bluuebunny/crossref_metadata_2025 huggingface dataset:
laboratory diagnosis of parvovirus B19 infection
Authors: Sevall J. Sanders , Ritenhous Judith , Peter J. B.
Published: March 2007

The sensitivity and application of the polymerase chain reaction (PCR) for the diagnosis of parvovirus B19 (B19) infection was investigated by simultaneously assaying a collection of 279 consecutively received samples for presence of anti‐B19 IgM and IgG antibodies by Western blot and for B19 DNA by PCR and dot‐blot hybridization (dot‐blot); samples were sera from patients with suspected B19 infection. PCR and dot‐blot detected B19 DNA in 9% (16/179) and 1% (2/179), respectively of Ab‐positive samples (IgM+/IgG‐,IgM+IgG+,IgM‐IgG+), and in 28% (15/54) and 2% (1/54), respectively, of IgM+ samples. PCR also detected B19 DNA in 2% (2/100) of IgM‐/IgG‐ samples, both of which had normal total IgG and IgM levels. PCR is of unique value because it permits diagnosis of B19 infection even in the absence of specific acute phase (IgM) and in the presence or absence of convalescentphase (IgG) Ab. © 1992 Wiley‐Liss, Inc.
Automated quantitative nephelometric latex immunoassay for determining ferritin in human serum
Authors: Borque Luis , Rus Antonio , del Cura Jorge , Maside Carlos , Escanero Jesus
Published: March 2007

We describe a rapid and sensitive latex nephelometric immunoassay for quantifying ferritin in human serum. This latex immunoassay procedure uses commercially available ready‐for‐use reagents [Tina‐Quant (a) Ferritin, Boehringer Mannheim] that have a long shelf life. The assay consists of incubating the diluted serum sample (5‐fold) for 12 min at room temperature with latex particles covalently coated with anti‐ferritin antibodies, and then quantifying the change of light‐scatter produced. The assay is fully automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. The method has an analytical range of 3 to 260 μg/l. Maximal intra‐ and inter‐assay CVs were 4.0 and 6.2%, respectively. Analytical recoveries ranged from 91.3 to 103.6%. Assay detection limit was less than 3 μg/l. Linearity of the test is given throughout the measuring range. There was no interference from bilirubin (up to 340 μmol/l), haemoglobin (up to 7 g/l), or rheumatoid factor (up to 1,100 IU/ml). Turbid and lipemic samples interfere. This interference may be avoided by pretreating these samples prior to assay. Results correlated well with those obtained by an automated ELISA test (r=0.995) and with those of two commercial RIA methods (r>0.97). This latex nephelometric procedure is a convenient method and represents an interesting alternative to other immunoassays for measuring ferritin in human serum. © 1992 Wiley‐Liss, Inc.
Immunoblot assay for detection of autoantibodies in autoimmune disease
Authors: Capín Norma G. , Rodriguez Monica R. , Lavalle Carlos M. , Ortiz‐Ortiz Librado
Published: March 2007

When detecting antinuclear antibodies (ANA) a similar indirect immunofluorescence (IIF) pattern may be produced by antibodies which correspond to different antigenic specificity or to diverse autoimmune disorders. To circumvent this problem, we used an immunoblotting against total antigen from HEp‐2 cells (TA‐HEp‐2‐C) which proved to be more specific and sensitive than IIF, since it detected up to 40 antigenic bands and an immunoblot pattern characteristic of each individual and unrelated to the fluorescence patterns of ANA.

The analysis of immunoblot patterns is of great diagnostic value, since in patients with systemic lupus erythematosus (SLE) the antigenic zone is found between 21 and 48 kDa, whereas in other rheumatic diseases with or without positive ANA antibodies by IIF it is foudn between 14 and 21 kDa. Bands which appear after 50 kDa may or may not have pathological significance since they are present in most normal individuals.

The use of reference sera allowed the identification in the extract of previously described antigens relevant to these diseases. Some patients showed bands which have yet to be reported and may be clinically significant on their own or in association with other autoantibodies. The long‐term follow‐up of these immunoblot patterns may prove to be of prognostic importance. © 1992 Wiley‐Liss, Inc.
Monoclonal antibody‐based ELISA to detect glycoprotein tumor‐associated‐antigen‐specific immune complexes in cancer patients
Authors: Gupta Rishab K. , Morton Donald L.
Published: March 2007

