bluuebunny/crossref_metadata_2025 huggingface dataset:
Quick measurement of serum unconjugated estrogens, and its clinical application to abnormal pregnancies and ovarian follicle maturation
Authors: Kondo Zenjiro , Makino Tsunehisa , Lzumi Shun‐Ichiro , Lizuka Rihachi
Published: March 2007

Using an automated HPLC system reported recently by us (J Clin Lab Anal 4:410–413, 1990), serum unconjugated estriol and estradiol (u‐E3 and u‐E2) can be measured within 22 min. The clinical value of this method was evaluated in this study with abnormal pregnancies, twin pregnancies, and pregnancies associated with fetal anencephaly or complicated by gestational toxicosis or diabetes mellitus. A significant correlation was demonstrated between an abrupt decrease in serum u‐E3 level and an abnormal pattern on fetal heart rate monitor. Since urinary E3 was not sensitive enough to detect any depressed fetal condition, serum u‐E3 is better biochemical parameter for assessment of fetal well‐being.

In addition, u‐E2 was measured in the sera of nonpregnant women with separation chromatography as a pretreatment in order to obtain higher sensitivity of the system. This modified HPLC system enabled us to determine the serum u‐E2 in about 60 min with the minimal detectable value of 80 pg/ml; the coefficient of correlation with RIA was 0.822. As a result, this quick measurement of u‐E3 and u‐E2 by our HPLC system revealed to be useful not only for evaluating fetoplacental function but also for monitoring ovarian follicle maturation.
Methodological pitfalls in serum IgG2 level measurements by immunoenzymatic assays with monoclonal antibodies
Authors: Aucouturier Pierre , Lacombe Corinne , Preud'homme Jean‐Louis
Published: March 2007

Four anti‐IgG2 monoclonal antibodies (Mabs) were evaluated for their reactivity with purified myeloma IgG2 of different light chain types and Gm allotypes in three distinct immunoenzymatic assays (ELISA). The reactivity of three Mabs with solid‐phase antigens was similar whereas an anti‐Fab antibody (clone HP 6114) predominantly bound IgG2k. In competitive and immunometric (sandwich type) assays, the binding of the two anti‐IgG2 Mabs (HP 6014 and HP 6114) reacting with epitopes located on the Fab fragment was strongly influenced by the light chain type of IgG2 and by other factors (probably including differences in the variable regions): the Mab HP 6114 reacted virtually only with IgG2k whereas the Mab HP 6014 displayed a much stronger affinity for IgG2λ than for IgG2k; for both anti‐Fab Mabs, important differences were found in their binding to individual IgG2 proteins. In addition, the Mab HP 6014 seemed to show a slightly better affinity for IgG2 bearing the Gm(23) allotype. These results urge much caution in IgG2 level measurement, especially with commerical kits, most of which use the Mab 6014 as the single anti‐IgG2 reagent.
Microparticle‐enhanced nephelometric immunoassay for human C‐reactive protein
Authors: Montagne P. , Laroche P. , Cuillière M. L. , Varcin P. , Pau B. , Duheille J.
Published: March 2007

Polyfunctional hydrophilic microspheres of 125‐nm diameter can be produced by copolymerization of acrylic monomers. Purified c‐reactive protein (CRP) was covalently bound to these new micropheres, and the conjugate obtained was used as reagent in a microparticle‐enhanced nephelometric immunoassay for human CRP. This assay was based on the measure, with a specially designed nephelometer, of the light scattered by aggregates formed during the immunoagglutination of the conjugate with anti‐CRP antiserum. Sensitive inhibition of this agglutination by free CRP (6 ng/ml) allowed CRP quantitation in highly diluted serum samples (1/500‐1/2,000), excluding any interference or sample pretreatment. The CRP assay was easy to perform (no washing or phase separation), reliable (coefficients of variation ranged from 1.3% to 9.3% for within‐run and between‐run determinations), and accurate (mean percentage of recovery: 104%; correlation coefficients with accepted analytical methods >0.97) over a large range of concentrations. The inhibition mode excluded errors in the antigen excess zone and provided total security at high concentrations.
Establishment of a sensitive radioimmunoassay for serum thymic factor with a special reference to extraction procedures
Authors: Mukaida Naofumi , Morimoto Taeko , Takanashi Naoki , Kawai Tadashi
Published: March 2007

A radioimmunoassay (RIA) for serum thymic factor (facteur thymique serique) was established. This assay could detect <0.5pg of serum thymic factor without the interference by other thymic hormones, including thymopoietin ii and thymosin α1. Both zinc‐free and zinc‐bound serum thymic factor showed a similar standard curve, indicating that this RIA could detect both forms of serum thymic factor in the same way. We observed that two procedures were required prior to the measurement in order to measure serum thymic factor content in plasmas; the first, the extraction procedure to remove substances interfering with the assay nonspecifically; the second, the storage of the sample with ethylenediamine tetraacetic acid dissodium salt to inhibit the proteases in the plasma to degrade serum thymic factor. By employing this assay system, we observed that cord blood plasmas contained significantly higher level of serum thymic factor than did adult blood plasmas.
Oxidative metabolism of polymorphonuclear leukocytes (PMN) in patients with IgA nephropathy
Authors: Chen Hung‐Chun , Tomino Yasuhiko , Yaguchi Yutaka , Fukui Mitsumine , Yokoyama Ken‐ichi , Watanabe Asako , Koide Hikaru
Published: March 2007

The production of hydrogen peroxide (H2O2) by neutrophilic polymorphonuclear leukocytes (PMN) after stimulation and the infiltration of PMN in glomeruli were determined in 20 patients with primary IgA nephropathy. The H2O2 production of PMN after the stimulation was measured with a spectrophotometer using horseradish peroxidase as substrate. The results were as follows: (1) when PMN were pretreated with cytochalasine B, H2O2 production after stimulation with heataggregated IgG (IgG) or serum‐treated zymosan (STZ) was significantly higher in patients with IgA nephropathy than in controls, and (2) there was an increased amount of PMN localized in glomeruli in patients with IgA nephropathy using immunofluorescence of monoclonal anti‐PMN antibody.

