A study on LH- and FSH-RIA kits by immunoradiometric assay

An evaluation of LH- and FSH-RIA BEAD kits based on IRMA was carried out. The results obtained with the methods characterized by the use of monoclonal antibodies, i.e., one is linked to solid phase, and another is isotopically labeled, were compared with those determined by the Daiichi LH- and FSH-kits. Intra- and inter-assay precision, recovery, linearity, and specificity of both methods were favorable without exceptions. The cross reactivity of the LH kit to 5,000 mIU/ml hCG revealed within the range of less than 3 mIU/ml. Significant correlations were observed between the results derived from conventional Daiichi LH- and FSH-kits. The results from the conventional kits exhibited 30 to 40% of those from the Daiichi kits, considered to be mainly due to the difference in standard calibrations used. Among the individuals within the normal menstrual cycle, the serum LH- and FSH-levels determined by the present kits gave a typical pattern with a peak in the preovulatory phase. On the other hand, the LH- and FSH-values of individuals in normal pregnancy revealed strikingly decreased in comparison with those of non-pregnant women.     

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.     

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.     

Fundamental studies on thyroid stimulating hormone immunoradiometric assay kit (TSH RIABEAD II)

We have reported fundamental studies on the TSH immunoradiometric assay, using TSH RIABEAD II kit (Dainabot). The sensitivity of the assay was 0.03 mu IU/ml and its C.V. was 27.2%. Intra- and inter-assay C.V. were less than 5%. Dilution test and recovery test were good. Serum TSH level was 0.3-4.0 mu IU/ml in normal subjects, less than 0.03 mu IU/ml in untreated Graves' disease and subacute thyroiditis. Therefore, it was found that the clear difference exist in serum TSH levels between normal subjects and patients with untreated Graves' disease. There was a well correlation on the serum TSH levels between this method and TSH radioimmunoassay kit (Amerlex TSH, r = 0.983). Especially, the measurement of serum TSH levels, using immunoradiometric assay kit, was useful for the diagnosis of patients with Graves' disease.     

The clinical evaluation of the serum SCC antigen levels with "SCC RIABEAD" by immunoradiometric assay

The clinical significance of serum SCC antigen level was evaluated by the monoclonal antibody method (SCC.RIABEAD Dinabot Co. Ltd). Patients with squamous cell carcinoma showed a high positive SCC antigen level and positive rate elevated with the advance of the clinical stage. The serum SCC antigen level was decreased by treatment, and it increased again before obvious clinical recurrence was recognized. The results suggest that measurement of serum SCC antigen level is useful as a follow up of cancer treatment.     

Solid phase inclusive immunoradiometric assay for human chorionic gonadotropin using monoclonal antibodies

Summary A simple, one step, inclusive immunoradiometric assay for human chorionic gonadotropin (hCG) employing monoclonal antibodies is described. Commercially available monoclonal antibodies from various commercial sources were screened. Identified “detection” antibody was radiolabeled with ¹²⁵I and the selected “capture” antibody was chemically coupled to magnetizable cellulose to form a solid phase. In the procedure developed, standard/sample, radiolabeled antibody and capture antibody were incubated together for 3 hours at room temperature with shaking. After incubation, the bound complex was quantitated for the associated radioactivity. The analytical sensitivity observed was 1.0 mIU/ml with a wide concentration range up to 1000 mIU/ml of hCG. “High dose hook” of the developed assay was observed beyond 2000 mIU/ml. Results showed that the developed assay had a good precision: intra-assay CV less than 8%, inter-assay CV less than 10% and good analytical recovery of 97-109%. The clinical samples analyzed by the developed procedure showed a good correlation with that of the commercial kit (r = 0.92; y = 0.99x+0.51).     

A spectrophotometric assay for quantification of artemisinin

A sensitive and rapid spectrophotometric method for determination of artemisinin concentration is described. The method is based on the measurement of a reaction product of the drug in strong alkali solution. The interaction produces a homogenous electronic transition band from 250 to 330 nm with maximum transition at around 291 nm. The absorption curve shows Gaussian distribution with identical half bandwidth, thus providing information for formation of a possible mono-type reaction product. The 291 nm absorption intensity increases with increasing concentration of artemisinin and obeys Beer's law in the range of 0.44-172 nmol (ml⁻¹). The optimum reaction conditions and other analytical parameters were evaluated including its recovery from human plasma and erythrocyte samples.     

