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Biomedical subjects

S Yoshitake

Publications and source records attributed to S Yoshitake.

At least 91 records · Page 5Linked to original sources
PubMed1982

A sandwich enzyme immunoassay of human growth hormone in serum using anti-human growth hormone IgG-beta-D-galactosidase conjugate.

A sandwich enzyme immunoassay for the measurement of human growth hormone in serum was developed using antibody IgG-beta-D-galactosidase conjugate and antibody IgG-coated silicone rubber rods. The sensitivity of the assay was 4 pg/tube, which corresponded to 0.4 ng/ml, when 10 microliters of serum was used. The assay system showed little cross-reaction to other human pituitary hormones including prolactin, thyroid-stimulating, follicle-stimulating and luteinizing hormones and human chorionic gonadotropin. The coefficients of variation for different levels of serum hGH ranged from 5.8-9.3% and 6.7-14.5%, for within and between run, respectively. The regression equation and coefficient for correlation to radioimmunoassay were gamma (EIA) = 1.06 kappa (RIA) -0.22 and 0.97 (n = 46), respectively.

Animals↗
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↗
PubMed1981

Highly sensitive sandwich enzyme immunoassay of human IgE with beta-D-galactosidase from Escherichia coli.

A highly sensitive sandwich enzyme immunoassay of human IgE was developed. Polystyrene balls were coated with goat anti-human IgE immunoglobulin (IgG) by physical adsorption. Goat anti-human IgE Fab' was purified by affinity chromatography and conjugated with beta-D-galactosidase from Escherichia coli. Using thus prepared anti-IgE-coated polystyrene balls and anti-IgE-beta-D-galactosidase conjugate, 0.2 mU (2 amol)--1 U of IgE per assay could be determined. When 0.1 microliter of serum per assay was used, the range of IgE levels in serum that could be determined was 2--10000 U/ml, and even 0.01 U/ml was measurable by using 20 microliters of serum. The regression equation and coefficient for correlation to radioimmunoassay were gamma (RIA) = 0.94 chi (EIA) + 18.2 and 0.96 (n = 81), respectively. The coefficients of within- and between-assay variations ranged from 5.4 to 8.5%. The mean levels of serum IgE determined by the present assay were 103 U/ml in 70 normal children and 1064 U/ml in 38 children with bronchial asthma.

Antibody Specificity↗
PubMed1980

Morphological changes of the liver in uremic patients treated with chronic hemodialysis--laparoscopic observations and light- and electron-microscopic studies.

In order to clarify morphological changes of the liver in the uremic state, 16 uremic patients treated with chronic hemodialysis were studied. Biopsy was performed in 14 cases under laparoscopic observation and in two on the occasion of renal transplantation. One uremic patients not being treated with dialysis was also studied for comparison. All biopsy specimens were examined by light and electron microscopy. The liver usually appeared mildly or moderately swollen under laparoscopic observations, which was considered at least partially due to the enlargement of the hepatocytes. All patients had hepatocytes with an Orcein-negative "ground glass" appearance, in which marked proliferation of smooth endoplasmic reticulum (SER) was found by electron microscopy. Since the patient not being on dialysis also had such hepatocytes, this finding may be characteristic of uremia. With electron microscopy, in addition to proliferation of SER, alteration of mitochondria and rough endoplasmic reticulum (RER) and an increase in cytoplasmic lipid droplets were observed. Hypertrophy of the Golgi apparatus containing electron-dense particles (VLDL) was often found in patients associated with hypertriglyceridemia. Amorphous electron-dense inclusions in microbodies were occasionally observed. Siderosis was observed in nine patients including three having parenchymal siderosis. With electron microscopy, various siderosomes were seen in the cytoplasm of hepatocytes in patients with parencymal siderosis. Conclusively, these histological and ultrastructral features of hepatocytes are rather associated with several metabolic abnormalities in uremia.

Adolescent↗
PubMed1980

A general and mild procedure for the purification of rabbit Fab' antibodies.

