bluuebunny/crossref_metadata_2025 huggingface dataset:
An immunoenzymometric assay for a CA 125‐like antigen, CA602
Authors: Mochizuki Hiroshi , Sato Ichiro , Nagoya Kimiko , Furusako Shoji , Yamauchi Tadakazu , Masuko Hideyuki , Sato Hiroshi , Morishita Hideaki , Yajima Masazumi , Sasaki Hirosuke , Udagawa Yasuhiro , Nozawa Shiro
Published: March 2007

An immunoenzymometric assay (IEMA) for a new CA 125‐like antigen, CA602, was developed. Five monoclonal antibodies raised against a human ovarian carcinoma cell line could detect their respective antigens in the sera of ovarian carcinoma patients. The antigen levels detected in serum by the various antibodies correlated significantly to each other, and to CA 125 levels. The results of epitope analyses and combined IEMAs suggested that the epitopes recognized by these antibodies are not same, but exist on the same antigen which bears the CA 125 epitope. A sensitive IEMA was developed with 602‐1 and 602‐6 antibodies which showed high reactivity to the CA 125‐like antigen. The antigen defined by these two antibodies was designated as CA602, and serum CA602 levels correlated well with CA 125 levels. The CA602 antigen is a CA 125‐like antigen. Furthermore, the serum CA602 levels did not correlate to CA54/61 levels. The combined assays of CA602 and CA54/61 may increase the detection of ovarian carcinoma.
An improved method for monitoring efficacy of anti‐retroviral therapy in HIV‐infected individuals: A highly sensitive HIV p24 antigen assay
Authors: Reddy Mohan M. , Winger Edward E. , Hargrove David , McHugh Thomas , McKinley George F. , Grieco Michael H.
Published: March 2007

Circulating human immunodeficiency virus (HIV) p24 antigen levels were measured by a highly sensitive HIV p24 antigen‐capture enzyme‐linked immunosorbent assay (ELISA) in patients with acquired immunodeficiency syndrome (AIDS) and AIDS‐related complex (ARC) otherwise negative for HIV p24 antigenmeasured by a commercial antigen‐capture ELISA. The assays were performed at baseline and at several intervals during treatment with either zidovudine (ZDV) or dideoxyinosine (ddl). To further enhance the rate of antigen detection, serum was pretreated with hydrochloric acid to denature antibody in immune complexes. Utilizing this assay system, we monitored these patients for drug efficacy. HIV p24 antigen levels obtained by using this sensitive assay decreased in 3 of 8 patients receiving ZDV during 8 weeks of ZDV treatment. Similarly, ddl administration was associated with a decrease of HIV p24 antigen levels in 3 of 5 patients. Thus, the use of the highly sensitive HIV p24 antigen assay permitted the monitoring of surrogate HIV p24 antigen as a measure of efficacy of anti‐retroviral therapy in all of these patients who were otherwise HIV p24 antigen‐negative at the onset of anti‐retroviral therapy. © 1992 Wiley‐Liss, Inc.
Elevation of platelet‐associated IgG in aplastic anemia
Authors: Kawaguchi Ryuji , Haruna Shoji , Hikiji Kazumasa , Higashi Yasuko , Tsukada Yutaka
Published: March 2007

We determined platelet‐associated IgG (PAIgG) levels in patients with aplastic anemia, idiopathic thrombocytopenic purpura (ITP), iron deficiency anemia, and systemic lupus erythematosus (SLE), as well as in normal healthy adults as a control group. To determine PAIgG levels, we used a competitive micro enzyme‐linked immunosorbent assay, which had excellent reproducibility, recovery, and dilution. We confirmed its reliability by comparing it to the immunoradiometric assay. Both the aplastic anemia group (n = 27, mean ± SD = 218.6 ± 244.6ng/107 platelets) and the ITP group (n = 82, mean ± SD = 212.5 ± 327.8 ng/107 platelets) had higher PAIgG levels than the SLE group (n = 4, mean ± SD = 38.4 ± 22.4 ng/107 platelets), iron deficiency anemia group (n = 10, mean ± SD = 16.1 ± 3.6ng/107 platelets), and normal control group (n) = 69, mean ± SD = 16.1 ± 3.6ng/107 platelets. The higher platelet associated IgG levels in aplastic anemia suggest that autoimmune mechanisms are involved. © 1992 Wiley‐Liss, Inc.
An enzyme immunoassay for determining plasma concentrations of didemnin B
Authors: Raybould T. J. G. , Grothaus P. G. , Simpson Samantha B. , Bignami G. S. , Lazo Carolyn B. , Newman R. A.
Published: March 2007

Didemnin A was conjugated at the amino terminus of the N‐methylleucine residue, via the linkers N‐succinimidyl‐3‐(2‐pyridyldithio)propionate and trans‐1,4‐maleimidomethylcyclohexane carboxylic acid, to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). The didemnin‐KLH conjugates were used to hyperimmunize rabbits. The resulting high titer antisera were employed with didemnin‐BSA conjugate‐coated microtiter plate wells to develop an indirect competitive inhibition enzyme immunoassay (CIEIA) that was fully cross reactive with didemnin B. A CIEIA is described that is capable of detecting the drug in plasma from didemnin Btreated patients at concentrations down to 1‐3 ng/ml. This simple, sensitive CIEIA has been employed to demonstrate plasma drug clearance profiles with samples from didemnin B‐treated patients. © 1992 Wiley‐Liss, Inc.
Development of a rapid microparticle‐enhanced nephelometric immunoassay for serum myoglobin in acute myocardial infarction
Authors: Cliquet F. , Montagne P. , Cuillière M. L. , Varcin P. , Duheille J.
Published: March 2007