This report describes the development and applicability of a tumor‐assocaited‐antigen‐specific immune complex (IC) detection assay to denote th presence oftumor cells in a cancer host. This assay utilizes a murine monoclonal antibody, AD1‐40F4, which was produced to a glycoprotein tumor‐associated antigen (TAA). In Western blot, the murine monoclonal antibody recognized a 90–10 kD subunit of the antigen. This antigen is immunogenic in cancer patients and induces formation of endogenous antigen‐antibody complexes. Analysis of 250 sera from normal individuals and 419 sera from cancer patients revealed that a significantly (P <.0005) greater proportion (234/419; 55.9%) of cancer sera was positive for the marker than the normal sera (8/250; 3.2%). The incidence of the 90 kD‐TAA‐specific‐IC was consistently and significantly higher in melanoma (58.5%; 38/65), Sarcoma (52.9%; 83/157), and carcinoma of breast (50.9%; 83/157), and carcinoma of breast (50.9%; 58/114), lung (68.2%; 30/44), and colon (64.1%, 25/39) than in the normal group. The age of serum donors did not affect the incidence of teh reactivity. Also, at least in the cancer group the gender ofthe serum donor did not affect the incidence of the 90 kD‐TAA‐specific‐IC positivity. In a retrospective study where sequential serum samples obtained postoperatively from 105 patients with melanoma were analyzed, the 90 kD‐TAA‐specific‐IC could be detected in 72 (69%) of patients several years before the appearance fo clinically detectable disease. These results indicate that the 90 kD‐TAA‐specific‐IC assay represents a new type of tumor marker of enhanced specificity and broad crossreactivity among a wide spectrum of different histological types of human neoplasms. This assay may be useful to assess prognosis and detect residual disease after surgical treatment. © 1992 Wiley‐Liss, Inc.
Comparison of various methods for the enumeration of blood cells in urine
Authors: McGinley Margaret , Wong Lois Lee , McBride James H. , Rodgerson Denis O.
Published: March 2007

This study compared the use of conventional urine microscopy, the KOVA system, Neubauer hemacytometry, and the Yellow IRIS for the determination of white blood cell (WBC) and red blood cell (RBC) counts in urine samples. Both KOVA WBC and RBC counts correlated better with the IRIS counts than with conventional microscopy. KOVA WBC counts correlated with Neubauer hemacytometry to the same degree as they did with IRIS WBC counts. The RBC count correlation was fairly similar between KOVA, hemacytometry, and the conventional method. It was concluded that the KOVA system is a suitable replacement for conventional urine microscopy. © 1992 Wiley‐Liss, Inc.
A homogeneous assay system of aspartate aminotransferase iso‐enzymes using proteases and application for clinical evaluation of myocardial infarction
Authors: Yoneda Koji , Katayama Yosiaki , Koike Toshiyo , Tanimizu Isao
Published: March 2007

We designed a rapid, homogeneous assay for human asparatate aminotransferase (AST) isoenzymes, by a selective proteolysis of soluble AST (s‐AST), using chymotryspin and protease 401. The linearity of mitochondrial (m‐AST) was elongated up to 4000 U/l. m‐AST values from the human liver, and determined by a homogeneous assay using protease 401 or chymotryspin, were relative to those obtained using an immunoprecipitaiton method.

In perioperative patients or those with an acute myocardial infarction, the peaks of s‐AST and m‐AST values were noted 13 h and at 57 h after ictus, respectively, whereas the peak of ratio between was seen 6 h after ictus. In the case of Budd‐Chiari syndrome, the maximum levels of the two AST activities were evident 14 days after hospitalization and the peak of ratio between them was seen after 7 days. We propose that htis homogeneous assay can serve as a diagnostic tool for early phase detection of myocardial infarction and of Budd‐Chiari syndrome. © 1992 Wiley‐Liss, Inc.
Influence of biological variations and sample handling on measured microalbuminuria in diabetic patients
Authors: Vermes I. , Spooren P. F. M. J.
Published: March 2007

Five immunochemical assays for determining low concentrations of albumin were investigated. These were a radioimmunoassay (RIA); turbidimetric immunoassays (TIA) both according to end‐point measuring principle on a Cobas Fara and Hitachi 717 analysers, and according to kinetic measuring principle on a Turbitimer instrument; and a nephelometric immunoassay (NIA). All achieved the analytical goal necessary for optimal patient care. The correlations between the albumin concentrations measured with the different techniques were very good. In vitro glycation of albumin did not influence albumin concentrations measured by the five assays. Urine albumin excretion measured over 3 consecutie days showed considerable day‐to‐day variation. This was highest for spot‐urine specimens and significantly lower for 24 h and timed‐overnight samples. Variation of storage temperature (room temperature, 4°C, −20°C), time (up till 3 months), and pH (within the range pH 5–8) of the urine samples did not change significantly the measured albumin concentrations. Different sample preparations (vortex‐mixing, centrifugation, and thawing) had no influence on the measured albumin concentration. In conclusion, a maximum standardization of the collection of timed‐overnight urine samples for screening and 24 h urine sampels for confirmation of microalbuminuria during 3 consecutive days is more crucial than the choice of the immunological technique. © 1992 Wiley‐Liss, Inc.
Streptomyces: A superior source for cholesterol oxidase used in serum cholesterol assay
Authors: Lolekha Porntip H. , Jantaveesirirat Yaovalak
Published: March 2007

The present study compared three cholesterol oxidase sources (Nocardia, Streptomyces, and Pseudomonas sps.) for serum cholesterol assay. We found cholesterol oxidase isolated from Streptomyces was superior thanthose isolated from Nocardia and Pseudomonas sps.