It appeared that the increased renal infiltration of PMN which have a high potential for production of reactive oxygen species might induce the glomerular injuries in patients with IgA nephropathy.
A monoclonal antibody‐based immunoassay for detecting tetrodotoxin in biological samples
Authors: Raybould T. J. G. , Bignami G. S. , Inouye L. K. , Simpson Samantha B. , Byrnes Jilanne B , Grothaus P. G. , Vann D. C.
Published: March 2007

Spleen cells from mice hyperimmunized with a keyhole limpet hemocyanin‐tetrodotoxin‐formaldehyde conjugate were fused with murine P3X63Ag8.653 myeloma cells. A single hybridoma clone was identified that secretes an IgG1,k monoclonal antibody (MAb), designated T20G10, against tetrodotoxin (TTX), with an estimated affinity of 1.2 × 108 L/M. Competitive inhibition enzyme immunoassays (CIEIAs) for detecting TTX were developed using this MAb. A direct CIEIA using alkaline phosphatase‐labeled MAb detected TTX with sensitivities at IC50 and IC20 of 6‐7 ng/ml and 2‐3 ng/ml, respectively. The accuracy of the direct CIEIA was comparable with the high‐performance liquid chromatography (HPLC) and the mouse bioassay systems, but the direct CIEIA exhibited greater sensitivity. The direct CIEIA was also more cost effective, as it required less sample preparation, a shorter assay time, and reduced investment in equipment than either of the other assay systems.
Comparison of the use of bulk to micro culture of cell preparations for lymphokine‐activated cytotoxicity assays
Authors: Anderson Loraine H. , McDonald Thomas L. , Thiele Geoffrey M. , Klassen Lynell W.
Published: March 2007

Different assay systems have been used to quantitate lymphokine‐induced natural cytotoxic activity as a measure of immune status. This study compares the effects of inducing cytotoxicity in a bulk culture system, where effector cells are transferred to a micro culture well for assay, to a micro culture system where the effector cells are not transferred. The effector/target ratio for both the bulk and micro culture systems was calculated using the number of viable effector cells present at the time of target cell addition. After over‐night incubation with interleukin‐2 (IL‐2), the lytic activity of murine spleen cells to targets using a micro culture system was increased two‐fold over the bulk culture method. This increase was amplified further after 5 days of activation with IL‐2, in that the micro culture system resulted in a four‐fold increase in cytotoxic activity. The loss of some adherent cells in the bulk culture system did not explain the overall decrease in recovered cytotoxicity. The difference appeared to be related to cell loss during centrifugation. Therefore, the E/T ratios are different in the two systems if not corrected for the number of viable cells. © 1992 Wiley‐Liss, Inc.
Delayed hypersensitivity skin tests in prognosis of human immunodeficiency virus infection
Authors: Zeballos R. S. , Cavalcante N. , Freire C. A. R. , Hernandez H. J. , Longo I. M. , Peixinho Z. F. , Moura N. C. , Mendes N. F.
Published: March 2007

Delayed hypersensitivity skin tests (DHST) with recall antigens were investigated as prognostic markers in five different approaches. In the first study, 42 acquired immunodeficiency syndrome (AIDS) patients (IVb, IVcl, IVd, and IVe; MMWR 35:334–339, 1986) 26 AIDS‐related complex (ARC) patients (IVa and IVc2), and 98 asymptomatic patients (II and III) were evaluated with candidin, tricophytin, PPD and streptokinase‐streptodornase. In the second study, 10 patients (II and III) were evaluated sequentially with the same antigens. In the third, 45 patients with at least two positive skin tests (“reactors”) were followed for one year and evaluated every 6 months with the same antigens. In the fourth, 16 “reactors” were followed and evaluated every 3 months with the same antigens. We measured the interval from the time at which patients first presented with only one or no positive DHST until the development of ARC or AIDS. In the last study, the correlation between absolute number of CD4+ lymphocytes and the number of DHST was studied in 151 patients. We found that the decrease in reactiveness to DHST correlated directly with the progression to AIDS, demonstrating the usefulness of this simple procedure as a valid prognostic marker. © 1992 Wiley‐Liss, Inc.
Effect of heat inactivation and sheep erythrocyte adsorption on the titer of anticardiolipin antibodies in primary antiphospholipid syndrome and healthy blood donor' sera
Authors: Nava Arnulfo , Bañales José L. , Reyes Pedro A.
Published: March 2007

The standard enzyme linked immunosorbent assay (ELISA) currently in use for detection of anticardiolipin antibodies (ACA) was used to evaluate the influence of heat inactivation and sheep erythrocyte adsorption on individual optical density (OD) of sera from healthy blood donors or patients with primary antiphospholipid syndrome. Each sample was tested after single or combined maneuvers as follows: adsorbed, adsorbed and inactivated, only inactivated, and compared to basal readings.

A significant increase of ACA titers did occur after inactivation of normal sera, but adsorption had no effect. In contrast, neither inactivation nor adsorption changed ACA titer in primary antiphospholipid syndrome sera as a group, although in certain sera there were changes.

