bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated microparticle enzyme immunoassay for neural thread protein in cerebrospinal fluid from alzheimer's disease patients
Authors: Chong Jonathan K. , Cantrell Lois , Husain Mazhar , Riesing Sue , Miller Barney E. , Wands Jack , de la Monte Suzanne , Ghanbari Hossein A.
Published: March 2007

An automated microparticle enzyme immunoassay (MEIA) with the IMx ® analyzer for the detection of neural thread protein (NTP) in cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients was developed. This assay uses monoclonal antibodies produced against the purified pancreatic from of the protein. The assay employs one monoclonal antibody covalently coupled to the microparticle to capture immunoreactive material in CSF or brain tissue. The second monoclonal antibody was conjugated to alkaline phosphatase and serves as detection antibody. The assay provides results in approximately 45 minutes with a sensitivity of 60 pg/ml (3 fmoles/ml). The titration curve of both normal and AD CSF resulted in a linear relationship with respect to the volume of CSF used. A similar relationship was observed when normal and AD brain tissue extracts were serially diluted. The molecular weight of NTP in CSF was approximately 20 kD as determined by gel filtration method under non‐denaturing conditions. The recovery for pancreatic threat protein (PTP) spiked in either normal or AD CSF was 104% and 108%, respectively. Intra‐, inter‐, and total assay CVs (coefficient of variation) for controls were less than 2.9%, 3.3% and 3.0%, respectively. This assay will provide a useful tool in the study of the Alzheimer's disease and may help research in diagnosis and prognosis of Alzheimer's disease and related disorders. © 1992 Wiley‐Liss, Inc.
A flexible, efficient, checkerboard immunoblot system for the detection and semiquantitation of specific antinuclear antibodies
Authors: Ritchie Robert F. , Collins Marilyn , Ledue Thomas B.
Published: March 2007

In this paper an uncomplicated method for the simultaneous detection and semiquantitation of 11 of the 12 commonly studied antinuclear antibodies (ANA) in a single run is described. This new application of checker‐board immunoblotting (CBIB) is based upon available technology and employs purified antigens which can be either purchased or produced in‐house. CBIB requires no electronic instrument, can be formatted to meet the needs of the user, is rapidly performed, and has acceptable labor and materials costs. Data on the use of the method to examine available reference antisera is presented. CBIB has also proven practical forthe clinical study of 18 sera, at two dilutions per membrane, for each set of specific antinuclear antibodies, also at two or more dilutions. © 1992 Wiley‐Liss, Inc.
Immunoelectron microscopic localizaiton of antigens which react with islet cell cytoplasmic antibodies within human pancreatic beta cells
Authors: Takino Hirofumi , Yoshimura Toshiro , Kawasaki Eiji , Chikuba Nobuhiro , Yokota Atsushi , Akazawa Shoich , Tsujihata Mitsuhiro , Nakane Paul K. , Nagataki Shigenobu
Published: March 2007

The presence of circulating autoantibody to islet cell cytoplasm is considered to be an important mrker of Type 1 (insulin‐dependent) diabetes mellitus. In the present study using islet cell cytoplasmic antibody positive patient sera as the first antibody, we studied the intracellulr distribution of its antigen at the electron microscopic level using the pre‐embedding immunoperoxidase method. Specific immunoreactivity was found in the membranes of beta cell‐secretory granules and cytoplasmic membranes. This result is compatible with the interpretation that the antigen(s) on the membranes of beta cell secretory granules is (are) the target of islet cell cytoplasmic antibody. © 1992 Wiley‐Liss, Inc.
Characterization of marine toxin(s) in Myripristis sp. by immunological, mouse toxicity, and guinea pig assays
Authors: Ganal Cheryle A. , Asahina Audrey Y. , Hokama Y. , Miyahara James T.
Published: March 2007

Myripristis sp. (squirrelfish) has been assessed for toxicity by 1) the stick‐enzyme immunoassay (S‐EIA); 2) mouse toxicity bioassay; and 3) guinea pig atrial assay. Analysis of Myripristis flesh with MAb‐CTX and MAb‐OA showed that with every fish examined, the reaction with MAb‐OA was considerably higher. The mean S‐EIA value for MAB‐OA was 2.9 ± 0.8 while the mean for MAb‐CTX was 1.7 ± 0.5. The data strongly suggests that Myripristis sp. appear to contain okadaic acid‐like toxins and/or mixed with CTX. Five fractions of the flesh extracts were obtained by silica gel chromatography. These included 100% CHC13, 10% MeOH/CHC13, 50% MeOH‐CHC13, 100% MeOH, and 80% MeOH/H2O. The 100% CHC13 eluate proved to be the most toxic (mouse killed in 32 minutes) and the 10% MeOH‐CHC13 fraction killed in approximately 48 hours. The remaining fractions showed a significantly lower toxicity level in mice. In the guinea pig atria examinations, extracts of Myripristis flesh, gut, and crustacea (from the gut) were studied. Extracts of the gut and crustacea showed strong sodium channel blockage, while the flesh extracts showed a weak inotropic effect, characteristic of okadaic acid. The latter response was only blocked by verapamil, but not with tetrodotoxin (10−5M) or the adrenergic blockers (10−5M). The data from this study suggest toxic compound(s) not previously reported, in the non‐polar fraction of Myripristis flesh having a sodium channel blockage effect. © 1993 Wiley‐Liss, Inc.
Flow karyotype analysis and sorting of the Chinese hamster chromosomes: Comparing the effects of the isolation buffers
Authors: Kuriki Hideki , Sonta Shin‐ich , Murata Kenjiro
Published: March 2007