A microparticle‐enhanced nephelometric immunoassay was developed for myoglobin quantitation in human serum. It uses rabbit antimyoglobin serum and hydrophilic polyacrylic microparticles covalently coated with baboon myoglobin in a competitive immunoagglutination system. The level of microparticle agglutination is assessed with a specially designed nephelometer. This sensitive (45 ng/ml of myoglobin detected in serum) and accurate (coefficients of variation from 3.0% to 8.2% in precision study and linear recovery of myoglobin in overloaded sera) immunoassay was evaluated with human sera from patients suffering from acute myocardial infarction. Myoglobin levels in patient's serum on admission appeared to be correlated with clinical and biological parameters assessed in emergency wards and later. This new, rapid, and easy microparticle‐enhanced nephelometric immunoassay could thus be useful in emergency conditions for the early quantitation of serum myoglobin.
Enzyme‐linked immunosorbent assay to measure apolipoproteins Al and B secreted by a human hepatic carcinoma cell line (Hep G2)
Authors: Hahn Susan E. , Parkes Joel G. , Goldberg David M.
Published: March 2007

We describe an enzyme‐linked immunosorbent assay (ELISA) to measure apolipoproteins Al and B secreted by Hep G2 cells and in cell homogenates. These assays utilize commercially available polyclonal antibodies, affinity‐purified to improve their specificity, thereby achieving a dramatic increase in the sensitivity of the assay. These affinity‐purified antibodies were also more sensitive than a series of monoclonal antibodies tested. We achieved a sensitivity of 0.4 ng in the apo Al assay, and a sensitivity of 5 ng in the apo B assay. By these methods, we measured secretion rates by Hep G2 cells of 358 ± 41 ng/mg cell protein/hr for apo B and 137 ± 8 ng/mg cell protein/hr for apo Al. These assays also allowed the measurement of intracellular apolipoproteins and thus can be used to facilitate investigations of human lipoprotein metabolism in cell culture systems. © 1992 Wiley‐Liss, Inc.
Chromatographic characterization of CA 19‐9 molecules from cystic fibrosis and pancreatic carcinoma
Authors: Wu James T. , Chang Jukay
Published: March 2007

We have compared the size, the binding to Concanavalin A (Con A), and the affinity for monoclonal antibody 1116NS‐199 (Mab 19‐9) among CA 19‐9 molecules from sera of cystic fibrosis (CF) and pancreatic carcinoma patients and from sputum extracts. CA 19‐9 molecules of two different sizes were found in all types of specimens by Sepharose 4B chromatography. While the smaller CA 19‐9 molecule was predominant in CF patient sera, the larger molecule was associated with most of the sera from patients with pancreatic carcinoma. The majority of the sputum extracts contained the larger CA 19‐9 molecule. All CA 19‐9 molecules studied by Con A chromatography did not appear to bind to Con A, and almost 100% were found in the nonreactive fraction. The CA 19‐9 molecules from sera of either CF or pancreatic carcinoma patients exhibited variable affinities for Mab 19‐9, some approaching that of the standard curve but many also having lower affinities. The lowest affinity was displayed by CA 19‐9 molecules from the sputum extract. It appears that development of more specific assays for CF and for carcinoma is possible if the correct CA 19‐9 molecule is selected for antibody preparation and for use as standards. © 1992 Wiley‐Liss, Inc.
Comparison of serum amyloid A and C‐reactive protein as indicators of lung inflammation in corticosteroid treated and non‐corticosteroid treated cystic fibrosis patients
Authors: Smith James W. , Colombo John L. , McDonald Thomas L.
Published: March 2007

Serum amyloid A (SAA) and C‐reactive protein (CRP) levels were compared in 830 serum samples from 155 cystic fibrosis (CF) patients. Correlation coefficients were calculated for all samples (r=0.73), for samples from non‐corticosteroid treated (CFNS) patients (n=698, r=0.80), and for samples from corticosteroid treated (CFS) patients (n=132, r=0.35). SAA was the more sensitive indicator of pulmonary inflammation when SAA and CRP were compared to pulmonary function tests of 49 hospitalized patients at admission and discharge. CRP levels were significantly (p<.05) lower at admission in CFS patients than in CFNS patients, whereas SAA levels were not significantly different between the two groups. All nine CFS patients hospitalized had elevated SAA levels (average 22 times above normal limits) at admission, while only six had elevated CRP levels (average 3.7 times above normal limits) at admission. In the 40 CFNS patients both SAA and CRP levels were significantly elevated at admission. In each case SAA and CRP levels declined as pulmonary functions improved with effective antimicrobial therapy. In three instances SAA levels increased during hospitalization while CRP levels did not. In each case, rising SAA levels indicated clinical deterioration associated with evolving resistance of P. aeruginosa which required a change in antibiotic therapy. © 1992 Wiley‐Liss, Inc.
Monoclonal antibodies to beta subunit of choriogonadotropin: Development and use in a sandwich ELISA pregnancy test
Authors: Gariglio Rosana , Toplikar Emilena , Carlomagno Amadeo , Rojkin Federico , Knecher Leonardo , Capriotti Gustavo , Lorenzo Luis
Published: March 2007

Balb/c mice were immunized with beta subunit isolated and purified from crude human chorionic gonadotropin preparations. Spleen cells from the higher titered mouse were fused with Sp 2/0 myeloma cells.

Four specific secreting hybridomas were obtained. Specificity, affinity, and suitability of secreted antibodies for use in enzyme immunoassays were studied. Ascites of the selected hybridoma was raised; the antibody was purified by protein A‐affinity chromatography and coupled to horseradish peroxidase.

This conjugate was employed in a simultaneous snadwich enzyme immunoassay on microtiter plates sensitized with goat polyclonal antibody to measure the hormone.

