Immunoradiometric assay for in-vitro determination of prolactin hormone in human serum or plasma using solid phase anti-PRL cellulose particles

The objective of the present study was oriented to produce and purify polyclonal anti-PRL antibody as the main key store in immunoradiometric assay (IRMA) using solid phase cellulose particles for determination of PRL in human sera. The preparation of ¹²⁵I-PRL was carried out by lactoperoxidase method for estimation of the titre of antibody production. The preparation of standards was undertaken. The activation of cellulose particles using 1,1-carbonyl diimidazole (CDI) and coupling of these solid phase particles with purified Rabbit anti-PRL were carried out. Optimization and validation of the assay were carried out. Results revealed that the produced PRL polyclonal antibodies have high titre. Cellulose particles IRMA system was highly sensitive and specific. The intra- and inter-assay variations were satisfactory. The recovery and dilution tests indicated accurate calibration and appropriate matrix. The present technique agreed well with IRMA commercial kit. These cellulose particles retained their characteristics during storage for 6 months at 4 °C. In conclusion, this low cost assay could be used as a useful diagnostic tool for diagnosis and follow up of galactorrhea, infertility and pituitary adenoma.     

Development of a functional impedimetric immunosensor for accurate detection of thyroid-stimulating hormone

Thyroid-stimulating hormone (TSH), which regulates the synthesis of thyroid gland hormones affecting the whole metabolism, is a pituitary hormone. Determination of TSH is crucial for monitoring thyroid gland-related disorders and some metabolic diseases.In this study, a nonlabeled immunosensor based on covalent immobilization of anti-TSH antibody by using the formation of self-assembled monolayers (SAM) of 4-mercaptophenylacetic acid (4-MPA) and functionalization of carboxyl ends with 1-ethyl-3-(3-dimetilaminopropil) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) was fabricated for detection of TSH. Immobilization steps including the concentration of 4-MPA, the concentration of anti-TSH antibody, and duration of anti-TSH antibody incubation were optimized by utilizing electrochemical impedance spectroscopy. Under optimal conditions, a sensitive, rapid, and accurate determination of TSH at a concentration range between 0.7 and 3.5 mIU/L was accomplished with a notable linearity and LOD value of 0.034 mIU/L, as well as reproducibility and repeatability. Moreover, for comparison, linear range experiments were also carried out by using other electrochemical methods, including linear sweep voltammetry, cyclic voltammetry, and capacitance spectroscopy. Finally, the constructed immunosensor was used for analyzing TSH levels spiked in the artificial serum samples.     

Zinc oxide nanoparticles based microfluidic immunosensor applied in congenital hypothyroidism screening

In this article, we present an innovative approach for congenital hypothyroidism (CHT) screening. This pathology is the most common preventable cause of mental retardation, affecting newborns around the world. Its consequences could be avoided with an early diagnosis through the thyrotropin (TSH) level measurement. To accomplish the determination of TSH, synthesized zinc oxide (ZnO) nanobeads (NBs) covered by chitosan (CH), ZnO-CH NBs, were covalently attached to the central channel of the designed microfluidic device. These beads were employed as platform for anti-TSH monoclonal antibody immobilization to specifically recognize and capture TSH in neonatal samples without any special pretreatment. Afterwards, the amount of this trapped hormone was quantified by horseradish peroxidase (HRP)-conjugated anti-TSH antibody. HRP reacted with its enzymatic substrate in a redox process, which resulted in the appearance of a current whose magnitude was directly proportional to the level of TSH in the neonatal sample. The structure and morphology of synthesized ZnO-CH NBs were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The calculated detection limits for electrochemical detection and the enzyme-linked immunosorbent assay procedure were 0.00087 μUI mL^?1 and 0.015 μUI mL^?1, respectively, and the within- and between-assay coefficients of variation were below 6.31 % for the proposed method. According to the cut-off value for TSH neonatal screening, a reasonably good limit of detection was achieved. These above-mentioned features make the system advantageous for routine clinical analysis adaptation.     