The effects of different swelling solutions on the univariate flow karyotype of whole chinese hamster embryo cells (WCHE K10) and cloned chinese hamster embryo fibroblast cells (CCHE 40) were compared using four methods of chromosome isolation. Chromosomes of each cell line were prepared by the Aten, Polyamine‐Digitonin, Tris‐Triton, and HEPES methods and analyzed with a FACStar flow cytometer. Polyamine‐Digitonin and Aten methods produced the most satisfactory flow karyotype. Structurally aberrant chromosomes in the cell lines were detected in the flow karyotype as extra peaks. CCHE 40 chromosomes 1,2,X and 3q were sorted into separate tubes and reanalyzed; and the coefficient of variation of each chromosome peak was near 5%. To apply the flow karyotype analysis to clinical cytogenetics, chromosome preparation was one of the most important factor, and it is necessary to sort the chromosome peak sharply. © 1993 Wiley‐Liss, Inc.
Quick assay of serum HBs antibody levels using latex agglutination‐integrating sphere turbidimetric assay (ISTA)
Authors: Yamashiki Masayoshi , Kosaka Yoshitane , Nishimura Akira , Kondo Isao , Tomita Akihito , Kishioka Hiroshi
Published: March 2007

A method for determining serum HBs antibody applying the principle of integrating sphere turbidimetric assay (ISTA) by latex agglutination was developed. The minimum detectable level of HBs antibody by this method is 12.5 IU/L, indicating that this method is 3 times or more sensitive with better reproducibility and specificity than the passive hemagglutination (PHA) method. With a cut‐off level of 25 IU/L, the possible highest simultaneous reading by this method was 1,000 IU/L Serum HBs antibody can be readily measured in 10 or so minutes by this method if a fully automated EL‐1000 analyzer is used. This rapid and simple method for determining serum HBs antibody will be useful not only clinically, but also in preventive medicine. © 1993 Wiley‐Liss, Inc.
Detection of hepatitis C virus infection with recombinant immunoblot assay, synthetic immunoblot assay, and polymerase chain reaction
Authors: Chaudhary R. K. , Andonov A. , Maclean C.
Published: March 2007

The newly developed immunoblot assay, RIBA SIA (recombinant and synthetic polypeptide immunoblot assay), Chiron, Calif., was compared with the commercially available second generation recombinant immunoblot assay (RIBA‐2) for the detection of antibody to hepatitis C virus (anti‐HCV). The two immunoblot tests were also compared with the polymerase chain reaction (PCR) for the detection of HCV RNA.

Ninety‐one percent of samples reactive by RIBA‐2 were positive for anti‐HCV by RIBA SIA. A total of 31% of RIBA‐2 indeterminate samples became reactive by RIBA SIA, 24% became non‐reactive, and 45% remained the same.

Samples reactive by RIBA‐2 or SIA from different risk groups, were mostly positive (67‐100%) by PCR for HCV RNA. All indeterminate samples from hemophiliacs and intravenous drug users were PCR positive. RIBA SIA is more sensitive and specific than RIBA‐2 and correlates well with PCR results © 1993 Wiley‐Liss, Inc.
Improved measurement of anti‐thyroglobulin IgG in urine of patients with autoimmune thyroid diseases by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay)
Authors: Yogi Yoshikuni , Hirota Kouichi , Hashida Seiichi , Toshimori Hirotaka , Matukura Shigeru , Setoguchi Toshiaki , Ishikawa Eiji
Published: March 2007

Previously, antithyroglobulin IgG was assayed in dialyzed urine from patients with autoimmune thyroid diseases by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay), and most of the assay results were useful as a diagnostic aid for autoimmune thyroid diseases. However, dialysis of urine was laborious and time‐consuming, and some results were less reliable due to low levels of anti‐thyroglobulin IgG in urine. This paper describes some improvements of the assay. Useful assay results could be obtained for most of urine samples without dialysis, although some interfering substance(s) was suggested to be present in some urine samples before dialysis. Accurate assay results with no interference could be obtained after gel filtration by only two min centrifugation in place of dialysis. More reliable assay results for urine samples containing low levels of antithyroglobulin IgG were obtained after concentration using a molecular sieve. © 1993 Wiley‐Liss, Inc.
Evaluation of vero cell lysate antigen for the ELISA of flaviviruses
Authors: Ansari Mohammad Z. , Shope Robert E. , Malik Sohail
Published: March 2007

The vero cell lysate antigen for the enzymelinked immunosorbent assay (ELISA) of flaviviruses was evaluated for sensitivity, specificity including cross‐reactions, and background by comparing with the standard ELISA. Human sera, in serial dilutions, were taken from subjects 14, 35, and 210 days postvaccination with 17D antigen. Early after injection, high sensitivity (82.9%) was shown by the cell lysate antigen method. Late after infection, high sensitivity was achieved by the standard method (96.2% and 94%), with significant difference (P = 0.0001). However, sensitivity achieved by the cell lysate antigen method was also acceptable (91.7% & 88.9%). The cell lysate antigen method showed high specificity and low cross reactivity early after infection. At 35 days postvaccination, no difference in specificity was observed between the two methods, but higher cross‐reactions were observed for the standard method. This pattern continued at 210 days postvaccination, with significantly higher cross‐reactions with the standard ELISA. The optical density differences by the two methods did not show significant relationship with the serial dilutions of human sera. No difference was observed in early and late infections in the background values of the negative control (Western equine encephalitis) between the two methods. The ELISA by the cell lysate antigen, within the limits of the experiments done, was found to be a good replacement for the ELISA by the standard method. © 1993 Wiley‐Liss, Inc.
Measurement of antimicrobial agents in cerebrospinal fluid using the abbott TDx analyzer
Authors: Jandreski Mark A. , Garbincius Jean
Published: March 2007