Performances fo the reagent contained Streptomyces cholesterol oxidase was excellent and comparable to the performances obtained from reagent contained Nocardia cholesterol oxidase. Moreover, the reagent containing Streptomyces cholesterol oxidase had the lowest cost and had the longest shelf‐life ($U.S. 0.50/mL, 10 weeks) compared to the reagent contained Nocardia ($U.S. 0.50/mL, 8 weeks) or Pseudomonas ($U.S. 0.20/mL, 6 weeks) cholesterol oxidase.
Single point quantification of antibody by ELISA without need of a reference curve
Authors: Dopatka H. D. , Giesendorf B.
Published: March 2007

A new method for the quantification of antibodies in the enzyme‐linked immunosorbent assay is described. This procedure replaces titer determinations based on end‐point dilution of the antibody under investigation. Here, the sample is tested in a single dilution and the optical density (OD) obtained is used in the equation

The titer can then be calculated by inserting into the formula the values for the constants α and β, which are specified by the manufacturer for each separate batch of kit reagents. This so‐called α‐method saves time and reagents while providing results which are equal to the titration method in accuracy and superior in precision. The α‐method is also a simpler and reliable alternative to the use of standard or reference curves for the quantification of the antibodies in I.U./ml. © 1992 Wiley‐Liss, Inc.
Application of Leu M5 (CD 11c) antibody in the cytodiagnosis of body fluids: Preliminary results
Authors: Berena Jelena , Sun Nora C. J.
Published: March 2007

Identification of metastatic malignant cells in body fluids and their distinction from reactive mesothelial cells is a common diagnostic problem. Application of the immunoalkaline phosphatase method using epithelial membrane antigen, cytokeratin, vimentin, carcinoembryonic antigen, lysozyme, and leukocyte common antigen (CD 45) is routinely performed in our Hematology Laboratory in conjunction with morphological examination of body fluids. Recently, we found that anti‐Leu M5 (CD 11 c), a monoclonal antibody to human monocyte/macrophage, is frequently reactive to mesothelial cells. We then undertook a prospective study, adding anti‐CD 11c to the above‐listed antibody panel and applying to cytocentrifuge preparations of body fluid. The neoplastic cells in only 2 out of 34 cases (5.9%) with the diagnosis of metastatic carcinomas reacted positively for CD 11c. The neoplastic cells in four cases of malignant lymphoma were non‐reactive for CD 11c. In addition, malignant cells on touch preparations of 25 carcinomas were also negative. Mesothelial cells in all 15 cases of reactive effusions and those mesothelial cells present in malignant effusions were positively stained with CD 11c, although the intensity of staining varied in different mesothelial cells of the same cases. We conclude that anti‐CD 11c can be considered as a useful adjunct to the differential diagnosis of malignant effusions. © 1993 Wiley‐Liss, Inc.
Evaluation of the Hawaiian reef fishes with the solid phase immunobead assay
Authors: Hokama Y. , Asahina A. Y. , Shang E. S. , Hong T. W. P. , Shirai J. L. R.
Published: March 2007

This study presents data on the evaluation of a laboratory ciguatera kit based on the solid phase immunobead assay (SPIA) for the detection of ciguatoxin in Hawaiian reef fish. The SPIA was performed on fish catches by volunteer fishermen throughout the State of Hawaii. A total of 1,067 fish of various species were tested for ciguatoxin (CTX) using the SPIA kit. Of the total 1,067 fish tested, 510 were from Oahu, 402 from Hawaii, and 75 from Maui. The number of fish from Molokai, Kauai, and Lanai were 23, 20, and 7 respectively. Twenty percent of the total fish tested were positive, 41% borderline, and 39% negative for ciguatoxin. The highest percentage of SPIA‐positive fish were from Hawaii (27%) followed by Oahu (19%) and Kauai (15%). These results correlate with the reported incidents from the Department of Health (DOH) of actual ciguatera poisoning in the State of Hawaii. Fish in all three categories of the SPIA test values were eaten. No false negatives were noted with individuals eating SPIA negative fish. Of the 232 SPIA borderline values eaten, 3 species of fish caused ciguatera poisoning. These fish included 2 papio, 1 mullet, and 1 po'ou. Of the 17 SPIA positive fish eaten, 5 caused ciguatera poisoning: 2 papio, a kole, an uhu, and a weke. The SPIA ciguatera test did protect the public when only SPIA‐negative fish were eaten. The borderline and positive SPIA fish were generally unsafe, especially the positive fish. The data indicated that the probability of getting ciguatera with a SPIA positive fish was 1 out of 3. The Caranx sp. (papio, ulua) appeared to be one of the major culprits in ciguatera poisoning in Hawaii. This was compatible with the known yearly reports from the DOH. © 1993 Wiley‐Liss, Inc.
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 , Kohno Takeyuki , Toshimori Hirotaka , Matsukura Shigeru , Setoguchi Toshiaki , Ishikawa Eiji
Published: March 2007