Enzyme-loaded liposome with biocatalytic precipitation for potentiometric immunoassay of thyroid-stimulating hormone in thyroid carcinoma

A simple and feasible potentiometric immunosensing platform based on enzymatic biocatalytic precipitation technique was designed for the sensitive detection of thyroid-stimulating hormone(TSH;a typical kind of biomarkers for thyroid carcinoma),using horseradish peroxidase(HRP)-loaded liposome for the signal amplification.To construct such an assay system,a sandwich-type immunoreaction was readily carried out on monoclonal anti-TSH capture antibody-coated electrode by using polyclonal antiTSH secondary antibody-conjugated HRP-loaded liposome.Accompanying the formation of sandwichtype immunocomplex,the carried liposome was lysed through the added Triton X-100 to release the entrapped HRP molecules,which catalyzed the oxidation of 4-chloro-1-naphthol to produce an insoluble and uncharged organic precipitation on the electrode surface,thereby causing the change of the local electrical potential.Two labeling protocols with and without the liposome were investigated for detection of target TSH,improved analytical features were achieved with HRP-entrapped liposome.Under optimal conditions,the potentiometric immunosensor had good responses for TSH detection within the linear range of 0.01-30 p IU/mL at a detection limit of 0.0067 p IU/mL.Good reproducibility,high specificity and long-time stability were acquired during the assay procedure.Importantly,a wellmatched accuracy between the potentiometric immunosensor and commercial human TSH ELISA kit was gave for the analysis of human serum samples.     

New adjuvant design using layered double hydroxide for production of polyclonal antibodies in radioimmunoassay techniques

Various adjuvants have been used to enhance the immune response against specific antigens. So the objective of this work describes the immune stimulating activity of layered double hydroxide (LDH) particles incorporate with mineral oil as a new formulation of adjuvant as compared to known Freund’s adjuvant for production of alpha-fetoprotein polyclonal antibody (anti-AFP) for estimation of alpha fetoprotein (AFP) in human serum by radioimmunoassay technique. In this concern, the study comprised two groups of white New Zealand rabbits, 2?2.5 kg body weight and each group comprised three rabbits. The first group vaccinated with AFP antigen emulsified with Freund’s adjuvant and the second group vaccinated with AFP antigen emulsified with LDH formulation. The obtained data show that the highest displacement using LDH adjuvant reached (74.2, 61.7 and 66.5 %) while the corresponding values with Freund’s adjuvant recorded (64.8, 60.3 and 54.6 %) which indicates that the use of LDH adjuvant as a cellular vehicle is a more suitable choice. Also, the preparation of AFP tracer using lactoperoxidase oxidation method and its purification using gel chromatography on PD-10 column were carried out. Different factors affecting the optimization of the assay process were studied. Validation testes of the assay were carried out. The reproducibility as measured by the intra- and inter- assay variations is satisfactory. The recovery and dilution testes indicated accurate calibration and appropriate matrix. The present technique agreed well with the currently used commercial kit (Siemens, IRMA kit). In conclusion, the liquid phase double antibody RIA technique proved to be sensitive, specific, precis and accurate for routine laboratory use.     

Solid phase radioimmunoassay for testosterone in human serum using antibodies coupled to magnetizable cellulose

Summary A simple user-friendly, solid phase radioimmunoassay for testosterone in human serum based on magnetic particles is described. IgG fractions precipitated from polyclonal testosterone antiserum were coupled to magnetizable cellulose particles using carbonyl diimidazole. The prepared antibody solid phase was stable for one year when stored at 4 °C. The optimized assay involves the incubation of 50 ml of testosterone standards (0.3-10 ng/ml), 100 ml of magnetizable cellulose particle coupled antibody suspension and 100 ml of ¹²⁵I-testosterone derivative for 4 hours at 37 °C. At the end of the incubation, the tubes were placed on a magnetic rack for 10 minutes after the addition of wash buffer and decanted. The sensitivity of the assay is 0.2 ng/ml. The intra-assay variation was <15% throughout the assay range. The recovery varied from 88 to 115%. On dilution of samples having high levels of testosterone, linearity ranged between 87 and 125%. Correlation coefficient of 0.978 was obtained when the developed solid phase assay was compared to the earlier established liquid phase assay.     

Liquid chromatographic determination of meloxicam in serum after solid phase extraction

A liquid chromatographic method for the determination of meloxicam in serum has been developed. The technique includes a solid phase extraction of the serum samples on [poly (divinylbenzeneco-N-vinylpyrrolidone)] as a solid phase extraction sorbent. After conditioning, the cartridge was loaded with 1 mL of acidified serum containing an internal standard. Elution was carried out using 1 mL of water-acetonitrile (φ _r = 1: 1) mixture. After evaporation of the eluate to dryness and reconstitution of the residue with 0.1 mL of methanol, the samples were analyzed on a Symmetry C18 column. Mobile phase consisted of 1 % aqueous acetic acid/THF/acetonitrile (φ _r = 60: 30: 10) + 0.1 mL of 1-octane sulfonic acid. Detection was carried out using a photodiode array detector. Full validation of the proposed method is provided. Linearity of the method was proven over the range of 0.01–10 φg mL⁻¹ of meloxicam. Meloxicam assay was accurate and reliable with average intra- and inter-day precisions lower than 5.0 % and the intra- and inter-day accuracy higher than 97 %. Limits of detection (LOD) and quantitation (LOQ) found were 0.003 μg mL⁻¹ and 0.01 μg mL⁻¹, respectively. The proposed method was successfully utilized to quantify meloxicam in serum.     