The test has a sensitivity of 10 mlU/ml either on urine, serum, or plasma samples when read in a microplate reader. The results can also be evaluated by the naked eye, with a sensitivity of 20 mlU/ml. No cross reactivity was detected with other human gonadotropins. © 1992 Wiley‐Liss, Inc.
Enzyme immunoaassay for specific analysis of pancreatic stone proteins in human pancreatic juice
Authors: Katsuzaki Tomoyuki , Tatemichi Noriyuki , Takeichi Chikako , Hayakawa Shinobu , Hayakawa Tetsuo , Shibata Tokimune , Harada Hideo , Ochi Koji
Published: March 2007

In order to study the concentration of pancreatic stone protein (PSP) in human pancreatic juice, we investigated the influence of the insoluble form of PSP‐S1 converted from PSP‐S2–5 on PSP determination and the assay method for PSP‐S1 precipitate after solubilizing PSP‐S1 was converted from PSP‐S2–5 and precipitated about 45–85% after 1 h. The precipitated PSP‐S1 was dissolved in 0.1 M sodium acetate buffer, pH4.0, and the concentration was measured by the enzyme immunoassay, with similar reactivity to PSP‐S1 and PSP‐S2–5. The proposed method can offer accurate and specific analysis of the PSP level in pancreatic juice. The results of the fractionation of pancreatic juice and duodenal juice on Mono S cation‐exchange chromatograhy suggested that the major coponent of PSP was PSP‐S2–5 in pancreatic juice and PSP‐S1 in duo‐denal juice. © 1992 Wiley‐Liss, Inc.
Experience with a provocative test of calcitonin release as a prospective screening for preclinical medullary thyroid carcinoma in men type 2A family members
Authors: van Lathem J. J. M. , Vermaak W. J. H. , Kuyl J. M. , Mollentze W. , Jansen S. , Wolmaran L. , Pelser H. , Barry R. , Kruger A. J. , Wolfaardt M. , Nel C. J. C.
Published: March 2007

We report our experience with a provocative test of calcitonin (CT) release using a combined stimulus of intravenous 10% CaCl2 solution and pentagastrin on 34 normal adults (15 females: age 41 ± 12.3 yers and range 22–65 years; and 19 males: age 43 ± 9.1 years and range 23–60 years) and in 44 family members of three proven multiple endocrine neoplasia type 2A syndrome (MEN 2A) patients. A commercial radioimmunoassay was used to determine the serum CT levels. Peak CT levels were reached within 2 to 5 minutes after administration of the stimulus in all subjects tested. In the group of normal subjects there was no significant difference in the mean basal CT levels between males (54.8 ± 21.7 pg/ml) and females (56.5 ± 34.8 pg/ml), whilst the mean peak response values ofr males was 146.3 ± 120.6 pg/ml, which was significantly different from the mean value of females, namely 71.6 ± 39.0 pg/ml. We did not find significant correlations between the basal CT level, peak CT response, and age.

Of the 44 family members tested, 9 showed an exaggerated CT response to the combined stimulus and subsequently had a total thyroidectomy. Histological examination confirmed C‐cell hyperplasia (CCH) in one and medullary thyroid carcinoma (MTC) in teh other 8. Three of the 9 had high basal plasma CT levels. The 9 patients were retested postoperatively and all showed a flat response to the combined stimulus. Those family members with histological proof of MTC or CCH were screened for genetic linkage to the disease gene for MEN 2A using probe MCK2, and showed correltion in each instance. Eight other members were identified as high‐risk genotypes. They are still asymptomatic and calcitonin negative.

Our study shows that the provocative test and the commercial calcitonin radioimmunoassay used is reliable in detecting family members of MEN type 2A patients having preclinical MTC or CCH. We found that peak response values higher than 250 pg/ml were highly suggestive of preclinical MTC in family members of MEN 2A cases (sensitivity 100% and specificity 94%). The test had minor side effects which did not las more than 2 minutes. © 1992 Wiley‐Liss, Inc.
Effect of anticardiolipin antibody in patients with recurrent fetal loss on thrombomodulin‐dependent protein C activation
Authors: Toyoshima Kiwamu , Makino Tsunehisa , Ozawa Nobuaki , Umeuchi Masakatsu , Nozawa Shiro
Published: March 2007

In order to approach an understanding of the mechanisms of fetal death in patients with antiphospholipid syndrome we investigated the association between protein C activity and anticardiolipin antibody. Protein C activity in vivo was decreased in some patients with recurrent fetal loss and a high ACA titer. This study suggests that anticardiolipin antibody in patients with recurrent fetal loss may inhibit activation of protein C by thrombomodulin. © 1993 Wiley‐Liss, Inc.
Proteinase K inactivation of cytosolic aspartate aminotransferase isoenzyme for measurement of human serum mitochondrial aspartate aminotransferase
Authors: Watazu Yoshifumi , Uji Yoshinori , Okabe Hiroaki , Shirahase Yasushi , Kaneda Nobuaki , Karmen Arthur
Published: March 2007

We studied a new proteinase K assay method for human serum mitochondrial aspartate aminotransferase. We found that proteinase K showed no inactivation of human mitochondrial aspartate aminotransferase isoenzyme and complete inactivation of cytosolic aspartate aminotransferase.

Previous studies have shown that selective proteolytic measurement for mitochondrial aspartate aminotransferase in serum using the protease 401 cleaved peptide bond at Leu 20 from the amino‐terminal bond shows complete inactivation of cytosolic aspartate aminotransferase and slight inactivation of mitochondrial aspartate aminotransferase isoenzyme, depending on protease concentration.

In this investigation, we found that the proteinase K method does not depend on protease concentration. The proteinase K enzyme inactivation of cytosolic aspartate aminotransferase is caused by the cleavage of the peptide bond at lieu 21 from the aminoterminal bond.