Occasionally, requests are made by our physicians for the measurement of gentamicin, tobramycin, or vancomycin in cerebrospinal fluid (CSF) specimens during the course of treating patients for bacterial meningitis. We evaluated CSF as a specimen type for the measurement of amikacin, gentamicin, tobramycin, and vancomycin on the Abbott TDx analyzer. Coefficients of variation for CSF spiked with these antimicrobial agents ranged from 0.8% to 6.5% for intra‐assay values and from 2.1% to 2.3% for inter‐assay values. Serum and CSF specimens were spiked at various levels with equal amounts of the antibiotics. Correlation coefficients for serum vs. CSF for these agents were 0.999. Recoveries ranged from 86% to 134%. Sensitivity for these assays is about fourfold better for CSF than for serum. CSF appears to be an acceptable specimen type for the measurement of these antibiotics using the Abbott TDx analyzer. © 1993 Wiley‐Liss, Inc.
Elevated levels of soluble CD54 (ICAM‐1) in human immunodeficiency virus infection
Authors: Reddy Mohan M. , Grieco Michael H.
Published: March 2007

Soluble CD54 levels in sera were quantitated in asymptomatic intravenous drug users, homosexuals, and patients with lymphadenopathy, AIDS‐related complex, or acquired immunodeficiency syndrome. Soluble CD54 levels were elevated in human immunodeficiency virus (HIV)‐seronegative asymptomatic intravenous drug users, reflecting infections like cytomegalovirus, Epstein‐Barr virus, and hepatitis B virus. The sera of human immunodeficiency virus‐seropositive groups of patients also had elevated levels of soluble CD54, reflecting infections like cytomegalovirus and human immunodeficiency virus infection. © 1993 Wiley‐Liss, Inc.
Development of an enzyme immunoassay for the detection of hepatitis B surface antigen employing monoclonal antibodies
Authors: Toplikar Emilena , Carlomagno Amadeo , Rojkin L. Federico , Gariglio Rosana , Lorenzo Luis E.
Published: March 2007

The use of monoclonal antibodies in the development of a third generation microtitration plate enzyme immunoassay for the detection of hepatitis B virus (HBV) is described. These antibodies were tested either alone or in combination with polyclonal antibodies raised in rabbit. Horseradish peroxidase was employed in the conjugates and 3,3′,5,5′‐tetramethylbenzidine was used as the enzyme substrate. Effect of type and concentration of antibodies used in the coating solution and in preparing conjugates as well as reaction time are discussed. The designed test employs a 100‐μl sample, with an overall incubation time of 3.5 hr. Serum or plasma (EDTA or citrate as anticoagulant) can be used. The sensitivity limit of the test was 0.4 ng/ml for subtype ad and 0.5 ng/ml for subtype ay. When used as screening test, 99.6% specificity was obtained; predictive value was 97.5% for a positive result and 99.8% for a negative result. According to the performance of the test, it seems to be suitable for diagnosis in routine laboratory and screening in blood banks. © 1993 Wiley‐Liss, Inc.
Detection of activated platelets in urinary sediments by immunofluorescence using monoclonal antibody to human platelet GMP‐140 in patients with IgA nephropathy
Authors: Tomino Yasuhiko , Tsushima Yoshimi , Ohmuro Hiroyuki , Shimizu Masahiko , Kuramoto Takao , Shirato Isao , Koide Hikaru
Published: March 2007

The presence of activated platelets in the urinary sediments was studied by indirect immunofluorescence using monoclonal antigranular membrane protein (GMP)‐140 antibody. GMP‐140 is generally expressed on the activated‐platelets and ‐vascular endothelial cells. The purpose of the present study was to determine if the presence of activated platelets in the urinary sediments is correlated with glomerular injuries in patients with IgA nephropathy. Fourteen patients with IgA nephropathy and 11 patients with diffuse mesangial proliferative glomerulonephritis without glomerular IgA deposition (PGN) were examined. The number of activated platelets in the urinary sediments was markedly increased in patients in the advanced stage of IgA nephropathy. The ratio of activated platelets to total platelets in the urinary sediments was also increased in such patients. It appears that the detection of activated platelets in the urinary sediments is useful in determining the degree of histological changes in IgA nephropathy. © 1993 Wiley‐Liss, Inc.
Assay for prostate specific antigen (PSA): Problems and possible solutions
Authors: Wu James T.
Published: March 2007