Preparation of magnetic particle antibodies for radioimmunoassay and immunoradiometric assay of thyroid related hormones

The International Atomic Energy Agency (IAEA), in cooperation with World Meteorological Organization (WMO) initiated in 1960 a world-wide survey of the isotope composition of monthly precipitation. The paper reviews the global distribution patterns of tritium and stable isotopes in precipitation and their relation to meteorological and climatic variables.     

Solid phase immunoradiometric assay for CA125 antigen levels in blood using monoclonal antibodies

We have developed a simple one step ‘sandwich’ immunoradiometric assay for CA125 using monoclonal antibodies directed against two different epitopes of the antigen. The detection antibody was radiolabeled with I-125 and the selected capture antibody was chemically coupled to magnetizable cellulose to form immobilized solid support. In the developed inclusive assay procedure, 200 μL of standard or sample was incubated with 100 μL of radiolabeled and capture antibody suspension for 18 h at room temperature with shaking. At the end of the incubation, the sandwich complex attached to solid phase is separated and counted for associated radioactivity. The analytical sensitivity for the developed assay procedure was observed to be 3.0 U/mL with an assay range up to 500 U/mL of CA125. The developed assay displayed acceptable precision; expressed in terms of percentage Coefficient of Variation (CV) estimated by repeated analyses of the quality control samples. Intra-assay CV was observed to be less than 5% whereas inter-assay CV was also less than 6%. The analytical recovery of the developed assay observed to be in the range of 88–107%. The clinical samples analyzed by the developed procedure showed a good correlation with that of a commercial kit (r = 0.99; y = 1.0052x − 38.942).     

Basic studies on an immunoradiometric assay system for human parathyrin (intact PTH1-84)

Two-site immunoradiometric assay for human parathyrin (PTH1-84) is specific for the intact, secreted, biologically active 84 amino peptide. This system incorporates two-different polyclonal antibodies to human intact PTH and has several technical advantages for use. This assay could detect a wide range of PTH in patients with hypo-, hyperparathyroidism, chronic renal failure and hypercalcemia with malignancy, especially distinguishing the level of human intact PTH in hypoparathyroidism from in normal.     

Pantothenic acid quantification by a stable isotope dilution assay based on liquid chromatography-tandem mass spectrometry

A stable isotope dilution assay for the quantification of free and total pantothenic acid has been developed by using [13C3,15N]-pantothenic acid as the internal standard. The three-dimensional specificity of liquid chromatography-tandem mass spectrometry enabled unequivocal determination of the vitamin. Due to the very simple extraction and clean-up procedure, free pantothenic acid could be analysed within 2 h, which is much faster than by microbiological or gas chromatographic assays. For quantification of total pantothenic acid, the vitamin was liberated from its conjugates by an overnight incubation with pigeon liver pantetheinase and alkaline phosphatase. In analyses of corn flour, the intra-assay coefficient of variation was 8.5% (n = 5) and 15.3% (n = 4) for free and total pantothenic acid, respectively. When pantothenic acid was added to corn starch at a level of 6 mg kg(-1), a recovery of 97.5% was found. Application of the stable isotope dilution assay to whole egg powder, hazel nuts and corn revealed similar data compared to those listed in nutrition data bases, whereas the content in mushrooms and porcine liver determined by the newly developed assay appeared to be lower and that of cocoa higher than reported in the literature.     