In studies with various animal cytosolic aspartate aminotransferase isoenzymes, proteinase K almost completely inactivated cytosolic aspartate aminotransferase. Precision and correlation using proteinase K for measurement of serum mitochondrial aspartate aminotransferase in human showed a good coefficient of variation (within‐run <4.45%) and a coefficient of correlation of r = 0.985 (N = 125). © 1993 Wiley‐Liss, Inc.
The levels of serum myoglobin in cardiac patients with elevated creatine kinase‐MB and suspected acute myocardial infarction
Authors: Moscoso Hugo , Kiefer Charles R. , Shyamala M. , Teabeaut Robert J. , Garver Fred A.
Published: March 2007

A monoclonal antibody enzyme immunoinhibition assay was used to quantitate serial serum myoglobin (Mb) levels in 121 patients who had ≫5% creatine kinase‐MB (CK‐MB) and suspected acute myocardial infarction (AMI). Serum Mb levels higher than 0.16 μg/ml were considered abnormal. In 94% of these patients who were finally diagnosed with AMI, Mb levels were higher than 0.16 μg/ml, whereas all 30 normal control blood donors had lower Mb levels. Patients with anterior or inferior wall infarcts had higher Mb levels (≫0.64 μg/ml) than patients with lateral or subendocardial infarction. Only 68% (82/ 121) of patients evaluated by elevated CK‐MB alone had a final diagnosis of AMI. In contrast, 94% (77/83) of patients who in addition showed elevated Mb had AMI. It is suggested that analysis of Mb levels allows a more accurate diagnosis of AMI in patients with elevated CK‐MB than does reliance on CK‐MB values alone. © 1993 Wiley‐Liss, Inc.
Dual organism infection in biopsy specimens from immunocompromised patients: Two cases demonstrated by immunocytochemistry
Authors: Reed Jon A. , Slater Leonard N. , Hemann Betty A. , Brigati David J.
Published: March 2007

We report two cases in which immunocy‐tochemistry demonstrated simultaneous infections within a single biopsy specimen obtained from each of two immunocompromised patients in whom multiple organism infection was not suspected. Using a rapid immunocyto‐chemical technique, Pneumocystis carinii and Cryptococcus neoformans were demonstrated in a transbronchial lung biopsy from the first patient; Candida and cytomegalovirus were demonstrated in an endoscopic biopsy of the esophagus from the second patient. Routine hematoxylin and eosin as well as special stains failed to establish the presence of more than one infectious agent in either case. Mycology cultures ultimately confirmed the presence of Cryptococcus in the first case, but cultures were not performed in the second case due to the scant amount of tissue obtained from the biopsy. The results indicate that concurrent infection by more than one opportunistic organism in immunocompromised patients can easily remain unrecognized. The use of immunocytochemistry may thus provide an important adjunct to routine and special stains or specific cultures. © 1993 Wiley‐Liss, Inc.
Antibodies to Sm, RNP and SSB detected by solid‐phase ELISAs using recombinant antigens: A comparison study with counter immunoeloctrophoresis and immunoblotting
Authors: Delpech Annie , Gilbert Daniéle , Daliphard Sylvie , Le Loet Xavier , Godin Michel , Tron Francois
Published: March 2007

Sera from 64 patients with systemic lupus erythematosus (SLE) were tested for the presence of anti‐Sm, anti‐RNP, and anti‐SSB antibodies using commercially available solidphase enzyme‐linked immunosorbent assays (ELISAs) and recombinant nuclear proteins as substrates. The results were compared to those obtained with counterimmunoelectrophoresis (CIE) and immunoblotting (IBT) using a rabbit thymus extract (RTE) as the substrate.

The ELSAs detected antibodies to Sm, RNP, and SSB in, respectively, 25%, 36%, and 15% of the SLE sera. Neither IBT‐positive/ ELISA‐negative nor CIE‐positive/ELISA‐negative sera were found, regardless of the specificity considered, suggesting that ELISAs using recombinant nuclear antigens are highly sensitive.

Discrepancies were observed between the results obtained with these different techniques. In addition to sera positive by both IBT and ELISA but negative by CIE, a substantial number of sera had ELISA‐detectable anti‐Sm, anti‐RNP, and anti‐SSB antibodies which failed to react with the corresponding polypeptides by IBT. The reasons for ELISA/IBT discrepancies were explored; however, no single explanation was found. Instead, a higher sensitivity of the ELISA to detect antibodies directed against certain polypeptides, the possible inability of IBT using RTE as the substrate to detect antibodies reacting with conformational antigenic determinants, and false‐positive reactions in the ELISAs were suggested.

Thus, it is still advisable to perform both IB and ELISAs simultaneously in well‐defined autoimmune diseases to further analyze the potential advantages of ELISAs using recombinant antigens. © 1993 Wiley‐Liss, Inc.
Rapid colorimetric detection of epidermal growth factor receptor mRNA by in situ hybridization
Authors: Iezzoni Julia C. , Kang Jin‐Han , Bucana Corazon D. , Reed Jon A. , Brigati David J.
Published: March 2007

We describe a rapid, formamide‐free, random oligomer‐enhanced in situ hybridization method in formalin‐fixed, paraffin‐embedded tissue sections using a biotinylated oligonucleotide probe for colorimetric detection of the mRNA transcript of the epidermal growth factor receptor (EGFR) gene, a putative protooncogene. Transitional cell carcinomas (TCC) of the urinary bladder and oral squamous cell carcinomas (SCC) were examined. A431, a human squamous carcinoma cell line that overexpresses EGFR mRNA, and mature skeletal muscle, Known not to express EGFR, served as control tissues. A biotinylated poly‐T oligonucleotide probe was used to evaluate the preservation of mRNA in the formalin‐fixed, paraffin‐embedded tissues. The EGFR mRNA and poly‐T oligonucleotide probes were constructed with a target‐specific, 5′ region and a 3′, non‐hybridizing, biotinylated tail. Random sequence oligomers nine bases long added to the probe cocktail eliminated the need for formamide and sheared carrier DNA in the method. The assay produced strong, specific staining for EGFR mRNA in all cases of TCC and SCC, and in the A431 tumors, but not in skeletal muscle. The assay was completed in approximately 90 minutes. This method may have widespread utility for rapid and specific detection of other mRNA sequences. © 1993 Wiley‐Liss, Inc.
Detection of antineutrophil autoantibodies by flow cytometry: Use of unfixed neutrophils as antigenic targets
Authors: Maher George M. , Hartman Kip R.
Published: March 2007