Standardisation of radioimmunoassay for human insulin employing magnetizable cellulose particles

We describe a convenient and flexible solid phase radioimmunoassay for human insulin employing magnetizable cellulose particles. Anti-porcine insulin antibody was covalently linked to magnetizable cellulose particles to form a stable and economical solid phase immunosorbent system. The tracer was prepared by radioiodinating insulin with ¹²⁵I using Chloramine-T oxidation method. The analytical sensitivity of assay observed was 5.5 μIU/mL. Intra-assay and inter-assay variations were found to be <12 % along with analytical recovery of 93–109 %. The developed assay can be used for the routine analysis of clinical samples. In addition, concentration of the solid phase magnetizable immunosorbent can be easily varied as per the specific requirement for research purposes.     

Evaluation and comparison of high-sensitivity immunoradiometric assay kits for thyroid stimulating hormone

Fundamental and clinical characteristics of 3 kinds of high-sensitivity immunoradiometric assay (IRMA) kits for thyroid stimulating hormone (TSH). i.e., RIA BEADS II (kit A), TSH kit Daiichi II (kit B) and Ab tube TSH 'Eiken' (kit C) and one conventional radioimmunoassay (RIA) kit, i.e., TSH kit Daiichi (kit D), were studied. In the recovery test and the reproducibility test, there was no significant difference between the 4 kits. The sensitivities of kits A, B and C were much higher than that of kit D, and those IRMA kits were sensitive enough to distinguish hyperthyroidism from normal samples. For low concentrations of TSH (less than 5 microU/ml), the data from kits D, B, C and A tended to show higher values in that order. The correlation between the data measured by kits B and D, and the tendency of kit A toward lower values agreed well with other reports.     

Development of a sensitive, rapid, biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone

A highly sensitive "two-site" chemiluminescent immunoassay specific for human thyroid stimulating hormone (TSH) was developed. The signal amplification was achieved via a biotin-streptavidin system (BSAS). The HRP-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. Biotinylated anti-TSH monoclonal antibody (MAb) and HRP-labeled streptavidin were first synthesized. Then the signal amplification was achieved through the interaction between the biotinylated anti-TSH MAb and the HRP-streptavidin conjugate. The light intensity developed was in proportion to the TSH present in the samples. The assay showed little cross-reactivity with three other glycoprotein hormones (human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH)) due to the high specificity of the antibody. The working range for human thyroid stimulating hormone was 0.1-40 mU L(-1). Both the intra-assay and inter-assay coefficients of variation were less than 10% for the BSAS based chemiluminescent enzyme immunoassay (CLEIA). The proposed assay had a sensitivity of 0.01 mU L(-1) which was 10-fold higher than the HRP-MAb conjugate based TSH immunoassay. Thus the higher sensitivity facilitated the clinical testing for thyroid states. The effects of several reaction parameters, such as incubation time, temperature, and reaction volume of the method, were also studied. This method has been successfully applied to the evaluation of TSH in human serum. Compared with the commercial enzyme chemiluminescent immunoassay, the correlation was satisfied.     

Radioimmunoassay of circulating schistosome antigen with a radiation-immobilized monoclonal antibody : Preliminary results

A two-site immunoradiometric assay with a mouse monoclonal antibody to a circulating schistosome antigen was comparatively investigated using the monoclonal antibody either absorbed to microtiter plates (reference IRMA) or immobilized by several techniques. Radiation polymerization methods were carried out at Takasaki Radiation Chemistry Research Establishment, Takasaki, Gunma (I. Kaetsu, M. Kumakura), using 2-hydroxyethyl methacrylate monomers and 1 Mrad irradiation. A significant correlation was obtained with the reference IRMA and the assay using radiation polymerization-immobilized antibody (r = 0.94), although non-specific binding to the polymer discs was higher (x 10) than with microtiter plates. Immobilization of the monoclonal antibody onto polypropylene/polyethylene copolymer films grafted with methacrylic acid irradiated at 0.68 Mrads and treated with carbodiimide/N-hydroxysuccinimide, was carried out at the Dept of Bioengineering, University of Washington, Seattle, Washington (A.S. Hoffman, W.R. Gombotz, S. Uenoyama). A significant correlation (r = 0.90) was obtained with the reference IRMA. Non-specific binding was also higher than with microtiter plates (x 6). An important result was the increased shelf life of the immobilized reagent.     

