A novel reagent for the screening of haptoglobin-deficient blood donors.

BACKGROUND AND OBJECTIVES: In Japan, the prevalence of haptoglobin deficiency is approximately 1 in 4000. Haptoglobin-deficient individuals may produce anti-haptoglobin from allo-immunization, leading to serious transfusion reactions. Therefore, implementation of a consistent supply of haptoglobin-deficient fresh frozen plasma is crucial. We developed a novel reagent to facilitate large-scale identification of haptoglobin-deficient individuals as potential donors of plasma products. MATERIALS AND METHODS: We established mouse monoclonal anti-haptoglobin-producing cell lines (three clones) using the hybridoma method by immunizing mice with the haptoglobin protein. Purified antibodies were conjugated with carboxylate-modified polystyrene latex beads and used for haptoglobin measurements by the latex agglutination method using an automatic analyser (LABOSPECT008). Samples with low protein concentrations were re-examined by enzyme-linked immunosorbent assay to confirm the results. Additionally, the haptoglobin gene was amplified by polymerase chain reaction to confirm the haptoglobin deletion allele (Hpdel ). RESULTS: From February to October 2022, 7476 blood donor samples were screened. Two haptoglobin-deficient and 21 low-haptoglobin-expressing individuals were identified. Two haptoglobin-deficient donors were found homozygous for Hpdel , and 19 (90%) of the 21 low-haptoglobin-expressing individuals were heterozygous for Hpdel , which includes the first reported case of heterozygous Hpdel /HpJohnson . CONCLUSION: We developed a new reagent for the detection of haptoglobin deficiency, which is automatable and inexpensive and appears useful for large-scale screening of blood donors.

PCB 118 and Aryl Hydrocarbon Receptor Immunoassays for Screening Dioxins in Retail Fish.

The efficacy of a combination of two enzyme-linked immunosorbent assay (ELISA) kits was examined for screening the toxic equivalent (TEQ) concentrations of dioxins in retail fish. The coplanar PCB-EIA system, which is a competitive immunoassay specific for polychlorinated biphenyl (PCB) 118, was tested as a screening method for mono- ortho PCBs. The Ah immunoassay (Ah-I), which is an ELISA-based aryl hydrocarbon receptor binding assay, was analyzed for its screening ability for non- ortho PCBs, polychlorinated dibenzo- p-dioxins (PCDDs), and dibenzofurans (PCDFs). Dilution and recovery tests using purified fish extracts revealed no major interference of the matrix in the PCB-EIA and suggested that the matrix effect was minimized in the Ah-I. Finally, the results for the fish samples ( n = 20) showed a strong correlation between this method and high-resolution gas chromatography coupled to high-resolution mass spectrometry for the determination of the TEQ concentrations of mono- ortho PCBs ( r = 0.99) and non- ortho PCBs and PCDD/Fs ( r = 0.97). These data indicate that our method is suitable for screening retail fish to determine the TEQ concentrations of dioxins.

Application of an ELISA for PCB 118 to the screening of dioxin-like PCBs in retail fish.

A commercially available enzyme-linked immunosorbent assay (ELISA) kit was evaluated for the determination of toxic equivalents (TEQs) of dioxin-like polychlorinated biphenyls (PCBs) in retail fish. The ELISA was highly specific for 2,3',4,4',5-pentachlorobiphenyl (PCB 118), which is generally the most abundant dioxin-like PCB isomer found in fish. The quantitative limit of the ELISA (using 3,3',4'-trichloro-4-methoxybiphenyl as a surrogate standard for PCB 118) was 10 ng ml(-1) (125 pg assay(-1)) in the standard curve, corresponding to 50 pg PCB 118 g(-1) in the tested sample. Good recoveries of PCB 118 (78.7-112.3%) were obtained for spiked purified fish extracts according to the ELISA. Good linearity was also obtained in dilution tests using purified fish extracts. No significant interference of the matrix was observed in the ELISA when this purification procedure was used. Recovery tests in which PCB 118 was added to fish samples also resulted in acceptable recoveries (60.2-82.3%) in the ELISA following purification. The ELISA results for fish samples correlated well with the TEQ concentrations of dioxin-like PCBs obtained by high-resolution gas chromatography/high-resolution mass spectrometry (r = 0.92, n = 26). These data indicate that the ELISA kit is suitable for screening retail fish for the TEQs of dioxin-like PCBs.