Antineutrophil antibodies may be found in the sera of patients with chronic neutropenia as well as in the sera of a variety of patients with neutropenia and associated autoimmune or infectious disorders. We evaluated an immunofluorescent flow cytometric technique for the measurement of antineutrophil antibodies in serum. Sera from patients with suspected immune neutropenia were studied and compared with a group of sera from normal healthy individuals, as well as with sera from patients with rheumatoid arthritis and systemic lupus erythematosus. Of 159 patients with suspected immune neutropenia and a variety of associated clinical disorders, 59 (37%) were found to have evidence for enhanced binding of IgG to normal target neutrophils, interpreted as positive for antineutrophil antibodies. Whereas 0/37 nonneutropenic patients with typical RA had positive results, 51/244 (21%) of sera from nonneutropenic patients with SLE or other collagen vascular disorders showed enhanced IgG binding to neutrophils. Living neutrophils were used to study the effects of cellular activation, and increased antibody binding was observed with certain sera that contained IgG directed against activation‐dependent antigens. We found that, under controlled conditions, flow cytometry can be reliably used to detect antineutrophil autoantibodies, with unfixed, living neutrophils as antigenic targets. © 1993 Wiley‐Liss, Inc.
Enzyme immunoassay and characterization of pancreatic stone proteins in human urine
Authors: Tatemichi Noriyuki , Takahashi Chikako , Hayakawa Shinobu , Hayakawa Tetsuo , Shibata Tokimune , Kitagawa Motoji , Sobajima Hiroshi , Nakae Yasuyuki
Published: March 2007

An enzyme immunoassay of pancreatic stone protein (PSP) in human urine was developed. Mean analytical recovery of pure PSP‐S2‐5 added to urine was 102.3% (SD 5.9%), and the precision of the assay was 2.0‐2.7% within an assay and 2.5‐2.9% between assays. In healthy volunteers (age 20‐55 years), the mean value of the PSP concentration, expressed as ratios to urine creatinine, was 129 ± 88 (mean ± SD) μg/g without any differences for sex. Urine PSP correlated with urine N‐acetylglucosaminidase (NAG) (r = 0.354). The molecular forms of immunoreactive PSP in urine were characterized by using cation exchange chromatography (Mono S), SDS‐PAGE, N‐terminal sequence, and enzyme immunoassay analysis. The urine PSP, eluted at the position corresponding to PSP‐S2‐5 on cation exchange chromatography, was converted to PSP‐S1 by trypsin digestion. The difference in mobility on SDSPAGE between urine PSP and PSP‐S2‐5 seems to be due to a glycosylated undecapeptide (N‐terminal 1‐11). The proposed method offers a sensitive, specific, and reproducible tool for laboratory analysis of human urine PSP levels. © 1993 Wiley‐Liss, Inc.
Reticulocyte count with maturation fractions in pancytopenic evaluation by a fully automated counter
Authors: Lin Chung K. , Hsu Hui C. , Chau Wing K. , Jiang Mei L. , Chiu Chang F.
Published: March 2007

Using a fully automated reticulocyte counter, the roles of the reticulocyte count with maturation in pancytopenia were evaluated. Different groups of pancytopenia including aplastic anemia, infiltrative marrow disorder, hypersplenism, and megaloblastic anemia were recruited. All patients had bone marrow examinations for morphological diagnosis and reticulocyte evaluation using an automated counter. The roles of these parameters were then analyzed statistically in the differential evaluation among these conditions. The following subjects were studied: 292 normal subjects, 67 cases of aplastic anemia, 69 cases of marrow infiltration by different malignancies, 35 cases of hypersplenism, and 13 cases of megaloblastic anemia. The results showed that the absolute retlculocyte counts were lowest in the groups of aplastic anemia and megaloblastic anemia and highest in hypersplenism. Both showed significant differences from the infiltrative groups. The maturation fractions were most immature in the group of marrow infiltration and are significantly different from the other groups. It was concluded that the highest absolute reticulocyte count (>0.09 1012/L) obtained in pancytopenic patients suggests it to be a case of hypersplenism. The lowest counts ( < 0.03 1012/L) with lowest immature fractions ( < 10%) suggest the groups of aplastic or megaloblastic anemias. The highest immature fractions ( >30%) with a nearly normal reticulocyte count favor the group of marrow infiltration. © 1993 Wiley‐Liss, Inc.
Human thyroid peroxidase: Autoantibody recognition depends on the natural conformation
Authors: Berthold Heike , Steffens Ute , Northemann Wolfgang
Published: March 2007

Thyroid peroxidase (TPO) purified from human thyroid glands was used to study the correlation between natural conformation and its ability for binding of autoantibodies in sera from patients with autoimmune lymphocytic thyroiditis (Hashimoto's thyroiditis). Treatment with heat (60°C), urea, sodium dodecylsulfate (SDS), and dithiothreitol (DTT) decreased the autoantigenicity of TPO on average by 73.3%, 74.1%, 86.3%, and 91.8% measured by enzyme‐linked immunosorbent assay (ELISA), respectively. These data demonstrate, that the binding of the majority of autoantibodies to human TPO depends on its natural conformation and modification. © 1993 Wiley‐Liss, Inc.
Quantitation of hepatitis B virus DNA in serum by ammonium sulfate precipitation and molecular hybridization
Authors: Wen Long T. , Henneberger Michael , Nguyen Nicole , McPherson Richard A.
Published: March 2007