Esterification of bagasse cellulose with metal salts as efficient catalyst in mechanical activation-assisted solid phase reaction system

The present study focused on investigating the catalytic mechanism of metal salts (sodium hypophosphite, sodium bisulfate and ammonium ferric sulfate) for esterification of bagasse cellulose carried out by mechanical activation-assisted solid phase reaction in a stirring ball mill. FTIR analysis of the products confirmed that these metal salts could catalyze the esterification of cellulose. XRD, SEM, FTIR, and ³¹P-NMR analyses of different samples indicated a synergistic effect between metal salt and ball milling, and the presence of metal salts enhanced the destruction on crystal structure of cellulose by mechanical force. The catalytic mechanism of three metal salts was difference: sodium bisulfate and ammonium ferric sulfate belonged to the catalytic mechanism of protonic acid and Lewis acid, respectively, while the catalytic mechanism of sodium hypophosphite was considered as that it could react with maleic acid to form active intermediates under ball milling.     

Basic evaluation of highly sensitive thyroid stimulating hormone immunoradiometric assay kits

Usefulness of three kinds of TSH kits by immunoradiometric assay (IRMA) was evaluated. They were able to measure low levels (less than 0.1 microIU/ml) in serum thyroid stimulating hormone (TSH) with incubation of short time (4 hours). In particular, RIABEAD II kit had a highly specific affinity for TSH and the normal range (+/- 2 S.D.) using it showed from 0.20 to 3.50 microIU/ml in 150 normal subjects. In patients with hyperthyroidism and in patients with hypothyroidism, the values of TSH were lower and higher than those of normal subjects, respectively. Another kits showed similar results. These results indicate that these TSH-IRMA kits are useful to evaluate serum TSH levels exactly.     

Quantitative determination and validation of octreotide acetate using 1H‐NMR spectroscopy with internal standard method

Quantitative nuclear magnetic resonance (qNMR) is a well‐established technique in quantitative analysis. We presented a validated ¹H‐qNMR method for assay of octreotide acetate, a kind of cyclic octopeptide. Deuterium oxide was used to remove the undesired exchangeable peaks, which was referred to as proton exchange, in order to make the quantitative signals isolated in the crowded spectrum of the peptide and ensure precise quantitative analysis. Gemcitabine hydrochloride was chosen as the suitable internal standard. Experimental conditions, including relaxation delay time, the numbers of scans, and pulse angle, were optimized first. Then method validation was carried out in terms of selectivity, stability, linearity, precision, and robustness. The assay result was compared with that by means of high performance liquid chromatography, which is provided by Chinese Pharmacopoeia. The statistical F test, Student's t test, and nonparametric test at 95% confidence level indicate that there was no significant difference between these two methods. qNMR is a simple and accurate quantitative tool with no need for specific corresponding reference standards. It has the potential of the quantitative analysis of other peptide drugs and standardization of the corresponding reference standards.     

Direct determination of major elements in solid plant materials by electrothermal vaporization inductively coupled plasma atomic emission spectrometry

The present work demonstrates the capability of electrothermal vaporization (ETV) to become an important tool of solid sample introduction in ICP-AES for plant sample analysis. Direct determination of Al, Ca, Fe, K, Mg, Mn, Na and Zn was investigated in powdered plant samples. Obtaining good results for major elements in plant samples was governed by some special operating conditions. The sensitivity of the method necessitated the use of ICP in radial view configuration. The behavior of elements during vaporization was studied between 500 and 2600 °C. External calibration was carried out using solid external (cellulose) spiked with aqueous standard solutions. However, performances of the analytical method were found dependent of argon flow rates. Analytical accuracy of the method was tested in three reference materials. Analytical results agreed with certified values when cellulose was used in calibration. However, K could not be determined because of excessive sensitivity. Without cellulose, it was found that Fe results were underestimated and Zn results overestimated. Relative standard deviations varied from 3 to 23%. Limits of detection varied from 1 to 80 ng g⁻¹ from one element to the other for a typical mass sample of 2 mg.     