The absolute tissue specificity of prostate specific antigen (PSA) allows the use of PSA test not only for detecting recurrence or metastasis at an early stage after radical prostatectomy but also for screening prostate cancer if combined with digital rectal examination. There is also a need to improve the current PSA test to better differentiate between prostate cancer and benign prostate hyperplasia (BPH). Because of these clinical applications, a much greater demand was placed on PSA test for extra sensitivity, accuracy, and precision even within the normal PSA concentration range. However, the current commercial assay kits for PSA do not provide correct PSA values. Many factors contributing to the problem include the specificity of the anti‐PSA antibodies, the composition of the calibrator, the PSA values assigned to the calibrator, the PSA isoform used for anti‐PSA antibody preparation, the test design, and the composition of the diluent. Most problems were derived from the failure of realizing earlier that the majority of the PSA exists in serum not as free PSA but as complexes with protease inhibitors. Other problems, such as constantly changing composition of various forms of PSA in serum specimens, and different clearance rates for various forms of PSA make almost impossible to develop an ideal assay for PSA. Therefore, we suggest that test should be designed for measuring PSA‐ACT (PSA‐α1‐antichymotrypsin) complex only. Changing the focus from the measurement of total PSA of various forms to the PSA‐ACT complex alone may improve the differentiation between prostate cancer and BPH but may also simplify the selection of anti‐PSA antibodies and the preparation of calibrator for the assay. © 1994 Wiley‐Liss, Inc.
Use of micro samples of finger prick blood dried on filter paper for a quick and simple dipstick dot‐EIA for diagnosis of amebic liver abscess (ALA)
Authors: Sharma Manoj , Ghosh S. , Singal A. K. , Anand B. S. , Talwar G. P.
Published: March 2007

Filter paper was used as a support to absorb micro samples of finger prick blood for detection of antibodies to Entamoeba histolytica (causative organism of amebiasis) by a rapid dipstick dot‐EIA technique; 8 μl of blood was sufficient to saturate discs (diameter 6 mm) of Whatman paper No. 3. Conditions of elution of blood from the discs were optimized and the best results were obtained when 0.4 ml of buffer was used for elution for 30 minutes at room temperature. The filter paper technique is extremely useful for field use and for diagnosis of amebic liver abscess on a large scale since it does not involve centrifugation of blood and use of sterile vials because blood dried on paper can be stored in polythene bags at room temperature for up to 3 months and for a week at 42°C without significant loss of antibody activity, thereby eliminating the need of refrigeration of samples for storage or transportation to a reference laboratory. Elution of blood from different discs of the same patient was found to be highly reproducible without appreciable loss of sensitivity or specificity. Twenty‐four confirmed ALA, 29 non‐ALA, and 25 apparently normal healthy controls, with no previous history of amebiasis, were tested. Sensitivity and specificity of the test was found to be 96% and 92%, respectively. © 1994 Wiley‐Liss, Inc.
Modulation of suppressor cell activities by cyclophosphamide in breast cancer patients
Authors: Kuroi Katsumasa , Sato Yukio , Yamaguchi Yoshiyuki , Toge Tetsuya
Published: March 2007

We investigated the immunomodulatory effect of cyclophosphamide on the induction of suppressor cell activities in peripheral blood lymphocytes. The sera from advanced breast cancer patients as well as concanavalin‐A (Con‐A) induced suppressor cell activities in lymphocytes from healthy volunteers. Pretreatment of these lymphocytes, including CD4+ T cells with 4‐hydroperoxycyclophosphamide (4‐HC), an active form of cyclophosphamide, abrogated the induction of suppressor cell activities by either cancer sera or Con‐A. A decrease in CD4+CD45RA+ suppressor/inducer T cells and reduction of Con‐A induced suppressor cell activities were observed in breast cancer patients with metastases that were treated with cyclophosphamide (CPM). These results suggest that cyclophosphamide may modulate the immune responses of breast cancer patients by interference with suppressor/inducer T cells. © 1994 Wiley‐Liss, Inc.
Anti‐HTLV‐I IgG in urine detected by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide, Cys‐env gp46(188‐224), as antigen
Authors: Hashida Seiichi , Hirota Kouichi , Kohno Takeyuki , Ishikawa Eiji
Published: March 2007

Antibody IgG to human T‐cell leukemia virus type I (HTLV‐I) in urine was detected by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide, Cys‐env gp46(188–224), as antigen, the sensitivity and specificity of which were 100 and 98.5%, respectively, using serum samples. Anti‐HTLV‐I IgG in urine was reacted simultaneously with 2,4‐dinitrophenyl‐bovine serum albumin–Cys‐env gp46(188–224) conjugate and Cys‐env gp46(188–224)‐β‐D‐galactosidase (Escherichia coli) conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG, eluted with ϵN‐2,4‐dinitrophenyl‐L‐lysine and transferred to polystyrene balls coated with affinity‐purified (anti‐human IgG γ‐chain) IgG. Finally, bound β‐D‐galactosidase activity was assayed by fluorometry. Thirty‐one urine samples from seropositive subjects and 100 urine samples from seronegative subjects were tested. The sensitivity and specificity were 87 and 100%, respectively, with unconcentrated urine samples and 94 and 100%, respectively, with approximately 10‐fold concentrated urine samples. These results were superior to those by the conventional ELISA and gelatin particle agglutination test. © 1994 Wiley‐Liss, Inc.
Failure of in vitro T‐cell assays to predict clinical outcome after human kidney transplantation
Authors: Steinmann J. , Kaden J. , May G. , Schröder K. , Herwartz C. , Müller‐Ruchholtz W.
Published: March 2007