We developed a method to quantitate hepatitis B virus (HBV) DNA in serum by ammonium sulfate fractionation and DNA hybridization. Serum samples were precipitated with 45% saturated ammonium sulfate, resuspended in buffer, and spotted on a nylon membrane. Following denaturation in alkali, HBV DNA sequences on the membrane were detected by hybridization with a 32P‐labeled DNA probe of the entire HBV genome. Bound radioactivity was measured with liquid scintillation counting. Ammonium sulfate fractionation of positive samples increased assay sensitivity by 10–30‐fold compared to no treatment. Sensitivity for detection of cloned HBV DNA added to negative serum was 0.2 pg. Recovery of cloned HBV DNA added to negative serum before fractionation was equivalent to direct spotting of DNA onto the membrane in the absence of serum. This method enhanced HBV DNA recovery from serum into small volumes, thereby expanding the potential analytic range of spot hybridization assays. © 1994 Wiley‐Liss, Inc.
Evaluation of bioassay for toxicity of ciguateric fish and associated toxins
Authors: Hokama Y. , Shirai J. L. , Islam M. A.
Published: March 2007

Evaluation of the mouse toxicity assay symptom of hind leg paralysis (HLP) with mouse death by statistical analysis is presented in this study. The fishes assessed were herbivores including Ctenochaetus strigosus (kole), Ctenochaetus hawaiiensis, Acanthurus sandvicensis (manini), and Mugil cephalus (mullet); and the carnivores, Cephalopholis argus (roi) and Cheilinus rhodochrous (po'ou). The latter can also be considered an omnivore. The extracts of both herbivore and carnivore species appeared to be most toxic when HLP occurred in the mice. Ninety‐three percent of the mice with HLP died, whereas when no HLP (NHLP) occurred, only 51% of the mice died. Carnivore flesh extracts (po'ou and roi) were least toxic with one death out of a total 22 mice. The unidentified toxin associated with HLP appears to differ in biological properties from that of ciguatoxin(s) in that it was not found in the flesh tissues of carnivores. Further chemical studies of this toxin(s) is being addressed presently. © 1994 Wiley‐Liss, Inc.
Mapping of antigenic epitopes within the recombinant human preproinsulin related to insulin‐dependent diabetes mellitus
Authors: Walter Michael , Berg Heike , Northemann Wolfgang
Published: March 2007

The human insulin domains, signal peptide, B‐chain, C‐peptide, and A‐chain, were highly expressed in Escherichia coli as recombinant proteins N‐terminally fused to glutathione‐S‐transferase and a histidinehexapeptide. The recombinant proteins were purified from insoluble cell fraction by affinity chromatography using metal chelating matrix, which was charged with Ni+2 iones. ELISA screening for autoantibodies directed to preproinsulin were performed with sera from patients with recently diagnosed insulindependent diabetes mellitus in order to localize the antigenic epitopes within the human preproinsulin. Of the patients, 14% had developed autoantibodies that recognized either the recombinant C‐peptide or the signal peptide. No reaction was observed with the A‐chain or B‐chain. © 1994 Wiley‐Liss, Inc.
Comparison of two Cobas Core enzyme immunoassays with other test systems for the detection of cytomegalovirus‐specific IgG and IgM antibodies
Authors: Steinmann J. , Weigel T.
Published: March 2007

The new Cobas Core CMV IGM and IgG enzyme immunoassays (Roche Diagnostic Systems, Basle, Switzerland) were evaluated for their ability to detect cytomegalovirus (CMV)‐specific IgM and IgG antibodies. Therefore, both were compared with some other commercially available and already established serological tests used in the laboratory diagnosis of CMV infection. These included the Abbott IMx CMV IgM and IgG assays, the Abbott CMV‐M EIA, the Gull CMV IgM and IgG immunofluorescence tests, the medac CMV‐IgM‐ELA, and the Enzygnost anti‐cytomegalovirus assay (Behringwerke). A total of 572 serum samples of various categories were examined and the results showed high concordances between all methods, ranging from 84.5% to 94.9%. In a follow‐up on renal transplant patients, the times of first detection of seroconversions were compared. Since a high overall agreement between the Cobas Core CMV IgM and IgG enzyme immunoassays and the other test systems were observed, these new assays represent useful and reliable tools for clinical CMV diagnosis. © 1994 Wiley‐Liss, Inc.
Comparison of techniques for minimizing interference of bilirubin on serum creatinine determined by the kinetic Jaffé reaction
Authors: Lolekha Porntip H. , Sritong Noppmats
Published: March 2007

This study compared the effect of sodium dodecyl‐sulfate, potassium ferricyanide, and preincubation technique to continuous‐flow analysis and prior deproteinization to correct the negative interference of bilirubin on serum creatinine found by the kinetic Jaffé reaction. Bilirubin increased to 684 μmol/L did not interfere with serum creatinine measured by the methods incorporated with dialysis or deproteinization. Trichloroacetic acid was the best protein precipitant. The reagent incorporated with sodium dodecyl sulfate was more appropriate to minimize bilirubin interference than reagent containing potassium ferricyanide. An increase in potassium ferricyanide concentration resulted in false positive creatinine values. Incorporation of both SDS and potassium ferricyanide in the reagent did not help in minimizing the bilirubin interference over use of each chemical alone. The 10 minutes of preincubation of the sample with alkaline buffer incorporating with either SDS or potassium ferricyanide before starting the Jaffé reaction was the appropriate way to overcome unconjugated bilirubin interference at a level of 342.0 μmol/L. However, the technique did not work uniformly with icteric patient sera containing conjugated, unconjugated, and delta bilirubin. This is a challenging problem that remains to be solved by the clinical chemist. © 1994 Wiley‐Liss, Inc.
Contribution of the host to test results in assays of staphylococcus epidermidis
Authors: Herndon Betty , Dall Lawrence , Suvarna Padma
Published: March 2007

From two human populations (one pediatric and one adult), clinically diagnosed with Staphylococcus epidermidis (S. epidermidis) sepsis of similar severity, bacteria were isolated from pre‐antibiotic blood samples and evaluated for virulence. The LD50 of the bacteria in a mouse model was performed, with evaluation of animals dying acutely following intravenous S. epidermidis administration. More simple assays of virulence were also performed, including bacterial adherence to a fibrin clot and carbohydrate specific lectin binding. The eight pediatric‐host S. epidermidis isolates required a significantly larger dose to produce lethality in dosed animals (LD50) when compared to the 20 adult‐host S. epidermidis isolates. The fibrin clot assay, a test that has corroborated bacterial virulence in endocarditis models, did not differentiate the groups: all but one of the 28 isolates were well above the adherence seen with the ATCC control, suggesting endocarditis‐producing potential. Glycocalyx (slime) from eight of the more virulent isolates showed reactivity with a glucose‐specific biotinylated lectin which was lacking in other isolates. Necropsy of mice dying at 12 hr showed S. epidermidis strain differences in specific organ effects.

