassays. Finally, new perspectives for antibody immobilization techniques in optical immunoassays are outlined.
