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Biomedical subjects

N Nakazawa

Publications and source records attributed to N Nakazawa.

At least 55 records · Page 3Linked to original sources
PubMed1984

Radioimmunoassay for neopterin in body fluids and tissues.

Specific antibodies against D-erythroneopterin have been prepared in rabbits using a conjugate of D-erythroneopterin to bovine serum albumin (D-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Comparison of beta-D-galactosidase from Escherichia coli and horseradish peroxidase as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique.

beta-D-Galactosidase from Escherichia coli and horseradish peroxidase were compared as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The anti-human ferritin Fab'-peroxidase conjugates gave lower nonspecific bindings and higher specific bindings than the corresponding Fab'-beta-D-galactosidase conjugates. As a result, the former provided more sensitive dose response curves for human ferritin than the latter. However, the peroxidase conjugates were required in a larger quantity, since peroxidase assay was much less sensitive than beta-D-galactosidase assay.

Antibodies↗
PubMed1983

A new direct solid-phase radioimmunoassay for carcinoembryonic antigen without pretreatment of serum samples.

We examined some fundamental conditions for establishing a new simplified radioimmunoassay system for CEA. The system consists of antibody-coated plastic beads and radiolabeled tracer antibody. The reactivity of this system with several purified CEA preparations can vary due to differences in the nature of antibody preparations used either for coating beads or for tracer antibody. Among purified CEA preparations, we also found some differences in reactivity with a given immobilized antibody preparation. The assay system that resulted, referred to as bead-direct-RIA, needs neither pretreatment of serum samples nor centrifugation in any of its steps, and has a wide assay range, measuring CEA concentrations of 1-300 ng/ml using only 50 microliters of a serum sample. Its accuracy, reproducibility, dilution effect, and analytical recovery proved the reliability of this method. The mean value +/- S.D. for 48 normal adults was 2.5 +/- 1.0 ng/ml. It was found by comparative studies that the Z-gel method of Hoffmann-La Roche generally gave the highest values, the antibody-disc method of Dainabot gave the lowest, and the values by bead-direct-RIA fell between those of the 2 other methods and correlated well with either value. Follow-up studies on patients with malignancy indicated that the values by bead-direct-RIA fluctuated in parallel with those by Roche and Dainabot kits.

Antibodies, Neoplasm↗
PubMed1982

Development of a highly sensitive sandwich enzyme immunoassay for human ferritin using affinity-purified anti-ferritin labelled with beta-D-galactosidase from Escherichia coli.

A highly sensitive sandwich enzyme immunoassay of human ferritin was developed. Polystyrene balls were coated with rabbit anti-human ferritin IgG by physical adsorption, and rabbit anti-human ferritin Fab' was purified by affinity chromatography and labelled with beta-D-galactosidase from Escherichia coli. Using the anti-ferritin-coated polystyrene balls and labelled anti-ferritin, the sensitivity obtained was 23 fg (0.05 amol) of ferritin per tube. The range of serum ferritin levels that could be determined using 0.1 microliter of serum was 0.23-4500 ng/ml, and even 2.3 pg/ml was measurable by using 10 microliter of serum. The coefficients of within-assay (n = 25) and between-assay (n = 10) variations were 5.9-8.8%. The regression equation and coefficient of correlation to a radioimmunoassay were Y(RIA) = 0.92 X(EIA) + 3.0 and 0.99 (n = 78), respectively. The corresponding sandwich radioimmunoassay was less sensitive, partly because the specific radioactivity of 125I-labelled anti-ferritin IgG used was not sufficiently high.

Antibody Specificity↗
PubMed1982

Mild and efficient conjugation of rabbit Fab' and horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay.

A mild and efficient procedure for conjugating rabbit Fab' and horseradish peroxidase using a maleimide compound was developed. The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl) maleimide to introduce maleimide groups. Then, the maleimide-enzyme was allowed to react with thiol groups of Fab', and the conjugate formed was separated from unreacted components by gel filtration with Ultrogel AcA 44. In the peak fraction of the separated conjugate, 98% of peroxidase was associated with Fab' and 90% of antibodies was associated with peroxidase. The recoveries in the conjugate of peroxidase and Fab' incubated for conjugation were 65-74%. The conjugate formed appeared to be largely monomeric. Both the enzyme activity and antigen-binding activity of Fab' were fairly well preserved in the conjugate. The cross-link formed was stable at 4 degrees C at least 6 months. Use of the conjugates obtained by this method gave greater sensitivity in sandwich enzyme immunoassay for human ferritin and human thyroid-stimulating hormone than conjugates prepared by the periodate method. The conjugation using N-hydroxysuccinimide ester of m-maleimidobenzoic acid provided a similar monomeric preparation but was less efficient.

Animals↗
PubMed1982

Major factors limiting sensitivity of sandwich enzyme immunoassay for ferritin, immunoglobulin E, and thyroid-stimulating hormone.

By using IgG-coated polystyrene balls and beta-D-galactosidase-labelled Fab', sandwich enzyme immunoassays for human ferritin, immunoglobulin E (IgE), and thyroid-stimulating hormone (TSH) were developed, and their sensitivities were shown to be largely limited by the purity, binding efficiency, and amount of beta-D-galactosidase-labelled Fab' used. (1) Their sensitivities were enhanced 10 to 50-fold by using affinity-purified Fab' labelled with beta-D-galactosidase. (2) Their sensitivities depended upon the efficiency of specific binding of the labelled Fab' to antigens adsorbed on antibody IgG-coated polystyrene balls. (3) Their sensitivities were improved by using the minimal amount of labelled Fab' that gave a reliable calibration curve. Under optimal conditions the sensitivities obtained for ferritin, IgE, and TSH were 0.09 pg (0.2 amol), 0.48 pg (2.4 amol, 0.2 mIU), and 3.2 millimicronU (5.7 amol) per tube, respectively.