Overall, this study demonstrates the utility of the LD50 to provide a highly sensitive quantification of bacterial virulence. Necropsy of test animals dying acutely has showed an apparent organ tropism of some of these isolates which are usually considered harmless commensals.©1995 wiley‐Liss, inc.
Colorimetrical rate assay for urinary dipeptidyl peptidase iv (dppiv) activity using a new substrate
Authors: Shibuya‐Saruta Hiroko , Sugiyama Masami , Kasahara Yasushi
Published: March 2007

We synthesized a new substrate glycyl‐L‐proline 3,5‐dibromo‐4‐hydroxyanilide (Gly‐Pro‐DBAP), for dipeptidyl peptidase IV (DPPIV). Its hydrolysis by DPPIV resulted in the formation of a chromophore, 2,6‐dibromo‐phenol‐indo‐p‐xylenol, and its maximal absorption wavelength (600 nm) was longer than that of p‐nitroaniline (415 nm) released from conventional substrate, glycyl‐L‐proline pnitroanilide (Gly‐Pro‐pNA). We also established the rate assay for urinary DPPIV activity using Gly‐Pro‐DBAP. The optimum pH was between 8.5 and 9.0. The apparent Km was 1.1 mmol/1. The detectable range was 2.5–350 U/I. No changes in blank values occured throughout the enzyme reaction in the optimum pH. Its value was also much lower than Gly‐Pro‐pNA. CVs for within‐run and between‐run were 1.1% (n=10) and 3.0% (n=10), respectively. Among tested peptidases, only DPPIV could hydrolyze Gly‐Pro‐DBAP. Among the protease inhibitors, only two, diprotin‐A and phenylmethylsulfonyl fluoride (PMSA), could inhibit DPPIV activity. The present method did not interfere with urinary ingredients such as hemoglobin. The correlation between the present (y) and conventional (x) methods is presented by the equation y=1.121×+0.096 (r=0.993). Thus the present method provides practical advantages over the conventional method for routine laboratory use.©1995 wiley‐Liss, inc.
Expression of matrix metalloproteinase matrilysin (mmp‐7) was induced by activated ki‐ras via ap‐1 activation in sw1417 colon cancer cells
Authors: Yamamoto Hiroyuki , Itoh Fumio , Senota Akinori , Adachi Yasushi , Yoshimoto Mitsuru , Endoh Takao , Hinoda Yuji , Yachi Akira , Imai Kohzoh
Published: March 2007

The matrix metalloproteinase matrilysin (MMP‐7) is a member of the matrix metallo‐proteinase gene family, which is believed to play an important role in tumor invasion and metastasis. We have previously found that matrilysin mRNA is specifically expressed in colorectal cancers and adenomas and that its message is localized in the tumor cells themselves. We examined the effects of activated Ki‐ras oncogene on the expression of matrilysin in colon cancer cells. We showed that both mRNA and the enzymatic activity of matrilysin were induced by the introduction of activated Ki‐ras into SW1417 colon cancer cells. To understand the mechanisms regulating this induction, we analyzed alterations of AP‐1 activity induced by activated Ki‐ras, using the chloramphenicol acetyltransferase assay. AP‐1 activity in SW1417 cells expressing activated Ki‐ras was higher than that in control cells. The gel‐shift assay also showed higher levels of AP‐1 binding protein in SW1417 cells expressing activated Ki‐ras than those in control cells. Our results suggest that activated Ki‐ras may play a role in inducing expression of matrilysin through an AP‐1‐dependent pathway in colon cancer cells.
Comparison of cathepsin d determinations in human carcinomas by enzyme immunoassay and immunoradiometric assay
Authors: Shaheen Raymond M. , Miseljic Slobodan , Doering David L. , Wittliff James L.
Published: March 2007

Overexpression of cathepsin D in several types of carcinoma in women appears to be associated with a poor clinical course. In this prospective investigation, cathepsin D levels in 170 specimens of normal and neoplastic human tissues were determined simultaneously by enzyme immunoassay (EIA) and immunoradiometric assay (IRMA) to allow comparisons in multicentric studies, such as cooperative clinical trials. Nonmalignant uteri and specially prepared reference powders were also evaluated. Linear regression analysis between the two assays for all specimens [EIA = 0.87(IRMA) – 3.18] demonstrated a correlation coefficient (r) of 0.99 (P <0.001). When malignancies were categorized by the tissue origin (i.e., breast, uterus, ovary, lymph node, and colon), highly significant correlations were also observed (regressions slopes ranged from 0.58 to 1.02). Intra‐ and interassay controls conducted for the new EIA procedure gave CV% ranging from 4.4 to 10.2, which was similar to the IRMA test for cathepsin D. The results of both assays correlated well and were highly reproducible. Either assay may be used with confidence that comparable cathepsin D values will be obtained in a wide range of tissue biopsies.
Problems in determining levels of free digoxin in patients treated with digoxin immune fab
Authors: Honda Stacey A. A. , Rios Carlos N. , Murakami Lourna , Morita Thelma , Scottolini Alfred G. , Bhagavan Nadhipuram V.
Published: March 2007