Development of MAb-based immunochromatographic assay for cadmium from biological samples.

Cadmium (Cd) is a general environmental pollutant of increasing global concern. In 2005 the joint FAO/WHO Codex Alimentarius Commission proposed new international food legislation for low-level Cd contaminants. In this study we demonstrate the use of novel monoclonal antibody (MAb) to Cd-EDTA in an immunochromatography (IC) format for the quick testing for trace Cd. This IC device could detect 0.3 microg kg(-1) (0-3 ppb) Cd. Contaminated Zn, Mn, Mg, and Cu, which would interfere the measurement of Cd by cross reaction to the MAb, could be removed by using a column that could separate trace Cd from other heavy metals in the extract of brown rice.

Inhibition of matrix metalloproteinases prevents cardiac hypertrophy induced by beta-adrenergic stimulation in rats.

Insulin-like growth factor (IGF) -I is one of the candidates for cardiac hypertrophy induced by beta-adrenergic stimulation. However, the mechanisms by which the biologic actions of IGF-I are regulated under this condition remain unclear. IGF-I becomes bioavailable for its receptors upon its dissociation from IGF-binding protein (IGFBP) through IGFBP degradation. Because matrix metalloproteinases (MMPs) have been implicated in the degradation of IGFBPs, the authors investigated the role of MMPs in the regulation of the IGF-I action through the degradation of IGFBPs in cardiac hypertrophy induced by beta-adrenergic stimulation. They examined the expression of MMPs in cardiac tissues of rats infused with isoproterenol (3 mg/kg per day), the effect of a MMP inhibitor, SI-27 (5 mg/rat per day), on cardiac hypertrophy, and the expression of IGF-I and IGFBP-3. MMP-1 and -2 activities increased and IGFBP-3 was degraded in heart hypertrophied by isoproterenol. MMP inhibition caused a regression in the myocyte hypertrophy in association with the suppression of both IGF-I protein in myocytes and the degradation of IGFBP-3 protein. These results suggest that the induction of myocyte hypertrophy by isoproterenol is mediated, at least in part, by a modulation of the IGF-I axis.

Prominent induction of cyclin B1 in G2/M renal cancer cells with butyrolactone 1.

BACKGROUND: Butyrolactone 1 (BL) is a cyclin dependent kinase (CDK) inhibitor derived from Aspergillus terreus. None of the present drugs are effective for the treatment of renal cell carcinoma. The use of BL is expected to promote a new type therapy of renal cancer. METHODS: We investigated three human renal cancer cell lines: ACHN, OS-RC-2 and RCC10RGB, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and two-color flow cytometry. Simultaneous measurements of DNA content and cyclin expression allowed us to perform cell cycle specific analysis. Western blot analysis was performed using ACHN to represent cell lines. RESULTS: BL inhibited cell proliferation and caused cell accumulation at G2/M phase associated with the emergence of the third peak. Moreover, BL induced cyclin B1 over-expression in G2/M cells. These changes were quite definite, whereas cyclins D1, E and A showed no changes at all. Cyclin B1 accumulation was confirmed by western blot analysis. The chronological observation revealed that the emergence of the third peak preceded the regression of the increased cyclin B1 positive G2/M cells. These results suggested that BL accelerated cyclin B1 accumulation in G2/M cells, which then shifted to G1 phase without cell division. New G1 cells started DNA synthesis most likely as endoreduplication to form the third peak and the mechanism of cyclin B1 accumulation converted into down-regulation. CONCLUSION: BL induced significant cell kinetic interference in the tested human renal carcinoma cell lines. This might indicate the possibility of a new medical treatment modality for renal cancer.