A mild procedure for the purification of rabbit Fab' antibodies is described. A small column of normal goat IgG-Sepharose 4B (3 cm x 5mm) was saturated with F(ab')2 prepared from rabbit anti-goat IgG, and anti-goat IgG Fab was eluted from the column by splitting the disulfide bond in the hinge of the F(ab')2 molecule using 10 or 12 mM 2-mercaptoethylamine at pH 7. The recovery of anti-goat IgG Fab' in the eluate was about 35% of anti-goat IgG F(ab')2 bound to the column, and the purity of anti-goat IgG Fab' eluted was more than 90%. This procedure may be applicable to most kinds of rabbit IgG antibodies and useful for various immunoassays.

Animals↗
PubMed1979

An enzyme immunoassay for the measurement of thyroglobulin in human serum.

An enzyme-linked sandwich immunoassay using silicone rods coated with rabbit (anti-human thyroglobulin) immunoglobulin G and rabbit (anti-human thyroglobulin) monovalent fragment of immunoglobulin F (Fab') conjugated with beta-D-galactosidase was developed for the measurement of thyroglobulin in human serum. The volume of serum needed for the assay was as little as 2 microliters. The sensitivity of the assay was 3.5 ng/ml, which is equal to or rather higher than that of radioimmunoassay. The specificity of the assay was demonstrated by the following observations: (1) The absence of crossreaction of thyroxine and triiodothyronine, (2) non-detectability of thyroglobulin in the sera of patients who underwent total thyroidectomy, (3) parallelism of the standard curve with dilutions of reference serum. The precision of the assay was proven by the demonstration of the sufficient recovery of human thyroglobulin added to sera (92--99%) and coefficients of variance in within and between assays were 6.2--9.3 and 2.5--5.3%, respectively. Furthermore, a highly significant correlation was observed between thyroglobulin concentrations measured by our enzyme immunoassay and those by radioimmunoassay (r = 0.99, p less than 0.001, n = 63). Human thyroglobulin in serum was detectable in 90% of 146 normal subjects, the concentration (mean +/- S.D.) being 13.3 +/- 10.3 ng/ml.

Female↗
PubMed1979

Efficient conjugation of rabbit Fab' with beta-D-galactosidase from Escherichia coli.

An efficient procedure for the conjugation of rabbit Fab' with beta-D-galactosidase from Escherichia coli using N,N-o-phenylenedimaleimide is described. Thiol groups of Fab' were stabilized by the presence of ethylenediaminetetraacetate, and malemide groups were shown to be stable at pH 5 at 4 degrees C. The stability of thiol and maleimide groups enabled an efficient introduction of maleimide groups into Fab' and the average number of maleimide groups introduced into Fab' was 0.76 (range 0.73-0.79; n = 10) per molecule. As a result, 43.4% (range 41.3-46.9%; n = 6) of Fab' used could be conjugated with most of beta-D-galactosidase used. The average number of Fab' molecules conjugated per enzyme molecule was calculated to be 4.2 (range 4.0-4.6; n = 6). Both the enzyme and antibody activities were well preserved in the conjugate. There was no self-coupling of Fab', although the enzyme was polymerized to some extent during the conjugation reaction. The enzyme activity and cross-link in the conjugate was stable at pH 6.0-7.0 at 4 degrees C for at least 3 months.

Adsorption↗
PubMed1979

Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide.

Glucose oxidase from Aspergillus niger was conjugated with rabbit immunoglobulin G or its monovalent fragments (Fab'). The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide to introduce maleimide groups, which were then allowed to react with thiol groups of reduced IgG or Fab'. More than 40% of immunoglobulin G, Fab' and enzyme used could be conjugated without self-coupling. The enzyme activity decreased about 26 and 15% upon conjugation with immunoglobulin G and Fab', respectively, and the ability of antibody to bind to antigen was well preserved in conjugates. Conjugate preparations purified by gel filtration contained little free form of immunoglobulin G, Fab' or enzyme. Both the cross-link and enzyme activity in Fab' conjugate were stable at pH 6-7 at 4 degrees C for at least 6 months.

Animals↗
PubMed1979

Improved procedure for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide.

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.

Animals↗