Synthesis,characterization, and application of griseofulvin surface molecularly imprinted polymers as the selective solid phase extraction sorbent in rat plasma samples

In present study, an investigation was carried out to develop and validate an analytical method for the selective extraction and determination of griseofulvin (GSF) from plasma samples. For this purpose, a rational approach was made to synthesize and characterize the surface molecularly imprinted polymers (SMIPs). The SMIPs were utilized as solid phase extraction (SPE) sorbents. The SMIPs were prepared by using GSF as template molecule on the surface of modified silica particles through a non-covalent technique. The particles demonstrated high adsorption capacity (119.1 µg/mL), fast adsorption equilibrium time (30 min) and good recognition selectivity for the template drug. The scanning electron microscopy and infrared spectroscopy were used to explain the structural and morphological characteristics of the SMIPs and surface non-imprinted polymers. The SPE method was combined with HPLC for plasma analysis. The method validation results demonstrated that the established method possessed good linearity for GSF ranging from 0.1 to 50 µg/mL (R² = 0.997). The limit of detection for this method was 0.02 µg/mL for rat plasma samples. The recoveries of GSF from spiked plasma samples were (90.7–97.7%) and relative standard deviations were (0.9–4.5%). Moreover, the SMIPs as selective SPE sorbent can be reused more than 8 times which is a clear advantage over commercial SPE sorbents. Finally, the usefulness of the proposed strategy was assessed by extraction and detection of GSF in real rat plasma samples.     

Enzyme Immunoassay of Thyroid-Stimulating Hormone Using Dried Blood Samples a Simple Technique of Screening for Congenital Hypothyroidism

Abstract

A method for enzyme imnunoassay of thyroid-stimulating hormone (TSH) in dried blood spotted onto filter paper has been developed. TSH was conjugated to horse-radish peroxidase according to Nakane's method. Separation of the bound and free fractions was obtained by a double antibody solid phase method using polyacetal beads which were coated with the purified IgG fraction from goat anti-rabbit IgG serum. p-Hydroxyphenyl propionic acid was used as substrate for the fluorophotometric assay of peroxidase activity. The assay sensitivity is 0.07, μU TSH/assay tube, which is equivalent to μU/ml when five 3 mm discs of dried blood spot are assayed. TSH values in dried blood samples obtained by this method correlate well with those of serum samples obtained by radioimmunoassay (r=0.89). The coefficients of variation were 6.8 to 13.4% (within assay) and 5 to 40% (between assay). The enzyme immunoassay of TSH presented here is applicable to the mass-screening for congenital hypothyroidism of neonate.     

Conformational study of isotactic poly[(S)-5-methyl-1-heptene] and poly[(S)-2-methylbutyl vinyl ether] by IR spectroscopy

Infrared (IR) investigation of structurally analogous optically active polymers such as poly[(S)-5-methyl-1-heptene (1)] and poly[(S)-2-methylbutyl vinyl ether (2)] was carried out in the solid state, in the molten state, and in solution. Vibrational data are in accordance with the existence of helical conformations in solution and in the molten state for both polymers, confirming the previous suggestions based on optical rotation measurements. In particular, an accurate examination of the spectral region between 850 and 700 cm^?1, where characteristic absorptions of the sec-butyl group occur, enables us to assign the medium intensity band at 827 cm^?1 (present in both polymers in the solid state) to the GTTG^?T conformation of the side chains.     

Lipopolysaccharide-stimulated interleukin-6 production for radioimmunoassay

Prepration of liquid phase radioimmunoassay (RIA) to assessment the interleukin-6 (IL-6) that produced in-house by microbial stimulation was the aim of the present study. The production of IL-6 through mouse and macrophage cell infections were caused by Escherichia coli lipopolysaccharide (LPS). Optimum dose of lipopolysaccharide was determined. Then, both ¹²⁵I- IL-6 tracer and polyclonal anti-IL-6 antibody were prepared. These reagents were used in detection of produced interleukin-6. Optimization and validation tests of local RIA were carried out. The results showed that the interleukin-6 produced by microbial macrophage or mouse infections gave positive qualitative detection using enzyme linked immunoassay sorbent assay. The local RIA was used in quantitative determination of local purified interleukin-6.     

Activity determination of <Superscript>88</Superscript>Y by means of 4πβ(LS)-γ coincidence counting

The activity concentration of an ⁸⁸Y solution was measured by means of a new custom-built 4πβ(LS)-γ coincidence counting system. Efficiency variation was carried out using chemical quenching and neutral density filters. A relative uncertainty of 0.57% was obtained for the activity concentration. Details of the measurements and of the analysis, as well as possibilities to reduce the uncertainty are discussed. Additional validation measurements were carried out by means of the well-established coincidence counting technique using a proportional counter (PC) in the β channel. The results of both coincidence methods were found to be in excellent agreement.