Allotransplant rejection is a T‐cell‐dependent reaction. Functional in vitro T‐cell assays are being used widely for donorrecipient matching in bone marrow transplantation and have recently been used in some centres for transplant monitoring. In order to assess tolerance induction after clinical transplantation, we measured the T‐cell response of the host against donor spleen cells of 33 kidney transplant patients before and every 3 months after transplantation over a period of 18 months. The T‐cell reactivity before transplantation was not significantly different in any of the assays in rejecting and nonrejecting patients. In the classical mixed lymphocyte culture (MLC), a donor‐specific loss of reactivity was seen only in a patient with a CMV‐associated irreversible transplant rejection. One patient with chronic rejection acquired a very high MLC response against donor spleen cells and a high response against third‐party cells. Little or nonspecific changes were seen in the MLCs of all other patients. Using the method of limiting dilution analysis (LDA), we found a significant reduction of donor‐specific cytotoxic T‐cell precursors (CTL‐p) within the first 3 months after transplantation in most patients with high antidonor CTL‐p frequencies before transplantation. The reduction of donor‐specific CTL‐p was seen in patients with rejection episodes as well as in patients without. Thus we conclude, in contrast to others, that MLC and CTL‐p LDA have no predictive value on the outcome of clinical transplantation. © 1994 Wiley‐Liss, Inc.
Detection of glycosylated protein in glomeruli of STZ‐induced diabetic rats using the nitroblue tetrazolium (NBT) reaction
Authors: Saitoh Akemi , Tomino Yasuhiko , Kuramoto Takao , Fukui Mitsumine , Ohmuro Hiroyuki , Shirato Isao , Koide Hikaru , Itoh Kiichi
Published: March 2007

Detection of glycosylated protein in renal tissues was determined in streptozotocin (STZ)‐induced diabetic rats using the nitroblue tetrazolium (NBT) reaction. The glycosylation of extra‐cellular matrix (ECM) components such as laminin and fibronectin was examined in vitro using the same method. Immunofluorescence staining of laminin or type IV collagen was also performed in renal tissues of STZ‐induced diabetic rats. There was no significant difference in the intensity of NBT in renal tissues between 4 week STZ‐induced diabetic rats and control rats of the same age. The intensity of NBT staining in the glomerular mesangial areas and capillary walls was marked in 12 week diabetic rats. The mean values of fructosamine measured by the NBT reaction in the glycosylated‐laminin and fibronectin were increased dose dependently. In immunofluorescence, laminin and type IV collagen were observed significantly in the glomerular mesangial areas and capillary walls of 12 week diabetic rats. However, there was no significant change in renal histopathology in 4 and 12 week diabetic rats. It appears that the non‐enzymatic glycosylation and expression of ECM components in glomeruli increased in the early stage of diabetic nephropathy prior to the appearance of marked histologic alterations. In conclusion, non‐enzymatic glycosylation of glomerular structural components may play an important role in the initiation of the early stage of renal injuries in diabetes. © 1994 Wiley‐Liss, Inc.
Measurement of free and total hydroxyproline by automated flow injection of serum or urine samples from maintenance hemodialysis patients with renal osteodystrophy
Authors: Uji Yoshinori , Karmen Arthur , Okabe Hiroaki , Hata Keishi , Miura Masakazu , Ozaki Kazuyuki , Minamizaki Mitsuo , Shibata Tetsushi , Inayama Seiichi
Published: March 2007

An automated measurement of total and free hydroxyproline in serum or urine is presented that uses flow injection analysis. After exclusion of nonspecific substances, hydroxyproline was oxidized by chloramine‐ T and L‐cysteine with Ehrlich's reagent.

The linearity obtained was from 3.8μmole/ L to 1.22 mmole/L with good precision (CV <3%). Comparison of the proposed method with HPLC yielded r = 0.939 as the correlation coefficient.

Reference intervals of free and total hydroxyproline are 1.4–9.7 μmole/L, 3.8–27.2 μmole/L for serum, and 10.0–72.5 μmole/L, 25.2–303.6 μmole/L for urine, respectively. Serum free and total hydroxyproline levels in renal osteodystrophy patients on maintenance hemodialysis (N = 71) were significantly higher than in controls (P<0.0001). This method is superior to the use of HPLC with regard to stability of the color reaction. The measurement of serum free and total hydroxyproline is a useful marker for therapeutic observation of renal osteodystrophy patients. © 1994 Wiley‐Liss, Inc.
Diagnosis and prediction of pediatric HIV‐1 infection and AIDS: Current status
Authors: Ugen Kenneth E. , Von Feldt Joan M. , Weiner David B. , Ziegner Ulrike H. M.
Published: March 2007

The increase in the incidence of HIV‐1 infection in women of child bearing age has resulted in a surge in the number of cases of pediatric AIDS. The World Health Organization (WHO) has estimated that the number of cases of pediatric AIDS worldwide will be at least 10 million by the year 2000. This alarming statistic underscores the need for accurate prediction and diagnosis of pediatric HIV‐1 infection which is of paramount importance for the initiation of effective therapeutic interventions. Since circulating maternal anti‐HV‐1 antibody persists in the baby for up to 21 months, early conventional serological diagnosis of infection is not possible. Other methods for diagnosis of HIV‐1 infection in a child less than 2 years of age have been utilized including the polymerase chain reaction (PCR), measurements of the HIV‐1 p24 core protein and anti‐HIV‐1 IgA, as well in vitro measurements of antibody producing cells. In addition, the ability to predict HIV‐1 infection in the child based upon maternal humoral immune responses to the envelope glycoprotein has also been suggested. This review summarizes the recent serological, biological and molecular methodologies used to predict and diagnose pediatric HIV‐1 infection and AIDS. © 1994 Wiley‐Liss, Inc.
Use of microplates and fluororeader for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti‐HTLV‐I IgG
Authors: Ishikawa Setsuko , Hashida Seiichi , Kohno Takeyuki , Ishikawa Eiji
Published: March 2007