This observation may suggest the presence in normal serum of a thermolabile factor which modulates ACA binding to its antigen and the reactivity of the anticardiolipin antibodies of the primary antiphospholipid syndrome with sheep erythrocyte membrane phospholipids. © 1992 Wiley‐Liss, Inc.
Acid pH‐induced changes in the immunoreactivity of specific antigen and antibody in circulating immune complexes from tuberculosis sera
Authors: Kumar Uday , Saxena R. K.
Published: March 2007

The effect of acid pH treatment of circulating immune complexes (CIC) derived from tuberculosis sera was studied on the relative titers of specific antibody (CIC Ab) and mycobacterial antigen (CIC Ag) in the complexes. While the specific antibody titers increased, the titers of CIC Ag declined as a result of acid pH treatment of CIC, both changes being highly significant statistically. Direct exposure of TB sera to pH 2.8 also resulted in significant enhancements in the titers of antibodies directed against Mycobacterium tuberculosis (M.tb.). Competition experiments indicated that the acid pH‐treated antibodies retained their antigen specificity. TB sera treated with pH 2.8 for 30 min and the neutralized back to pH 7.4 retained the enhanced antibody reactivity even after 7 days of storage at 4°C. Our results indicate that acid pH treatment induces higher antigen binding ability in anti‐M.tb. antibodies, present in free or complexed form in TB sera. These changes appear to be irreversible. Dissociation of CIC and analyzing the titers of released antibody and antigen components offered no advantage with respect to the immunodiagnosis of tuberculosis, as compared to the levels of undissociated CIC components or the serum antibody. © 1992 Wiley‐Liss, Inc.
Specificity of a Tumor Marker (CA54/61) and Its Individual Epitopes Recognized by Monoclonal Antibodies, MA54 and MA61, in Human Tumor Patients
Authors: Mochizuki Hiroshi , Nagoya Kimiko , Yamauchi Tadakazu , Takao Yoshimi , Masuko Hideyuki , Sato Hiroshi , Sasaki Hirosuke , Itoh Kotaro , Tsukazaki Katsumi , Udagawa Yasuhiro , Kobayashi Toshifumi , Nozawa Shiro
Published: March 2007

The specificity of a tumor marker (CA54/61) and its individual epitopes (CA54 and CA61) recognized by monoclonal antibodies (MA54 and MA61) and expressed by the same tumor marker were studied. Serum levels of CA54 and CA61 were compared with that of CA54/61. In lung adenocarcinoma and ovarian carcinoma, the positive rates of CA61 (42% and 68%) were higher than those of CA54 (32% and 32%) and similar to those of CA54/61 (45% and 74%). The serum levels of CA54 and CA61 showed a significant correlation (r=0.78), but 22% of tested sera were positive for CA54 and negative for CA61 or negative for CA54 and positive for CA61. It was demonstrated that the tumor specificity between CA54 and CA61 was not same and that the tumor specificity of CA54/61 was similar to that of CA61 rather than CA54. Moreover, the difference in the tumor specificity between CA54 and CA61 was considered to be reflected in the difference in their epitope structure. © 1992 Wiley‐Liss, Inc.
Sera from simian immunodeficiency virus‐infected rhesus macaques inhibit lymphocyte proliferation
Authors: Tomar Russell H. , Hinds Paul , Katz David , Schultz Kevin
Published: March 2007

Rhesus macaque monkeys infected with the simian immunodeficiency virus (SIV) develop a syndrome mimicking acquired immunodeficiency syndrome (AIDS) in humans. We had demonstrated previously that sera from individuals infected with human immunodeficiency virus (HIV) inhibit the proliferation of lymphocytes from healthy non‐infected subjects and that this phenomenon was associated with the development of clinical AIDS. Thus, we sought to determine whether sera from SIV‐infected monkeys would also inhibit lymphocytes from healthy humans and SIV‐negative rhesus monkeys.

Sera from SIV‐infected monkeys were compared with sera from uninfected animals and cultured with cells from healthy human volunteers or SIV‐negative monkeys in the presence or absence of phytohemagglutinin (PHA). Cell proliferation was determined by measuring the incorporation of radiolabeled thymidine into cellular DNA.

Sera from SIV‐infected monkeys suppressed the proliferation of human and non‐human primate lymphocytes. This activity appears to be similar to that described for sera from HIV‐1‐infected humans. Therefore, rhesus macaques infected with SIV provide a model for the study of serum inhibitory factors previously reported in AIDS patients. © 1992 Wiley‐Liss, Inc.
Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti‐human T‐cell leukemia virus type I) IgG in serum using recombinant gag p24(14–214) as antigen
Authors: Kohno Takeyuki , Hirota Kouichi , Sakoda Iwane , Yamasaki Motoo , Yokoo Yoshiharu , Ishikawa Eiji
Published: March 2007

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti‐human T‐cell leukemia virus type 1) IgG (anti‐HTLV‐l IgG) in serum using recombinant gag p24 (14–214) of HTLV‐l is described. The recombinant gag p24(14–214) is soluble in the absence of detergents and allows the use of enzymes other than horseradish peroxidase as a lbel in teh assays.

The usefulness of recombinant gag p24(14–214) was examined with 305 sera characterized by other methods including gelatin particle agglutination, enzyme‐linked immunosorbent assay (ELISA) using HTLV‐l, and Western blotting.

This Assay was more sensitive than other methods using HTLV‐l as antigen. The specificity could be tested by preincubation of test serum with excess of the recombinant protein. Most of negative and positive sera were discriminated. However, some results appeared to be false‐positive or false‐negative, and recombinant gag p24(14–214) was suggested to be useful, when used with other recombinant proteins and/or peptides, for improving the reliability of serodiagnosis by separately demonstrating antibodies against as many different epitopes of HTLV‐l as possible.