Anti‐thyroglobulin IgG in urine of patients with Graves' disease and chronic thyroiditis and healthy subjects was measured by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay). Anti‐thyroglobulin IgG in dialyzed urine was reacted simultaneously with 2,4‐dinitrophenylated thyroglobulin and thyroglobulin‐βT‐D‐galactosidase conjugate. The immune complex formed consisting of the three components was trapped onto polystyrene balls coated with (anti‐2,4, dinitrophenyl group) IgG, eluted with ∈N‐2,4‐dinitrophenyl‐L‐lysine, and transferred onto polystyrene balls coated with (antihuman IgG γ‐chain) IgG. β‐D‐Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. Anti‐thyroglobulin IgG was detected in most of the patients, but not in most of the healthy subjects; levels of anti‐thyroglobulin IgG in urine of the patients were well correlated to those in serum of the same patients. The measurement of anti‐thyroglobulin IgG in urine by the immune complex transfer enzyme immunoassay was suggested to be useful as a diagnostic aid for autoimmune thyroid diseases. The conventional standard ELISA was not sufficiently sensitive for measuring anti‐thyroglobulin IgG in urine. © 1993 Wiley‐Liss, Inc.
Stability studies of the components of a prototype penicillinase (β‐lactamase)‐linked ELISA kit
Authors: Khatkhatay M. I. , Desai M. P. , Sankolli G. M. , Pardhe D. K. , Joshi U. M.
Published: March 2007

Penicillinase (β‐lactamase) enzyme‐linked immunosorbent assay (ELISA) for various reproductive hormones developed in the laboratory were found to have wide applicability in the fertility check clinic of the Institute. A need was thought to transform these assays into ready‐to‐use kit forms. Therefore, prototype ELISA kits for these hormones were developed and stability of the individual component was ascertained at various temperatures (room temperature, 37°C and 2‐8°C).

Stability studies were conducted on previously validated assay for pregnanediol‐3α‐glucuronide (PdG). The studies showed that immunosorbents (antibody coated plates) are stable at room temperature for a period of 2 weeks, at 37°C for 1 week and at 2‐8°C for a period of 9 months when preserved after treatment with glycerol solution. The lyophilised conjugate, standard and immunoassay buffer, colour reagent, and its substrate were stable at 37°C up to 1 week and at room temperature up to 2 weeks and at 2‐8°C for a period of 6 months, during which the stability was studied. © 1993 Wiley‐Liss, Inc.
Immunologic significance of increased soluble CD8/CD4 molecules in patients with active systemic lupus erythematosus
Authors: Sawada S. , Hashimoto H. , Iijima S. , Tokano Y. , Takei M. , Shida M. , Obara T.
Published: March 2007

This study attempted to estimate soluble CD4(sCD4)/CD8(sCD8) molecules in active systemic lupus erythematosus (SLE) patients. Measurements were made by solid‐phase enzyme‐linked immunosorbent assay. sCD8 or sCD4 molecules were significantly increased in the patients as compared to control subjects. sCD8 correlated with the erythrocyte sedimentation rate. sCD4 correlated with the anti DNA antibody titer, the IgG concentration, and negatively with the complement titer. An association of these molecules with immunologic abnormalities and disease activity exists in SLE patients. © 1993 Wiley‐Liss, Inc.
Production of hydrogen peroxide by neutrophilic polymorphonuclear leukocytes in patients with diabetic nephropathy
Authors: Watanabe Asako , Tomino Yasuhiko , Yokoyama Ken‐Ichi , Koide Hikaru
Published: March 2007

The production of hydrogen peroxide (H2O2) by neutrophilic polymorphonuclear leukocytes (PMN) after stimulation with phorbol myristate acetate (PMA), n‐formyl‐I‐methionyl‐IIeucyl‐I‐phenylalanine (FMLP), aggregated human IgG, or Staphylococcus aureus was determined in 36 patients with non‐insulin dependent diabetes mellitus (NIDDM). H2O2 production by PMN after stimulation was measured using flow cytometry. Thirty‐six patients with NIDDM were divided into four stages as follows: 1) stage I: non‐microalbuminuric stage;2) stage II: microalbuminuric stage; 3) stage III: proteinuric stage without impairment of renal function; and 4) stage IV: proteinuric stage with impairment of renal function. H2O2 production after PMA stimulation in all stages of NIDDM patients was higher than that in healthy controls. This increase of H2O2 production by PMN was particularly observed in stage IV of NIDDM patients after stimulation. Furthermore, H2O2 production in patients in stage IV was higher than that in patients with non‐diabetic disease with impairment of renal function.