Previously, an ultrasensitive solid phase enzyme immunoassay (immune complex transfer enzyme immunoassay) was described to detect low levels of anti‐HTLV‐I IgG in serum below those detectable by conventional methods. In this method, polystyrene balls as solid phase were transferred from test tube to test tube with tweezers. This was not only tedious but also causative of false‐positivity by carryover, unless tips of the tweezers were washed carefully after each transfer of polystyrene balls. Bound enzyme activities for many samples were measured one by one by fluorometry using a spectrofluorophotometer. As a result, the assay of many samples was difficult. In the present study, microplates and a fluororeaderwere used in place of test tubes and a spectrofluorophotometer. Polystyrene balls were transferred quickly and easily from well to well by placing a microplate upside down on that containing polystyrene balls, and turning the two plates together upside down. Tweezers were not used for transfer of polystyrene balls, minimizing the possibility of false‐positivity. Fluorescence intensities of bound enzyme activities for 96 samples were measured within a minute by using a fluororeader. Thus, it became easy to test many samples, although the sensitivity was lowered to some extent. © 1994 Wiley‐Liss, Inc.
Measurement of serum Lp(a) by COBAS MIRA using a latex immunoturbidimetric assay kit
Authors: Fujita Seiichi , Sano Takahiro , Katayama Yoshiaki
Published: March 2007

We had an opportunity to test a reagent kit for serum lipoprotein (a) [Lp(a)] assay, which was based on the latex immunoturbidimetric assay method, and applied it to COBAS MIRA (Roche). Reproducibility of the assay procedure was 0.92–4.0% CV of three samples which contained 12.58–60.4 mg/dl of Lp(a). Minimum detectable concentration was 1.5–2.0 mg/dl. It was confirmed that interference of co‐existing substances, i.e. hemoglobin, bilirubin, and intralipid, was negligible. No cross reactivity was seen with plasminogen. Correlation with a ELISA method was excellent. Frequency distribution of Lp(a) in healthy Japanese was similar to that found in white population. © 1994 Wiley‐Liss, Inc.
Magnetophoresis: I. detection of magnetically labeled cells
Authors: Winoto‐Morbach S. , Tchikov V. , Müller‐Ruchholtz W.
Published: March 2007

A new method for the detection of magnetically labeled cells in suspension using an optical microscope is presented. The movement of individual cells in a nonuniform magnetic field (magnetophoresis) can be studied and the direction and velocity of their movement can be determined. Cell‐associated magnetic particles (e.g., attached magnetic immunomicrospheres) influence these parameters. We developed a magnetophoresis chamber for measuring the movement of single cells in a high‐gradient magnetic field. Paramagnetic erythrocytes were used to monitor the level of magnetic force in the chamber. Bw6 antigen‐positive REH cells were used as a biological model. Magnetic microspheres coupled with anti‐Bw6 antibody were used as magnetic labels. The movement of magnetically labeled REH cells was investigated versus the intensity of magnetic field. The magnetization of microspheres was experimentally determined by the Faraday balance method. Experimental and theoretical results substantiate the feasibility of detecting a single cell associated with a single magnetic microsphere. Thus, magnetophoresis provides a sensitive biometrical system for magnetically labeled entities. © 1994 Wiley‐Liss, Inc.
Soluble MHC class I molecules in human body fluids
Authors: Zavazava N. , Wobst B. , Ferstl R. , Müller‐Ruchholtz W.
Published: March 2007

Apart from their well‐known function of antigen presentation in the form of peptides, major histocompatibility antigens (MHC) have been found to be unique markers of individual body odors in murine experimental models. In the current study we examined the nature and expression of soluble human MHC class I molecules in body fluids. Biochemical analysis of affinity purified serum class I molecules revealed a variety of molecules within the molecular weight region of 45 to 21 kD. SDS‐Western blotting of HLA derived from hepatocytes and spleen cells suggested that much of the small molecular mass fraction of sHLA (<45 kD) found in serum is derived from the liver. sHLA were detected and quantitated in serum, plasma, urine, cerebrospinal fluid, and sweat. No sHLA were detectable in cerebrospinal fluid (n = 20). In addition, sHLA was measured in serum of women during the menstrual cycle. A significant increase in sHLA was observed during the first half of the cycle, suggesting that sexual hormones may increase sHLA concentration. The observed increase was most prominent in women that were HLA‐A24. © 1994 Wiley‐Liss, Inc.
Cytokine production in whole blood cell cultures of patients with crohn's disease and ulcerative colitis
Authors: Elsässer‐Beile U. , von Kleist S. , Gerlach S. , Gallati H. , Mönting J. Schulte
Published: March 2007

Levels of the cytokines interleukin‐1‐α, ‐1‐β, and ‐2 (IL‐1‐α, IL‐1‐β, IL‐2), tumor necrosis factor‐α (TNF‐α), and interferon‐γ (IFN‐γ) were measured in the mitogen‐stimulated whole blood cell cultures from 96 patients with Crohn's disease (48 untreated, 12 treated with sulfasalazine, 36 treated with corticosteroids), 74 patients with ulcerative colitis (21 untreated, 25 treated with sulfasalazine, 28 steroid treated), and 360 healthy controls. The cytokines were measured 4 days after induction by a sensitive immunoenzyme assay.

In the blood cell cultures of the untreated and sulfasalazine treated patients with Crohn's disease and ulcerative colitis higher levels of TNF‐α, IL‐1‐α and IL‐1‐β were found whereas IL‐2 production was decreased and IFN‐γ‐production was not significantly different as compared to the controls. Leukocytes of the corticosteroid‐treated patients with both diagnoses showed a lower production of all measured cytokines compared to the untreated patients.