Anti‐HTLV‐l IgG in test serum, which had been incubated with excess of inactive β‐D‐galactosidase to eliminate interference by anti‐β‐D‐galactosidase antibodies, was reacted simultaneously with 2,4‐dinitrophenyl‐bovine serum albumin‐recombinant gag p24(14–214) conjugate and recombinant gag p24(14–214)‐β‐D‐galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity‐purified (anti‐2, 4‐dinintrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the β‐D‐galactosidase conjugate, the complex was eluted from the polystyrene balls with ϵN‐2,4‐dinitrophenyl‐L‐lysine and transferred to polystyrene balls coated with affinity‐purifiedc (anti‐human IgG γ‐chain) IgG. β‐D‐Galactosidase activity bound to the (anti‐human IgG γ‐chain) IgG‐coated polystyrene balls was assayed by fluorometry. © 1992 Wiley‐Liss, Inc.
Histocompatibility screening by molecular techniques: Use of polymerase chain reaciton products and heteroduplex formation
Authors: Rubpcki Rlonald J. , Wisecarver James L. , Hook Darla D. , Cox Susan M. , Biesel Kirk W.
Published: March 2007

Novel molecular approaches have recently become available allowing improved major histocompatibility complex (MHC) matching of potential allogeneic bone marrow donors and recipients. Current cellular and serological assays are hindered by aberrant cell populations and limited reagents which only detect an individuals' phenotype. Therefore, a molecular screening protocol which discriminates at the genotypic level would be advantageous. Here we describe a two‐step DNA‐based approach that can be applied to large‐scale screening of potential donors. A primiary screen, utilizing polymerase chain reaction (PCR), reduces the potential donor population, whereas a secondary or fine resolution screen uses DNA heteroduplex analysis to determine identity or non‐identity at specific loci. Heteroduplex analysis generates a DNA migration pattern that is unique for alleles at a given locus, and is more sensitive than serology in discriminating among individuals. Here we demonstrate the potential feasibility of htis approach by analyzing results at one MHC locus, HLA‐DQ. Since this mehod does not rely on typing sera or viable lymphocytes, it is not subject to the variability found in the traditional methods. In contrast to traditional methods, these molecular techniques can provide the critical information needed to select a potential bone marrow donor. © 1992 Wiley‐Liss, Inc.
Chemically humanized murine monoclonal antibody against a cell nuclear antigen: Usefulness in autoimmune diagnostics
Authors: Miyachi Junko , Doi Kazuyuki , Kitamura Kazuyuki , Jitsukawa Tomofumi , Watanabe Hiroshi
Published: March 2007

The cellular nuclear antigen SS‐B/La is known to be a major antigenic target to an autoantibody in patients with Sjogren's syndrome and systemic lupus erythematosus. It is useful to detect an anti‐SS‐B/La antibody from patients' sera in a clinical point of view. We purified SS‐B/La from rabbit thymus acetone powder by affinity chromatography with a murine anti‐SS‐B/La monoclonal antibody (1C3‐H7). An enzyme‐linked immunosorbent assay method, in which SS‐B/La was used to coat a plate, was also successfully established. It is difficult to obtain a large volume of patients' serum with high antibody titer and high specificity as a positive control. We investigated whether or not a positive control from human could be replaced by a murine monoclonal antibody to SS‐B/La. The 1C3‐H7 was conjugated with a human IgG Fc' fragment using N‐γ‐maleimidobutyryloxysuccinimide as a cross‐linker. The chemically humanized murine monoclonal antibody (1C3‐Fc') was recognized by antiserum specific for human IgG Fc fragment. 1C3‐Fc' reacted to SS‐B/La but not to other antigens. Furthermore, the titration curve of this conjugate ran parallel with those of patients' sera specific for SS‐B/La. It is concluded that a chemically humanized murine monoclonal antibody is useful as a positive control in place of a human patient's serum. © 1992 Wiley‐Liss, Inc.
Simultaneous expression of type I procollagen mRNA and albumin in cirrhotic human liver
Authors: Yamada Shuhei , Kohi Takehiko , Hata Ryu‐Ichiro , Hirohashi Setsuo , Kurata Shun‐Ichi , Senoo Haruki , Sakakibara Kooko , Hayashi Toshihko , Nakane Paul K.
Published: March 2007

The gene expression of human type 1 procollagen was investigated in cirrhotic human liver by using in situ hybridization with nonradioactive DNA probes. Using in situ hybridizaiton can provide direct evidence for the cell type capable for type I collagen synthesis in tissues. T‐T dimerized DNA probes were used and DNAs hybridized in situ were detected immunohistochemically using specific antibodies against T‐T dimer. The data demonstrated that type I collagen is synthesized in hepatocyles and stellate cells in pseudolobules and in fibroblasts in Glissons capsules in cirrhotic human livers. we indicated hepatocytes morphologically and functionally by using immunohistochemical localization of albumin, which ws used as a marker of hepatocyte, since albumin is synthesized exclusively by hepatocytes. © 1992 Wiley‐Liss, Inc.
Comparison of two automated nephelometers
Authors: Emancipator Kenneth , Elin Ronald J. , Fleisher Thomas A.
Published: March 2007

The intercalibration precision and linearity were determined for two representative analytes, apolipoprotein A1 (apo A1) and immunoglobulin G (IgG), on the Beckman Array® and the Behring Nephelometer 100® (BN‐100). For two of nine samples analyzed, poor precision was observed for IgG with the Array. The poor precision for these two samples is attributed to large systematic shifts. A statistical non‐linearity was observed for apo A1 with the BN‐100, but this non‐linearity does not exclude use of this assay for clinical diagnosis. Method comparisons were done for 12 analytes: apo A1, apo B, IgG, IgA, IgM, IgE, C3, C4, albumin, C‐reactive protein, alpha‐1‐antitrypsin, and ceruloplasmin. Thjese comparisons showed proportional biases of >10% for seven of 12 analytes. Furthermore, correlation coefficients were <0.96 for seven of 12 analytes. We conclude that comparing results obtained from th two different nephelometers is of only limited value for the individual patient. © 1992 Wiley‐Liss, Inc.
Lymphoproliferative disease in scid mice reconstituted with human Sjögren's syndrome lymphocytes
Authors: Fox Robert I. , Pisa Pavel , Pisa Eva K. , Kang Ho‐Ii
Published: March 2007