It appears that reactive oxygen species produced by PMN after stimulation under some conditions may play an important role in the progression of diabetic nephropathy. © 1993 Wiley‐Liss, Inc.
Specificity of an immunochemical reagent for quantifying the isoforms of creatine kinase‐MB
Authors: Christenson Robert H.
Published: March 2007

Fractionation of the creatine kinase‐MB isoforms is promising for use in diagnosing acute myocardial infarction and for monitoring myocardial perfusion status after thrombolytic therapy. An immunochemical reagent intended for use in fractionating the MB1 and MB2 isoforms of creatine kinase‐MB was examined before and after immunoextraction, qualitatively by visually examining electrophoresis separation of various MB1 and MB2 mixtures, and quantitatively by comparing the observed and predicted enzymatic activity ofvarious MB1 and MB2 mixtures. Qualitatively the reagent showed greater reactivity for MB1 than for MB2, as demonstrated by a marked decrease in the MB1 electrophoretic region following immunoextraction. Quantitatively, the reagent consistently eliminated about 75% of MB1 activity; however, the assay also eliminated about 40% of MB2 activity from isoform mixtures. Although the performance of the immunochemical reagent was not ideal, the greater reactivity for MB1 may have clinical use. © 1993 Wiley‐Liss, Inc.
Double immunochemical screening test (hemoglobin and albumin) for the detection of occult intestinal bleeding
Authors: Otto Szabolcs , Nemeth MÁRia
Published: March 2007

The authors have developed a combined electrophoretic‐immunoprecipitation technique suitable for the simultaneous demonstration of two blood proteins, i.e., hemoglobin and albumin, in the feces. The analytical sensitivity of the method is 2 μg hemoglobin and albumin/ml. The technique is simple, inexpensive, and suitable for the reliable demonstration of occult intestinal bleeding for either inpatients or mass screening. This technique is an aid not only to the fact that bleeding can be detected, but to its intensity, as well. This new technique improves the diagnostic accuracy of early detection of carcinomas and polyps, presumably without raising significantly the number of “false‐positive” reactions. © 1993 Wiley‐Liss, Inc.
Development of radioimmunoassays on microplate: Application for CA‐GI and CA‐Br tumor markers
Authors: Wu James T. , Erali Maria
Published: March 2007

By taking advantage of a newly available microplate counter for radioactivity and the organic solvent‐resistant, pigmented microplates, we have successfully established radioimmunoassays (RIA) for both CA‐GI and CA‐BR on microplate for routine clinical use. In the process of assay development, we found that both pigmented PicoPlate, made of acrylonitrile, and polystyrene Microlite 2 can be coated with antialpha fetoprotein (AFP) and antinerve growth factor (NGF) and used for setting up immunoassays for AFP and nerve growth factors. There were no problems following a test format of either competitive binding or sandwich design. Microlites 2 is recommended over PicoPlate because Microlites 2 is made of polystyrene, which is less expensive and separable into 8‐well strips or even single wells. Single‐well separation allows for the use of regular gamma counters in case Topcount is unavailable. We also found that the sensitivity of these tests was not significantly affected even though Topcount counts the weaker beta emissions.

Similar dose‐response curves could also be generated between original Biomira tube assays and assays using PicoPlate or Microlite 2 coated with protein antigens CA‐Br and CA‐GI. Excellent correlations were also obtained between the microplate assays and the Biomira tube assays for CA‐GI and CA‐Br using groups of serum specimens from cancer patients. We recommend the development of various RIAs on the microplate: it requires less reagents and less sample handling by the technologists and it can be essentially automated. © 1993 Wiley‐Liss, Inc.
Detection of antibody IgG to HIV‐1 in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens for diagnosis of HIV‐1 infection
Authors: Hashida Seiichi , Hirota Kouichi , Hashinaka Kazuya , Saitoh Atsushi , Nakata Atsuo , Shinagawa Hideo , Oka Shinichi , Shimada Kaoru , Mimaya Jun‐Ichi , Matsushita Shuzo , Ishikawa Eiji
Published: March 2007