Determination of free digoxin levels in patients treated with digoxin immune FAb has long been a problematic area in clinical laboratory testing. The older radioimmunoassays resulted in inaccurate and variable results due to the competition of the administered drug with the radioactively labelled forms. The 1995 Physicians' Desk Reference continues to state that digoxin immune FAb will interfere with digitalis immunoassay measurements. This statement, however, is based primarily on the RIA methods. We evaluated the Stratus digoxin assay and the TDX digoxin II assay. Increasing amounts of immune FAb were added in a stepwise fashion to 12 patient samples containing high normal to elevated digoxin levels. Results showed a progressive decrease in digoxin levels when assayed with the Stratus kit. However, five patient samples tested with the TDX kit resulted in constant digoxin values despite the presence of increasing digibind levels. These results suggest that the Stratus method measures free digoxin, whereas the TDX method measures the total digoxin. Measurement of digoxin by the Stratus method is simple and quick. The Stratus digoxin assay may be an accurate and objective way of measuring free digoxin levels in patients on digoxin immune FAb.
Anticoagulant response to activated protein c: Method validation and assay comparison
Authors: Calkins Tom , Greengard Judy , Griffin John H. , Bylund David
Published: March 2007

Poor anticoagulant response to activated protein C (APC), present in 20–60% of thrombophilic patients, is most often caused by abnormal factor Va due to the mutation of Arg506 to Gln, and DNA sequencing confirms this finding. At Scripps Reference Laboratory (SRL), we have validated an in‐house assay to detect APC resistance. A study of 80 normal subjects (40 males and 40 females, 21–60 years old) showed that adult males and females have statistically significant differences in their anticoagulant response to APC. Furthermore, APC response is increased in older individuals. APC responses of the same 80 normal samples determined using the SRL assay were compared to a commercial kit (Chromogenix®, Coatest). Although both procedures are similar, the SRL assay gave a greater difference between male and female normal ranges. Eight of 18 (44%) thrombophilic patients were identified as APC‐resistant. Samples from individuals having the factor V Arg506Gln mutation yielded low responses to APC in both assays. Determination of APC resistance assists physicians in determining the cause of thrombotic disorders and may eventually help in predicting the risk of familial thrombosis.
Splitting and dehulling lentil (Lens culinaris): Effects of genotype and location
Authors: Erskine William , Williams Philip C , Nakkoul Hani
Published: March 2007

Most red‐cotyledon microsperma lentils (Lens culinaris Medikus) are utilised after postharvest processing into split dehulled seed. This study examined the effects on dehulling of 23 diverse genotypes of red‐cotyledon microsperma lentil, each grown at three sites ranging from 245 to 663 mm total seasonal rainfall in Lebanon and Syria. Dehulling efficiency (%) was measured as the sum of split dehulled seed, whole dehulled seed, and whole hulled seed. Despite a range in location means of 464–2018 kg ha−1 for grain yield, the corresponding mean dehulling efficiencies varied only from 84.8 to 85.9%, indicating that the location effect on dehulling efficiency is of minor importance. There was more variation among genotypes for dehulling efficiency (80.8–87.7%) and for preparation of split dehulled seed (62.1–80.2%) than between locations. Seed processing variables showed medium to high broadsense heritabilities. Correlations between seed processing characters and agronomic variables were generally low. An approach to seed selection for optimum processing variables within the ICARDA breeding programme is discussed.
Selection of the most appropriate reference crop for the assessment of nitrogen fixation by soya bean in Anambra state, Nigeria
Authors: Okereke G U , Dauda D , Ayama D
Published: March 2007

Nitrogen fixation by various soya bean cultivars was estimated using upland rice, maize and sesame as reference non‐legume crops to enable the selection of the most appropriate reference crop for Nigerian soil. The per cent 15N atom excess of rice was consistently higher than that of the three soya bean cultivars at all sampling dates, indicating that the soya bean cultivars fixed N whereas rice did not. By contrast, the per cent 15N atom excesses of sesame and maize were smaller than those of the soya bean cultivars at certain days after planting, giving a false impression of nitrogen fixation by sesame and maize. Using rice to calculate the per cent nitrogen derived from the atmosphere (NDFA) gave larger and consistently positive values. The use of maize and sesame as reference crops gave inconsistent negative and positive values for the NDFA. Thus, upland rice was a better reference crop for the calculation of NDFA under the prevailing conditions of the experimental site.
Effect of water extracts of neem (Azadirachta indica) on the activity of hydrolytic enzymes of mixed rumen bacteria from buffalo
Authors: Agarwal Neeta , Kewalramani Neelam , Kamra Devki N , Agarwal Dinesh K , Nath Kedar
Published: March 2007

The effect of cold and hot water extracts of neem (Azadirachta indica A Juss) seed kernel cake on the hydrolytic enzymes of mixed rumen bacteria was studied. The activities of carboxymethylcellulase and xylanase were stimulated, and those of α‐amylase, protease and urease were inhibited by the addition of neem cake extract to the reaction mixture. The stimulation and inhibition became more intense with increasing concentration of extract. The hot water extract was more effective than cold water extract in stimulation of carboxymethylcellulase and xylanase activities, but ä‐amylase was inhibited equally by both the extracts.
Effects of heat treatment and storage temperature on the biogenic amine content of straw mushroom (Volvariella volvacea)
Authors: Yen Gow‐Chin
Published: March 2007

The changes in the biogenic amines tryptamine, 2‐phenylethylamine, putrescine, cadaverine, histamine and tyramine of straw mushroom Volvariella volvacea (Bull, ex Fr) Sing produced by heat treatment and during storage at different temperatures were studied. About 80% of the original content of these amines was lost during cooking. Commercial canned straw mushroom contained low amounts of amines. The amine contents of straw mushroom increased during storage at 4°C, with particularly notable increases in the concentrations of 2‐phenylethylamine and tyramine after 5 days' storage. However, the levels of all amines increased more markedly during storage at 25°C, and the increases in the putrescine and cadaverine concentrations were much greater than those reached at 4°C.