Patients with Sjögren's syndrome (SS) have increased frequency of non‐Hodgkin's B‐cell lymphoma. These lymphomas frequently use a specific subclass of kappa light chain (encoded by variable region gene segment Hum KV325) and exhibit bcl‐2 protooncogene translocation t(14;18). In order to determine whether expansion of this B‐cell subset could be reproduced in an animal model, immunodeficient SCID (CB‐17) mice were reconstituted with lymphocytes from 4 different SS patients at high risk of the development of lymphoma. Tumor‐like nodules developed in all 11 SCID mice that received at least 5 × 105 lymphocytes from SS salivary glands or peripheral blood samples. However, the tumorlike nodules in the SCID mice differed from SS lymphomas in vivo in that they (1) exhibited multiple immunoglobulin gene rearrangements; (2) did not have expansion of B‐cells expressing the Hum KV325 K‐light chain; and (3) lacked detectable t(14;18) translocations. Characterization of the SCID tumor‐like nodules revealed a high level of Epstein‐Barr virus (EBV) DNA, EBV‐associated antigens (EA‐R, EBNA‐2, AND LMP), and the EBV‐encoded cytokine BCRF‐1 that is structurally similar to IL‐10. These results demonstrate that the lymphoproliferation occurring in the salivary glands of SS patients is not reproduced in the SCID/hu chimeric mouse. It is likely that specific factors in the human salivary gland are required for development of lymphoma in SS patients and that such factors are not present in the SCID/hu chimeric mouse. Furthermore, EBV‐induced lymphoproliferation, as seen in the SCID/hu chimera, does not lead to expansion of the same lymphoid subsets that occurs in vivo. © 1993 Wiley‐Liss, Inc.
Primary structure of the variable regions of a monoclonal antibody MUSE11 recognizing the tandem repeat domain of a mucin core protein, MUC1
Authors: Hinoda Yuji , Arimura Yoshiaki , Itoh Fumio , Adachi Masaaki , Tsujisaki Masayuki , Imai Kohzoh , Yachi Akira
Published: March 2007

A monoclonal antibody (MAb) MUSE11 recognizes an epitope in the tandem repeat domain of a mucin core protein, MUC1. We show that the epitope of MAb MUSE11 could be within the continuous amino acid sequence PDTRPAPG. Since there is increasing evidence indicating that this region is highly immunogenic, cDNA cloning of the variable regions of heavy‐chain (VH) and of light‐chain (VL) of MAb MUSE11 was performed by using RT‐PCR to provide a basis for analyzing the structure of the antibody‐antigen complex and for producing anti‐idiotypic antibodies. The deduced amino acid sequence revealed that the VH and VK of MAb MUSE11 could be assigned to subgroups IIIA and II of mouse immunoglobulin heavy and light chains, respectively. When compared with the V regions of other MAbs in the same subgroup, the complementary determining region 3 (CDR3) in the VH region of MAb MUSE11 consisted of a unique sequence that may be important in defining the specificity of MAb MUSE11. © 1993 Wiley‐Liss, Inc.
Review of diabetes: Identification of markers for early detection, glycemic control, and monitoring clinical complications
Authors: Wu James T.
Published: March 2007

The hallmark of diabetes mellitus, whether type I or type II, is hyperglycemia. Clinical complications associated with diabetes are most likely the consequence of hyperglycemia via both altered metabolic pathways and nonenzymatic glycation of proteins.

The nonenzymatic glycation of proteins is accelerated in diabetes due to elevated blood glucose concentration. The Amadori product of nonenzymatic glycation will further crosslink with other proteins to form advanced glycosylation end products (AGEs). The reaction of AGEs with long‐lived proteins, such as collagen, and the uptake of AGEs by the receptors on macrophages, endothelial cells, and platelets are major reasons for the development of various clinical complications in diabetes.

Several markers have been identified for the screening, diagnosis, and monitoring of the disease. Autoantibodies against beta cells are the best markers for mass screening and for early detection of type I diabetes. In addition to glycated hemoglobin, AGEs and blood glycated proteins of various half‐lives could be used for monitoring glycemic control. Several abnormal metabolites have been identified as potential markers for monitoring the severity of various clinical complications. The most interesting findings in diabetic markers could be AGEs. The amount of AGEs found in the tissues could be related to the extent of micro‐ and macrovascular damage and might prove useful for monitoring the treatment of patients at early stages of either nephropathy, atherosclerosis, retinopathy, or neuropathy. © 1993 Wiley‐Liss, Inc.
Principle and applications of ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for antibodies in body fluids
Authors: Ishikawa Eiji , Hashida Seiichi , Kohno Takeyuki , Hirota Kouichi , Hashinaka Kazuya , Ishikawa Setsuko
Published: March 2007