For diagnosis of HIV‐1 infection, attempts were made to detect anti‐HIV‐1 IgG in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT) and p 17 as antigens. Anti‐HIV‐1 IgG in urine was reacted simultaneously with 2,4‐dinitrophenyl‐bovine serum albumin‐recombinant protein conjugate and recombinant proteinenzyme conjugate. The enzymes used as labels were horseradish peroxidase for RT and Escherichia coli β‐D‐galactosidase for p17. 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 ∈N‐2,4‐dinitrophenyl‐L‐lysine and transferred to polystyrene balls coated with affinity‐purified (anti‐human IgG γ‐chain) IgG. Finally, bound enzyme activity was assayed by fluorometry. Urine samples were collected from 100 seronegative subjects and 70 seropositive subjects. The sensitivity and specificity were both 100% with unconcentrated urine samples. The positivity was confirmed by preincubation of urine samples with excess of the antigens. The positivity and negativity with one of the two antigens could be confirmed with the other antigen. The positivity with low signals could be confirmed by concentration of urine samples. Detection of anti‐HIV‐1 IgG in urine by the immune complex transfer enzyme immunoassay using different antigens would make diagnosis of HIV‐1 infection possible. © 1993 Wiley‐Liss, Inc.
Variable‐region subgroup distribution among λ‐type immunoglobulins in normal human serum
Authors: Abe Masahiro , Ozaki Shuji , Wolfenbarger Dennis , Debram‐Hart Mary , Weiss Deborah T. , Solomon Alan
Published: March 2007

The distribution of the major VL subgroups (VλI, VλII, VλIII, VλIV, VλVI, and VλVIII) among λ‐type immunoglobulins (Igs) in normal serum was determined by a sandwich enzymelinked immunosorbent assay (ELISA) using a panel of murine anti‐human Vλ‐subgroup‐specific monoclonal antibodies (MoAbs) and appropriate reference standard proteins. The mean concentration of λI, λII, λIII, λIV, λVI, and λVIII Igs in serum specimens obtained from 23 adults was 2158, 162, 1958, 264, 225, and 169 μg/mL and represented 44, 3, 40, 5, 5, and 3% of the total Igλ population, respectively. The low percentage of λII Igs in normal serum was in marked contrast to the ∼40% incidence of this Vλ subgroup found among λ‐type Bence Jones proteins and monoclonal serum Igs obtained from patients with multiple myeloma and AL amyloidosis, or ∼60% in Waldenström's macroglobulinemia. The non‐random expresson of the VλII subgroup in these diseases implies a relationship between Vλ‐gene usage and plasma cell, as well as certain types of lymphocytic dyscrasias. © 1994 Wiley‐Liss, Inc.
In situ localization of transforming growth factor‐β 1 mRNA in the rat kidney with Masugi nephritis
Authors: Oguchi Satohiro , Yamada Shuhei , Oguchi Hisao , Nakane Paul K.
Published: March 2007

Masugi nephritis was induced in rats by the administration of nephrotoxic duck serum and, in their kidneys, the distributions of transforming growth factor‐β 1 (TGF‐β 1) mRNA, TGF‐β 1, and macrophage antigenic marker were investigated. Kidney specimens were obtained at 3, 7, and 14 days after the injection. By in situ hybridization, TGF‐β 1 mRNA was found to be localized in the cells of mesangial region from the early stage of nephritis. By the immunohistochemical stainings, TGF‐β 1 was found to be localized in the cells of distal convoluted tubulus throughout the experiments and the macrophage antigenic marker was found to be localized in the occasional cells which were different from that contained TGF‐β 1 mRNA. Our data indicate that TGF‐β 1 mRNA is expressed by the resident glomerular cells and not by the migrating macrophages in rat kidney with Masugi nephritis. © 1994 Wiley‐Liss, Inc.
Hormonal modulation of herpes simplex virus replication in a mouse neuroblastoma cell line
Authors: Sawiris Gamil P. , Sydiskis Robert J. , Bashirelahi Nasir
Published: March 2007

In this study, the effect of the synthetic glucocorticoid hormone dexamethasone (DXM) on HSV replication was studied in a DXM receptor‐positive mouse neuroblastoma (NB) cell line. In cells treated with 10−7M DXM and then infected with HSV, there was a statistically significant 9–18‐fold increase in the amount of virus produced in these cells compared to untreated controls. Adsorption kinetic studies with HSV were performed in DXM‐treated NB cells and untreated controls. It was found that there was a significant increase in the adsorption rate of HSV in the DXM‐treated cells as compared with the controls. During the course of these studies, a strain of NB cells was noted to have lost its ability to stimulate HSV replication following DXM treatment. Receptor binding assays were performed on cytosols prepared from NB cells that responded with an increase in HSV titers to DXM treatment and the new strain of NB cells that was DXM refractile. These latter cells were found to have lost their DXM receptors. These results indicate that the modulation of HSV replication of DXM treated cells was regulated by the presence of DXM receptors in these cells. Once lost, the cells do not respond to DXM treatment with increased HSV replication. These observations may lead to a clinical assay to determine patients with high glucocorticoid levels who may be at risk of recurrent herpes infections. © 1994 Wiley‐Liss, Inc.
Adenylate kinase mimics creatine kinase‐MM isoenzyme in a CK isoenzyme electrophoresis assay
Authors: Murthy Vadiraja V.
Published: March 2007