The same results were obtained, when the somewhat different counts of mononuclear cells in the peripheral blood of the patients and controls were taken into account. The elevated production of proinflammatory cytokines in the blood cell cultures suggests a systemic immune activation in patients with inflammatory bowel disease. © 1994 Wiley‐Liss, Inc.
Solid‐Phase polymerase chain reaction
Authors: Kohsaka Hitoshi , Carson Dennis A.
Published: March 2007

The polymerase chain reaction (PCR) has facilitated the diagnosis of infectious diseases and genetic disorders, because of its ability to amplify minute amounts of nucleic acids. To distinguish genes of interest from nonspecifically amplified DNA, PCR products commonly are fractionated by electrophoresis, transferred to membranes, and then probed with a labeled internal sequence‐specific oligonucleotide. Alternatively, the PCR products have been labeled directly, and hybridized to immobilized oligonucleotide probes. These methods require the tedious physical transfer of the PCR products. In order to amplify and immobilize genes simultaneously, we have developed a simple solid‐phase PCR method. The technique enabled us to detect the HIV envelope gene readily without any transfer of amplified DNA. © 1994 Wiley‐Liss, Inc.
Racial differences in serum immunoglobulin levels: Relationship to cigarette smoking, t‐cell subsets, and soluble interleukin‐2 receptors
Authors: Tollerud David J. , Weiss Scott T. , Brown Linda Morris , Blattner William A. , Maloney Elizabeth M. , Kurman Carol C. , Nelson David L. , Hoover Robert N.
Published: March 2007

To investigate the influence of race, cigarette smoking, and immunologic parameters on serum immunoglobulins, we analyzed serum IgG, IgA, and IgM levels in 455 healthy adults. The study population ranged in age from 20 to 69 years, including 282 whites and 173 blacks, 181 never‐smokers, 93 ex‐smokers, and 181 current smokers. Race and smoking were independently associated with alterations in serum IgG levels. Blacks had significantly higher IgG levels than whites (1,587 vs. 1,209 mg/dl; P<0.001), and never smokers had significantly higher levels than current smokers (1,426 vs. 1,287 vs. mg/dl; P<0.001). IgA and IgM levels were unrelated to race or smoking. Serum IgG was also found to be directly related to the proportion of HLA‐DR+ cells and the level of soluble interleukin‐2 receptors (sIL‐2R) and inversely related to the proportion of CD4+ cells. Investigation of this racial heterogeneity may provide insights into the pathogenesis of immunologic diseases that exhibit unexplained racial variation.©1995 wiley‐Liss, inc.
Magnetophoresis: II. quantification of iron and hemoglobin content at the single erythrocyte level
Authors: Winoto‐Morbach S. , Müller‐Ruchholtz W. , Tchikov V.
Published: March 2007

The iron and hemoglobin content of individual erythrocytes was determined using a method based on parallel velocity measurements during magnetophoresis and gravitational sedimentation of individual erythrocytes in suspension. In previous publications we have suggested employing cell magnetophoresis, a biophysical phenomenon characterized by cell movement in a fluid under magnetic field influence, for cytometry. The paramagnetic ferric iron in methemoglobin is used as a magnetic label. The iron content is estimated from the magnetophoresis velocity, and hemoglobin content from the gravitational sedimentation velocity of erythrocytes. Blood samples are also analyzed in a Coulter counter to determine their mean corpuscular hemoglobin. The time course of the reaction of methemoglobin reduction is quantified at the single erythrocyte level. The methemoglobin content in individual erythrocytes is determined following the oxidation reaction. Erythrocytes from patients with normo‐, hypo‐, or hyperchromic anemia exhibit magnetophoresis and gravitational sedimentation velocities that correlate closely with mean corpuscular hemoglobin. We propose the utilization of magnetophoretic cytometry for detailed diagnostic studies at the single erythrocyte level. Furthermore, the magnetophoresis velocity to gravitational sedimentation velocity ratio is proposed as a standard value for comparative study of magnetically labeled cells in future investigations, as it was found to be constant and independent of hemoglobin content.©1995 wiley‐Liss, inc.
Reassessment of the two‐site enzyme immunoassay for human epidermal growth factor (hegf) and measurement of immunoreactive hegf in human sera and urine
Authors: Takei Yoshifumi , Ezawa Hisayo , Hayashi Kyozo , Kurobe Masayuki
Published: March 2007

We reassessed the enzyme immunoassay (EIA) system for hEGF previously developed by our laboratory (Clin Chim Acta 156:51–60, 1985), since it appeared that the reported EIA system detected not only hEGF but also pS2 protein owing to minor contamination by pS2 protein in the hEGF sample used for immunization. In this study, we purified the hEGF sample using Benzamidine‐Sepharose 6B column chromatography as a critical step for purification and newly developed an EIA system with specificity for hEGF. We also measured the hEGF level in serum, plasma, and urine from normal subjects by our new EIA system and found that the values measured by the previous system were 1.2–5.8‐fold higher than the new system values. These results suggest that the previous system detected “hEGF” in excess owing to the nonspecificity of the antibody used. We investigated the molecular nature of immunoreactive hEGF detected in serum using our new system and confirmed that considerable amounts of immunoreactive hEGF exist as a high molecular weight form through S‐S linkage with some macromolecule(s) in human blood as reported previously (Biochem Int 12:677–683, 1986).©1995 wiley‐Liss, inc.
Acid citrate dextrose (acd) formula a as a new anticoagulant in the measurement of in vitro platelet aggregation
Authors: Pignatelli Pasquale , Pulcinelli Fabio M. , Ciatti Filippo , Pesciotti Marzia , Sebastiani Silvia , Ferroni Patrizia , Gazzaniga Pier Paolo
Published: March 2007