The sensitivity and specificity of enzyme immunoassay for antibodies in body fluids have been improved considerably by transferring the complex of labelled antigen and antibody to be detected from one solid phase to another to eliminate interfering substance(s) in the samples (immune complex transfer enzyme immunoassay). Usefulness of the new method has been tested for antibodies in serum as well as in urine. Anti‐thyroglobulin IgG could be measured not only in serum of all patients with autoimmune thyroid diseases and almost all healthy subjects but also in the urine of most of the patients. Anti‐HTLV‐I IgG was unequivocally demonstrated in some of sera, which were indeterminate or negative by Western blotting, and diagnosis of HIV infection by detecting anti‐HIV IgG in urine and saliva would be possible with higher reliability than by conventional methods. © 1993 Wiley‐Liss, Inc.
Clearance of Histoplasma capsulatum variety capsulatum antigen is useful for monitoring treatment of experimental histoplasmosi
Authors: Kohler Stephen , Blair Robinette , Schnizlein‐Bick Carol , Fojtasek Marvin , Connolly‐Stringfield Patricia , Wheat Joseph
Published: March 2007

We sought to determine whether measurement of Histoplasma capsulatum var. capsulatum antigen concentration in tissues and blood provided a marker for antifungal effect of itraconazole in a nonlethal murine model of histoplasmosis. Treatment with itraconazole (Sporanox), in cyclodextrin, was evaluated in a pulmonary model of histoplasmosis. Mice infected with 4.0 × 107 yeast‐phase organisms by endotracheal inoculation were treated with itraconazole, 1.5 mg twice daily by gavage, for 10 consecutive days, beginning on day 4 of infection. All mice were sacrificed on day 15 of infection. Blood, spleen, and lung tissues were removed for culture and quantification of antigen. Numbers of organisms were significantly lower in spleens from the treated group: 20.8 ± 41.8 vs. 65.8 ± 39.1 in the control group, P = 0.017. Numbers of organisms in lung were 9.6 ± 27.3 colonyforming units in treated versus 24.2 ± 36.3 in control animals, P = 0.267. Antigen concentrations in spleen tissue and serum were lower in treated versus control mice: spleen, 1.8 ± .6 units in treated versus 11.0 ± 2.3 in controls, P < 0.001; serum, 0.8 ± 0.2 units in treated versus 2.2 ± 1.0 units in controls, P < 0.001. Lung antigen concentrations were similar in the two groups, 19.2 ± 1.4 units in treated compared to 17.9 ± 3.0 units in control mice, P = 0.142. The cyclodextrin formulation of itraconazole (Sporanox) demonstrated antifungal activity in experimental histoplasmosis. Antigen detection was a useful marker for antifungal effect. © 1994 Wiley‐Liss, inc.
Evaluation of protein‐denaturing immunoassays for avidity of immunoglobulin G to rubella virus
Authors: Polanec J. , Seppälä I. , Rousseau S. , Hedman K.
Published: March 2007

Immunoassays have been recently developed that measure the avidity of IgG antibodies to complex microbial antigens and are suitable for serologic diagnosis of infectious diseases. In these avidity ELISAs, proteindenaturing agents are applied either as diluents of patient sera to prevent the immune complexing of IgG (diluting principle), or the preformed complexes are treated with the protein denaturants (eluting principle). We compared four protein denaturants previously used in such assays, in a diagnostic avidity ELISA for rubella IgG. Diethylamine, guanidine, thiocyanate, or urea were applied, by either principle at various concentrations, and thiocyanate, or urea were applied, by either principle at various concentrations, and thiocyanate at an optimum pH. Patient sera obtained recently after primary infection were distinguished from sera representing past rubella immunity by any protein denaturant tested by the eluting principle, which was superior to the diluting principle. © 1994 Wiley‐Liss, Inc.
Two‐color analysis of peripheral lymphocyte surface antigens in inherently healthy adults
Authors: Yamashiki Masayoshi , Nishimura Akira , Kosaka Yoshitane , James Stephen P.
Published: March 2007

We studied 134 “healthy persons” with no past history of any serious or latent diseases, and observed differences in their 25 lymphocyte subsets by conducting two‐color analyses using 12 useful combinations of dye‐labeled monoclonal antibodies.

CD4+Leu8+ cells and CD25+CD3+ cells were significantly higher in males than in females, whereas CD4+ Leu8− cells and CD23+ CD19+ cells were significantly higher in females. In comparison of the 25 lymphocyte subsets among four age groups, CD45RA− CD4+ cells and CDw29+ CD4+ cells significantly increased with age, and CD8+ CD11b− cells and CD57− CD8+ cells significantly decreased with age.

In females, helper T cells significantly increased and helper inducer T cells increased, while cytotoxic T cells decreased with age. These are important findings that should be considered in studies of immune function and autoimmune disorders. © 1994 Wiley‐Liss, Inc.
Analysis of monoclonal antibodies reactive with meconium‐ and amniotic fluid‐derived mucin
Authors: Kobayashi Hiroshi , Ohi Hidekazu , Terao Toshihiko
Published: March 2007

The present study was undertaken to determine whether monoclonal antibodies (moABs TKH‐2, MA54, MA61, B72.3, and CC49) directed toward O‐linked mucin‐type glycoprotein detect the NeuAcα2‐6GalNAc (sialyl Tn) epitope in meconium‐ and amniotic fluidderived mucin. Fetal colonic mucosal cells express the sialyl Tn antigen, particularly in goblet cell mucin. The reactivities of these moABs with a perchloric acid extract of meconium (meconium extract) and different native and neuraminidase treated glycoproteins were examined by solid‐phase enzyme‐linked immunosorbent assay (ELISA). All the moABs react with the meconium supernatant and meconium extract. The reactivities of TKH‐2, MA54, and MA61 are neuraminidase sensitive, and the reactivity of TKH‐2 with meconium extract was specifically inhibited by ovine submaxillary mucin (OSM). A NeuAcα 2‐6GalNAc epitope is the characteristic component in meconium. Mucin released from the fetal respiratory tract could, in part, provide an alternative source in the amniotic fluid. TKH‐2 is the most sensitive antibody directed to sialyl Tn antigen in meconium supernatant. The likelihood of TKH‐2 serving as the basis for a sensitive assay to detect sialyl Tn in meconium‐ and amniotic fluid‐derived mucin is provided. © 1994 Wiley‐Liss, Inc.
A rapid simple in situ hybridization method for herpes simplex virus employing a synthetic biotin‐labeled oligonucleotide probe: A comparison with immunohistochemical methods for HSV detection
Authors: Wang John Y. , Montone Kathleen T.
Published: March 2007