Adenylate kinase activity (AK) originating from erythrocytes, present in hemolyzed serum behaves like creatine kinase MM isoenzyme (CK‐MM) in some CK electrophoresis assays that employ, in their visualization reagent kits, adenosine monophosphate (AMP) as the sole inhibitor of AK, rather than a combination of AMP and a more potent inhibitor of erythrocyte AK, diadenosine pentaphosphate (Ap5A), to inhibit all contaminating‐AK activities in serum and quantify only the CK isoenzyme activities in serum following electrophoretic fractionation on agarose gel. This can spuriously overestimate the CK‐MM fraction and thereby result in underestimation of CK‐MM or CK‐BB isoenzymes if present. A hemolyzed serum sample obtained from an elderly patient was erroneously reported as containing low CK‐MB due to such overestimation of CK‐MM fraction in the sample. Supplementing the AMP already present in the visualization reagent formulation, used to estimate CK isoenzyme concentration in serum, with Ap5A can eliminate or effectively minimize AK interference, especially that caused by hemolysis, and thereby prevent reporting false‐negative CK‐MB result obtained with CK isoenzyme electrophoresis assays. © 1994 Wiley‐Liss, Inc.
Activity of antiphospholipid antibody ELISA cofactor in different animal sera
Authors: McCarthy Jennifer M. , Wagenkencht Dawn R. , McIntyre John A.
Published: March 2007

A plasma protein cofactor, β2‐glycoprotein I (β2GPI), also known as apolipoprotein H, is necessary to detect certain antiphospholipid antibodies (aPA) to negatively charged phospholipids (PL) in the ELISA. Inasmuch as sera are diluted 1:100 before testing, the concentration of native β2GPI may be insufficient to provide an optimal aPA ELISA signal. Therefore, many laboratories add adult bovine serum (ABS) to the diluent buffer to provide a consistent level of cofactor for optimal aPA binding. To determine if other animal sera can provide the cofactor, cat, chicken, dog, horse, goat, guinea pig, mouse, pig, rat, and sheep were tested as diluent supplements in the aPA ELISA. To measure cofactor activity in these animal sera, ELISA for aPA to anionic phospholipids were performed. Two aPA positive patient plasmas were selected for study; one with cofactordependent and one with cofactor‐independent aPA. Only four of the animal sera tested (bovine, pig, sheep, and cat) supported the cofactor‐dependent aPA in ELISA. The cofactor‐independent aPA was positive in the presence of each animal serum except bovine and rat. In order to determine whether these animal sera contain a β2GPI‐like molecule, Western blot analyses were performed. By using a polyclonal antiserum produced to human β2GPI, specific β2GPI‐like cross‐reactivity was observed with all animal sera except the chicken. In summary, cofactor activity in animal sera varied significantly; however, bovine and pig sera appear to allow optimal binding of cofactor dependent aPA. © 1994 Wiley‐Liss, Inc.
Effects of radiolabelled murine antibody infusion on TNF‐α, IL‐1β, and soluble IL‐2 receptor in cancer patients
Authors: Gridley Daila S. , Hammond Samantha N. , Slater James M.
Published: March 2007

This study evaluates the plasma levels of tumor necrosis factor‐α (TNF‐α), interleukin‐ 1β (IL‐1β), and soluble IL‐2 receptor (sIL‐2R) in cancer patients infused with radiolabelled murine monoclonal antibodies (MAbs) for the purposes of imaging and dosimetry. Blood samples were collected from 13 patients (10 with colon cancer and 3 with lung cancer) before and at 4 and 7 days after infusion of either conventional intact 111In‐ MAb or a bifunctional antibody delivery system. For all subjects, except one, this was the first exposure to murine MAb. Before infusion, higher levels of TNF‐α, IL‐1β, and sIL‐2R than the average expected in the plasma of healthy individuals were found. A significant decrease was noted in TNF‐α when preinfusion concentrations were compared to 4 day (P < 0.01) or to 7 day (P < 0.05) postinfusion values. A 50% or greater decrease in IL‐1β was also observed in most individuals with time after infusion. In contrast, sIL‐2R concentrations remained relatively stable during the 1 week follow‐up period. However, strikingly different patterns in the IL‐1β and sIL‐2R levels were noted in the subject who had received two previous murine antibody infusions. Our data show that the administration of radiolabelled murine antibodies, either conventional or bifunctional, can significantly alter plasma levels of TNF‐α and IL‐1β. These cytokines are important in immunoregulation and, perhaps also, in modulation of neoplastic growth. © 1994 Wiley‐Liss, Inc.
Diagnosis of HIV‐1 infection by detection of antibody IgG to HIV‐1 in urine with ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens
Authors: Hashida Seiichi , Hashinaka Kazuya , Saitoh Atsushi , Takamizawa Akihisa , Shinagawa Hideo , Oka Shinichi , Shimada Kaoru , Hirota Kouichi , Kohno Takeyuki , Ishikawa Setsuko , Ishikawa Eiji
Published: March 2007