Simple fluorescence assay for quantification of OSCS in heparin

In 2008, heparin contaminated with oversulfated chondroitin sulfate (OSCS) penetrated the worldwide market and was associated with severe adverse effects. Feasible and reliable methods to test heparin for adulteration are needed. The objective was to develop a simple approach based on a microplate assay for quantification of heparin and sulfated glycans using the fluorescent heparin sensor polymer-H (polymer-H assay). However, both heparin and OSCS concentration-dependently increase the fluorescence intensity (FI) of polymer-H, so that OSCS in heparin cannot be detected. The idea was a two-step procedure including, first, separation of heparin by degradation with heparinase I, and then measurement of the remaining OSCS. To achieve complete heparin (unfractionated heparin (UFH), enoxaparin) degradation, several conditions (e.g. incubation time and heparinase I concentration) were optimized by using the aXa assay for monitoring. Defined UFH/OSCS mixtures incubated in this way showed a concentration-dependent FI increase in the polymer-H assay (λ _(em) 330 nm, λ _(ex) 510 nm). The sensitivity was unexpectedly high with an LOD/LOQ of 0.5%/0.6% OSCS content in heparin. Further experiments testing UFH/OSCS mixtures in the aXa assay confirmed our hypothesis: OSCS inhibits heparinase I resulting in incomplete heparin degradation and thus an additional FI increase of polymer-H by intact heparin. This two-step microplate fluorescence assay is a sensitive, rapid, and simple method for quantification of OSCS in heparin. In contrast with ¹H NMR and CE, neither expensive equipment nor much experience are required. It could be applied not only in the quality control of heparin, but also in clinical practice, to check the applied heparin preparation when a patient suffers any adverse effect.     

A new method for the production of a magnetic immunosorbent used in radioimmunoassay and immunoradiometric assay

A method has been developed enabling the direct coupling of first or second antibody to finely dispersed magnetite (Fe₃O₄). The immunosorbent thus produced was applied in various radioimmunoassay systems (T3, T4, TSH, Cortisol) for the separation of bound and free antigens. The elimination of the need for precoating the magnetic particles with a polymer has several advantages. One of them lies in the ease of one-step production of the immunosorbent and other is the high antibody/magnetite ratio. The influence of the concentration of the immunosorbent and detergent (TWEEN 20 or TRITON X-100) on the assay parameters (Bo, NSB, etc.) has been systematically investigated and the optimum concentration of the magnetizable particles and detergent has been determined. The reliability of magnetic separation has been validated by comparing it with the conventional PEG separation method.This work was performed in the scope of the Coordinated Research ProgrammeAntibodies Immobilized on Magnetic Particles for Radioimmunoassay and Immunoradiometric Assay (No6 487CF) organized and supported by the International Atomic Energy Agency, Vienna. The authors express their thanks to the Agency for supporting this work.     

Direct assay techniques for comprehensive radionuclide quantification of radwaste packages

Battelle, Pacific Northwest Laboratories has recently developed, tested and field-demonstrated a technology for the direct assay of transuranic radionuclides (TRU), fission products, and activation products in a variety of radwaste packages generated at commercial nuclear power plants. This technology involves non-destructive passive neutron counting for determination of nanocurie/gram quantities of the TRU radionuclides. Direct gamma spectrometry combined with thermoluminescent dosimetry (TLD) and correlation analysis is also utilized to determine the concentrations of the fission and activation products present in the radwaste packages. Employing counting times of 10 to 20 minutes, a complete analysis of all radionuclides specified for assay by the U.S. Nuclear Regulatory Commission (in 10CFR61) prior to shallow-land disposal of commercial radwastes can be measured at concentrations at least tenfold below the least restrictive Class A waste catagory.     

An immunoradiometric assay for human follicle stimulating hormone using a monoclonal antibody coupled to magnetisable cellulose

A simple immunoradiometric assay for human follicle stimulating hormone (hFSH) was developed using a pair of monoclonal antibodies obtained from commercial sources. The system developed makes use of a capture antibody covalently coupled to magnetisable cellulose, which is a more economical and stable immunosorbent as compared to the other solid phases. The detector antibody is labeled with¹²⁵I using the chloramine-T oxidation method and purified by gel filtration. After initial cross-matching of the capture and detector antibodies, various assay parameters have been optimised. This assay does not show any significant cross reactivity with homologous hormones. A number of serum samples from men and women from reproductive age group was screened and compared with another commercially available kit (r=0.98). Sensitivity of the assay is 1.4 mIU/ml, interassay variation is <5% and intraassay variation around 15%. The assay is reproducible and sensitive enough for regular estimation of serum hFSH and is relatively inexpensive.