We examined 28 paraffin‐embedded tissue specimens with histologic evidence of herpes virus infection by in situ hybridization (ISH) utilizing manual capillary action technology (Micro Probe Staining System) and a 21 base synthetic multibiotinylated oligonucleotide probe from the HSV glycoprotein C region. The results were compared to a rapid simple immunohistochemical (IHC) protocol for detection of HSV proteins. HSV was detected by ISH and IHC in all but one specimen which was shown to be positive for varicella zoster virus by direct fluorescent antibody studies. Hybridization signal was confined to the nucleus in all cases. Staining was identified in cells with early as well as late cytopathic effect. IHC produced intense nuclear and/or cytoplasmic signal in infected cells and stained in areas of necrosis which were otherwise spared by ISH. HSV was detected by IHC and/or ISH in 3/5 specimens with histology suggestive of, but not diagnostic for, HSV infection. Both techniques were sensitive and specific for HSV, resulted in rapid detection of the pathogen in routinely processed tissues, and may be useful in cases where the histologic impression is equivocal for HSV infection. ISH for HSV may be preferred because it can identify early HSV infection, which in turn can be treated with antiviral agents. © 1994 Wiley‐Liss, Inc.
Primed in situ DNA amplification (PIDA)
Authors: Moss Ronald B. , Kaliner Michael A.
Published: March 2007

A novel PCR in situ protocol is described which can be implemented in one day. An initial amplification step with a single primer pair is followed by an amplification with an internal oligonucleotide probe with labeled nucleotides (dig‐dUTP). Detection is then accomplished with an antidigoxigenin antibody and substrate solution. This rapid and sensitive method may be useful in diagnosing low copy DNA in tissue specimens and cells. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Automated determination of glycated hemoglobin: Comparative evaluation of five assay systems
Authors: Tiran Andreas , Pieber Thomas , Tiran Beate , Halwachs‐Baumann Gabriele , Dobnig Harald , Grubelnig Horst , Wilders‐Truschnig Martini M.
Published: March 2007

We evaluated four newly introduced assays for determination of glycated hemoglobin allowing the processing of large amounts of samples in a clinical routine laboratory. These methods were compared to the Bio‐Rad Diamat system. The investigated methods were the Merck Hitachi L‐9100, a fully automated HPLC analyser, the Abbott IMx glycated hemoglobin ion capture assay, the DAKO HbA1c enzyme linked immunosorbent assay (ELISA), and the Boehringer‐Mannheim HbA1c Tinaquant turbidimetric assay. All methods showed generally acceptable precision and good accordance with the Diamat system. Interference study showed influence of anaemia, polycythemia, rheumatoid factor, and chronic hemodialysis on the values of the DAKO ELISA and of anaemia and polycythemia on the values of the Boehringer‐Mannheim Tinaquant assay. All of the investigated methods allow referring of results either as measured or standardised HbA1c values, the latter obtained after calibration with reference to an ion exchange high‐pressure liquid chromatography method. Our data confirm the feasibility of this kind of standardisation of glycated hemoglobin assays, allowing direct comparison of results from various determination methods. © 1994 Wiley‐Liss, Inc.
Clinical significance of serum high‐molecular‐mass alkaline phosphatase, alkaline phosphatase–lipoprotein‐X complex, and intestinal variant alkaline phosphatase
Authors: Wolf Paul L.
Published: March 2007

This paper is a study to identify the clinical significance of high‐molecular‐mass alkaline phosphatase (ALP:E:C.3.1.3.1.), ALP–lipoprotein–X complex (LP‐X) and intestinal variant ALP. We used cellulose acetate and agarose gels and techniques including wheat germ lectin, cetavlon‐diethyl ether, thermostatability, neuraminidase and L‐phenylalanine to improve the electrophoretic separation of the alkaline phosphatase isoenzymes. Patients' serum samples were electrophoresed from a diverse group of individuals ill with cholestasis, neoplastic disease metastatic to the liver, hepatocellular carcinoma, cirrhosis, diabetes mellitus, and chronic renal disease. Agarose gels provided better separation of ALP isoenzymes than cellulose acetate gels. The results also indicated that high‐molecular mass ALP is present in patient's serum in conditions associated with cholestasis especially caused by hepatic malignancy. High‐molecular mass ALP was frequently found to co‐exist with the liver isoenzyme and LP–XALP complex. The intestinal variant was identified in patients with malignancy, cirrhosis, chronic renal disease and diabetes mellitus. Intestinal ALP coexisted concomitantly with a variant intestinal ALP. Intestinal variant ALP is most likely composed of intestinal ALP attached to a cellular membrane‐binding domain, or may be an artifact produced by neuraminidase incubation © 1994 Wiley‐Liss, Inc.
A new method for detecting anti Helicobacter pylori antibodies: An analytical and clinical evaluation
Authors: Plebani M. , Basso D. , Brigato L. , Cassaro M. , Farinati F. , Di Mario F. , Rugge M.
Published: March 2007