To evaluate whether the use of ACD Formula A may affect in vitro platelet function, blood samples were obtained from 21 healthy blood donors and anticoagulated in ACD (acid‐citrate dextrose, NIH Formula A), Na citrate 3.8%, and K3EDTA. Platelet count, mean platelet volume, and in vitro platelet aggregation were evaluated on each sample. No significant difference was observed in platelet count and mean platelet volume among the different samples. Conversely, the ACD treated platelets showed a higher reactivity to the agonists as demonstrated by a significant increase of the maximum percentages of aggregation induced by ADP, epinephrine, and collagen, as well as a significant decrease of secondary aggregation thresholds to ADP and epinephrine. In conclusion, it may be speculated that ACD Formula A is capable of better maintaining the intraplatelet signal transduction mechanisms during PRP preparation, thus improving the overall responsiveness of platelets.©1995 wiley‐Liss, inc.
Identification and characterization of c‐erbb‐2 proteins in serum, breast tumor tissue, and sk‐br‐3 cell line
Authors: Wu James T. , Zhang Ping , Astill Mark E. , Lyons Bradley W. , Wu Lily H.
Published: March 2007

We have identified and characterized c‐erbB‐2 protein molecules in sera from patients with carcinomas, in both cytosol and cell membrane extract from breast tumor tissue and in both the culture medium and cell extract of the SK‐BR‐3 cell line. These proteins were characterized by various chromatographic techniques and identified by the use of two immunoassays; one measures both the c‐erbB‐2 oncoprotein (p 185) and its ectodomain (p 120), and the other in‐house assay reacts specifically for p 185. We found that the majority of the immunoreactivity detected in the serum, tumor tissue cytosol, and conditioned cell medium was derived from the ectodomain molecule (p 120) of the c‐erbB‐2 oncoprotein (p 185), whereas only p185 was detected in the extracts from cell membrane of both tumor tissue and the SK‐BR‐3 cell line. The ectodomain molecules (p 120) found in the serum, cytosol, and cell medium were very similar in terms of molecular size and charge property. The molecular weight was determined to be 120 kDa by the size exclusion HPLC method. Both p 120 and p 185 are glycoproteins and were retained by the ConA Sepharose column. Both molecules are also heterogeneous in charge and multiple peaks could be identified in the elution profiles of anion exchange HPLC and chromatofocusing. This information should not only facilitate the isolation of these molecules, but also improve preparation of specific antibodies, preparation of calibrators, and development of improved assays for these proteins.©1995 wiley‐Liss, inc.
Measurement of c‐erbb‐2 proteins in sera from patients with carcinomas and in breast tumor tissue cytosols: Correlation with serum tumor markers and membrane‐bound oncoprotein
Authors: Wu James T. , Astill Mark E. , Gagon Shane D. , Bryson Lynn
Published: March 2007

Using a commercial kit with antibodies against the ectodomain of c‐erbB‐2 protein, we detected c‐erbB‐2 immunoreactivity in human serum. We found that the percentages of patients with elevated serum c‐erbB‐2 immunoreactivities were 35, 21, and 9% in breast, prostate, and ovarian carcinoma, respectively. The majority of the elevated immunoreactivities were associated with sera containing highly elevated tumor markers with the highest in breast carcinoma (35%) and lowest in ovarian cancer (9%). Excellent correlations were also observed between the serum levels of c‐erbB‐2 immunoreactivity and the dominant tumor markers in serial specimens from individual cancer patients. We could also detect the c‐erbB‐2 immunoreactivity in the cytosols prepared from the breast tumor tissue for estrogen and progesterone receptor (ER&PgR) measurements using the same commercial kit for serum studies, and the intact c‐erbB‐2 oncoprotein (p 185) in the extracts of the tissue membrane fractions with a different kit designed for tissue extract. The level of c‐erbB‐2 immunoreactivity in the cytosol from 124 human breast tumor specimens had an excellent correlation with the cell membrane concentrations of p 185 (γ = 0.89). Most of the elevated cytosol c‐erbB‐2 immunoreactivities were also found to associate with breast tumor specimens containing low concentrations of ER&PgR. It appears that measuring the c‐erbB‐2 immunoreactivity potentially could be used as a prognostic marker without performing tissue biopsies and also as a serum tumor marker for managing cancer patients.©1995 wiley‐Liss, inc.
Comparative study of the genepath group 4 reagent system and other chef systems for karyotype analysis of candida spp
Authors: Holmberg K. , Feroze F.
Published: March 2007

The commercial GenePath Group 4 Reagent Candida kit (BioRad), designed to simplify the electrophoretic karyotyping of Candida spp. was evaluated against several other established contour‐clamped homogeneous electric field (CHEF) systems for Candida. This comparison allowed assessment of both the GenePath system and the other CHEF systems regarding the sources of technical variability of the assays and variation in karyotypic analysis.

The GenePath system appeared to be a simple, rapid and reliable tool for karyo‐typing of Candida spp. with a discriminatory power comparable with established CHEF systems. The evaluation showed that the variability of the CHEF systems for subtyping of Candida is largely a function of technical variabilities in the assay system (reagents, sample preparation, running conditions, and test performance), and of analytical variabilities due to imprecision or observers bias. Lack of standardization of these factors may contribute to variability among investigators and have an impact on the ultimate conclusions of an epidemiological study using CHEF methods.©1995 wiley‐Liss, inc.