Ferritins↗
PubMed1980

An improved preparation of antibody-coated polystyrene beads for sandwich enzyme immunoassay.

An improved preparation of antibody-coated polystyrene beads for sandwich enzyme immunoassay of human thyroid-stimulating hormone (TSH) was described. Rabbit anti-TSH IgG was purified by eluting at pH 2.5 from a TSH-Sepharose column, diluted 3 or 9 fold with normal rabbit IgG and used for coating polystyrene beads by physical adsorption. In a sandwich enzyme immunoassay of TSH using rabbit (anti-TSH) Fab'-beta-D-galactosidase conjugate, beta-D-galactosidase activities specifically bound to thus prepared polystyrene beads in the presence of TSH was 2.8-6.3 fold higher than those bound to polystyrene beads coated with anti-TSH IgG before purification. A similar effect was observed when guinea pig anti-pork insulin IgG, rabbit (anti-human IgE) IgG and goat (anti-human IgE) IgG were treated at pH 2.5. This improvement may be based on a conformational change of Fc in IgG molecule which was caused by the treatment at pH 2.5. Other sandwich immunoassays such as fluoro- and radio-immunoassays may also be improved in the same way.

Animals↗
PubMed1978

Radioimmunoassay of human calcitonin using labeled [Asu1, 7]-human calcitonin analogue.

Application of synthetic human calcitonin analogue ([Asul,7]-hCT) was attempted, since it is chemically more stable than native hCT. [Asu1, 7]-hCT was successfully labeled with 125I to a specific activity ranging from 410 to 540 microCi/microgram. Sensitivity, recovery and reproducibility of the assay system using 125I-labeled [Asu1,7]-hCT analogue, standard synthetic hCT and its antibodies, were 49 pg/ml, 99.8 +/- 4%, and 7.95% (C. V. within assay) or 21.9% (C. V. between assays), respectively. The dilution curve of the sera obtained from patients with medullary carcinoma paralled well with the standard curve. 125-I-labeled [Asu1, 7]-hCT analogue can be used for routine radioimmunoassay of hCT.

Animals↗
PubMed1977

[Human proinsulin. C-peptide radioimmunoassay method. 125I labeling of human proinsulin. C-petide].

125I-labelled human-C-peptide was prepared by chloramin T method, enzymic method and active ester method, respectively. Using respective 125I-labelled human-C-peptides in human proinsulin-C-peptide RIA, we compared the binding (Bo/T%) to antibody, displacement by standard human-C-peptide, the recovery test and stability. The usable 125I-labelled antigen for human proinsulin-C-peptide RIA could be prepared by chloramin T method and enzymic method wich labelled 125I to tyrosyl human proinsulin connecting peptide, and active ester method which conjugates 125I-labelled active ester to human proinsulin connecting peptide. The differences among those 125I-labelled antigens was not observed in displacement (B/Bo%) by standard human-C-peptide and the recovery test. In the case of constant preparation of 125I-labelled antigen for RIA, the enzymic method was the best from the viewpoint the reaction ratio is stable and stability of Bo/T% is good.

C-Peptide↗
PubMed1977

[Studies on human proinsulin C-peptide radioimmunoassay method--preparation of antiserum and its specificity-- (author's transl)].

The antisera using at final dilution of 1 : 10,000 have been prepared by immunizing synthetic human proinsulin connecting peptide to rabbits for human proinsulin C-peptide radioimmunoassay. The cross reactivities of human proinsulin C-peptide derivatives with the prepared antisera were reduced by leaving amino acid residues from N terminal, although this phenomenon was a little different among antisera. Those results suggested that main antigen determinant in N terminal 31-38 of human proinsulin connecting peptide. The cross reactivities of other animal proinsulin C-peptide and other peptide hormones with the prepared antissera were not recognized at 10(3) p mole/ml.

C-Peptide↗
PubMed1977

[Human proinsulin-C-peptide radioimmunoassay method; stability of the assay kit].

We have been studies time sequent stability each on standard human-C-peptide, human-C-peptide antiserum, 125I-tyrosyl human-C-peptide and the assay kit (all reagent) which is necessary in human proinsulin-C-peptide radioimmunoassay(RIA). Also we measured using this assay system, human proinsulin C-peptide in blood after oral administration of glucose to normal subject. Standard human-C-peptide and human-C-peptide antiserum were very stable on storage at 4 degrees C, 125I-tyrosyl human-C-peptide was unstable as compared with the former two, The stability of the assay kit was influenced by the stability of 125I-tyrosyl human-C-peptide, and was stable at 4 degrees C for ten weeks after preparation. Three lots of the assay kit prepared at different period showed almost same stability. We think this assay system using the assay kit is satisfactory in respect of stability. The measured values of human proinsulin-C-peptide in blood, using this assay system, showed insulin secretory reaction after oral administration of glucose.

C-Peptide↗
PubMed1975

Radioimmunoassay of main urinary metabolite of prostaglandin F2alpha.

Radioimmunoassay of 5alpha, 7alpha-dihydroxy-11-keto-tetranorprosta-1,16-dioic acid, main urinary metabolite of prostaglandin F2alpha (PGF2alpha), was performed using an antiserum produced in the rabbit. The antibody in 100 mu1 of 1,600-fold diluted antiserum binds with 60 picograms of metabolite. The main urinary metabolite level fell when flufenamic acid, a prostaglandin synthetase inhibitor, was given to rats. In contrast, it was significantly elevated when PGF2alpha was administered.

Animals↗