The diagnosis of Helicobacter pylori (Hp) infection is an important goal in clinical practice. In this paper we evaluated 1. the analytical reliability of a new second‐generation antigen based enzyme immunoassay (Cobas Core Anti Helicobacter pylori EIA) in detecting anti‐Hp IgG antibodies, and 2. the behaviour of anti‐Hp IgG in patients with chronic atrophic and non‐atrophic gastritis as compared to healthy controls. The findings from the dilution curve, the values of intra and inter assay coefficients of variations (never above 10%) and of the recovery test (between 96 and 109%), confirm that the method is reliable. Serum IgG anti‐Hp levels were found to be significantly higher in patients with histologically identified Hp infection, than in those negative at histology. Furthermore, the grade of histological positivity was correlated with serum IgG levels. However, we found a discrepancy between a low prevalence of Hp staining and a high prevalence of Hp seropositivity in patients with chronic atrophic or non‐atrophic gastritis, but not in controls. This suggests that IgG serum determination may be more useful than histology in determining a present or previous infection in patients with chronic atrophic or non‐atrophic gastritis. © 1994 Wiley‐Liss, Inc.
Serum pseudocholinesterase and very‐low‐density lipoprotein metabolism
Authors: Kutty K. M. , Payne R. H.
Published: March 2007

Serum pseudocholinesterase (PChE) was discovered in 1932. Since this protein mimics many of the catalytic properties of acetylcholinesterase, it has traditionally been referred to as PChE, even though its true biological function is unknown. Serum PChE is synthesized in the liver and secreted into the circulation as a sialated glycoprotein. Although no convincing evidence of biological function exists, a significant number of obese and diabetic patients have elevated levels of PChE. The same phenomenon is found in experimental animal models of obesity, diabetes and hyperlipoproteinemia. Streptozotocin‐induced diabetic mice showed increased serum PChE activity concomitant with increased serum triacylglycerol and PChE activity declined with treatment. Iso‐OMPA, a nontoxic inhibitor of serum PChE, reduced serum and liver triacylglycerols and serum VLDL in streptozotocininduced rodent diabetes. These findings suggest that PChE may have a role in VLDL metabolism. © 1994 Wiley‐Liss, Inc.
Comparison of interferon‐γ and interleukin‐4 production by peripheral blood mononuclear cells and isolated T cells after activation with polyclonal t cell activators
Authors: Gonzalez Salvador , Beck Lucinda , Wilson Nevin , Spiegelberg Hans L.
Published: March 2007

Controversial data have been reported regarding the ability of peripheral blood T cells to secrete interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) from atopic patients as compared to nonatopic healthy controls. In most of these studies, T cells in peripheral blood mononuclear cell preparations (PBMC) were stimulated with polyclonal T cell activators. Some of these activators are able to activate cells other than T cells in the PBMC preparations which may influence the lymphokine levels in supernatants of PBMC. To evaluate this, we compared the IFN‐γ and IL‐4 levels in PBMC and isolated T cell preparations after activation with phytohemagglutinin (PHA), Concanavalin A (ConA), anti‐CD3 plus phorbol myristate acetate (PMA), or ionomycin plus PMA. The IFN‐γ and IL‐4 levels in the supernatants were calculated based on the percent T cells in the preparations. Whereas all activators induced significant IFN‐γ secretion, only ionomycin plus PMA stimulation induced large IL‐4 secretion. In virtually all cases, the IFN‐γ levels calculated on a per T cell basis differed for PBMC versus isolated T cells. Whereas in some donors the IFN‐γ levels were higher in PBMC preparations than in T cells, in others it was the opposite. Similarly, in about one half of both normal and atopic donors tested, the IL‐4 levels of activated PBMC were 2‐ to 7‐fold lower than levels in isolated T cells. The data suggest that non‐T cells have a significant effect on the IFN‐γ and IL‐4 levels in supernatants of polyclonally activated PBMC. This indicates that isolated T cells rather than PBMC should be analyzed for determining possible differences in the ability of T cells from patients or normals to secrete lymphokines. © 1994 Wiley‐Liss, Inc.
Detection of autoantibodies against M2, LKM‐1, and SLA in liver diseases by standardized uniform ELISA‐techniques
Authors: Gruber R. , Felber E. , Pape G. R. , Höchtlen‐Vollmar W. , Riethmüller G.
Published: March 2007

Antibodies directed against soluble liver antigen (SLA), liver kidney microsomal antigen (LKM‐1‐AG), and antimitochondrial antigen M2 (M2‐AMA) are critical serological markers for the differential diagnosis of autoimmune chronic active hepatitis (AI‐CAH) and primary biliary cirrhosis (PBC). The exact diagnosis of autoimmune hepatitis and PBC is of great clinical relevance, as it leads to different therapeutic strategies.

In the present work, a simple and reliable ELISA test system is described, which applies the same test principle for the detection of three different species of autoantibodies important for the diagnosis of chronic liver disease. The ELISA assays are based on a competitive inhibition of binding of positive standard antibodies by patients sera containing antibodies of unknown specificity. The purified immunoglobulins of clinically and serologically clearly defined patients with SLA or LKM‐1 positive AI‐CAH and with M2 positive PBC were used as coating‐ and detection antibodies in the ELISAs. From homogenized rat liver the fractionated 100,000g supernatant was employed for the SLA ELISA, the microsomal preparation served as antigen for the LKM‐1 ELISA and the mitochondrial preparation was used for the M2 ELISA. In 1,500 sera of patients with the differential diagnosis of a hepatobiliary disease, 17 gave a positive signal in the SLA ELISA, 12 in the LKM‐1‐ELISA, and 72 in the M2‐ELISA. The results of the ELISAs were compared with Western blotting and immunofluorescence staining pattern on cryostat sections and Hep2 cells. The antibody profiles of several patients are described in detail. © 1994 Wiley‐Liss, Inc.