Anti‐HIV‐1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT), p17 and p24 as antigens, and β‐D‐galactosidase from Escherichia coli as label. Anti‐HIV‐1 IgG in urine was reacted simultaneously with 2,4‐dinitrophenyl‐bovine serum albumin‐recombinant protein conjugate and recombinant protein‐β‐D‐galactosidase conjugate. The immune complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity purified (anti‐2,4‐dinitrophenyl group) IgG. After washing, the immune complex was eluted from the polystyrene balls with excess of ϵN‐2,4‐dinitrophenyl‐L‐lysine and transferred to clean polystyrene balls coated with affinity‐purified (anti‐human IgG γ‐chain) IgG. Finally, the enzyme activity bound to the last solid phase was assayed by fluorometry. Using recombinant RT as antigen, the sensitivity and specificity for 83 seropositives and 100 seronegatives were both 100%, and the lowest signal for 60 asymptomatic carriers was 8.2‐fold higher than the highest signal for the seronegatives. The positivity with recombinant RT as antigen could be confirmed by using recombinant p17 and p24 as antigens. The sensitivity could be improved by a longer assay of bound β‐D‐galactosidase activity, by using concentrated urine samples and by the combined use of recombinant RT, p17, and p24. Thus, reliable diagnosis of HIV‐1 infection was possible for asymptomatic carriers. © 1994 Wiley‐Liss, Inc.
Simultaneous automated measurement of serum total CK and CK‐MM lsoform ratio in serum
Authors: Uji Yoshinori , Karmen Arthur , Sugiuchi Hiroyuki , Okabe Hiroaki , Hata Keishi , Miura Masakazu
Published: March 2007

We automated a two‐step kinetic procedure for determining serum CK‐MM isoform ratio using an immunoinhibition method. By measuring the total CK activity and the residual CK activity (serum CK‐MM isoform) remaining after the inhibition by tissue CK‐MM isoform specific monoclonal antibody reagent (CK‐M01) the CKMM isoform ratio is calculated using the difference between total CK and residual CK activities divided by the residual CK activity.

Linearities of total CK and residual CK assays were⩽7750 U/L and 2,500 U/L, respectively; within‐run CVs of isoform ratio (N = 10) were 2.8 and 7.0% (mean 0.14 and 0.60), respectively. The MM3/MM1 isoform ratio obtained with the proposed method (X) correlated well with the results of electrophoretic method (Y) according to the equation: Y = 0.98X −0.3, r = 0.988. The normal reference range of isoform ratios obtained by assaying 1,222 serum samples from healthy subjects was 0.09–0.75. The isoform ratio increased after onset of chest pain, peaking at 2–6 hr thereafter. A mean isoform ratio of 1.86 was obtained with serum sample from 86 patients diagnosed as having an acute myocardial infarction (AMI). This method is accurate and highly sensitive, as the detection and early diagnosis of AMI can be completed in 10 min. © 1994 Wiley‐Liss, Inc.
Prostate‐specific antigen in screening of prostate cancer
Authors: Chu T. Ming
Published: March 2007

Prostate‐specific antigen (PSA) is a wellcharacterized human prostate‐specific glycoprotein. PSA has been shown to be the most effective immunohistologic marker for prostate cancer, as well as the most useful serologic test in staging and monitoring prostate cancer and in early detection of recurrent disease. The greatest clinical value of PSA is as an aid for early detection of prostate cancer. Recent studies have indicated that PSA‐based screening of the older population for organ‐confined early‐stage prostate cancer is an acceptable, practical, and reliable modality. The accuracy of PSA screening is within the same range as the mammogram. The cost‐effectiveness of PSA is comparable to other cancer screening tests. Although the increase in the patient's survival due to PSA‐based detection of early prostate cancer remains to be documented, it is generally agreed that the PSA test along with digital rectal examination (DRE) should be included in the annual physical examination for men 50 years of age or older. Highrisk men are urged to commence at age 40. Asymptomatic men who have both a negative DRE and normal PSA blood test need only to continue an annual DRE and PSA check‐up. Men who have a negative DRE and elevated PSA, and all those who have a suspicious DRE regardless of PSA results, should undergo further diagnostic workup, such as transrectal ultrasonography with biopsy of visible lesions. The cure rate is high with timely treatment, when prostate cancer is detected while still confined to the prostate. © 1994 Wiley‐Liss, Inc.
Evaluation of free psa isoforms, psa complex formation, and specificity of anti‐psa antibodies by hplc and page‐immunoblotting techniques
Authors: Wu James T. , Zhang Ping , Wang Tian , Wilson Lorraine , Astill Mark
Published: March 2007

Both high performance liquid chromatographic (HPLC) and polyacrylamide gel electrophoresis‐immunoblotting (PAGE‐immunoblotting) procedures have been established for the study of isoforms of free prostate‐specific antigen (PSA) and the complex formation between free PSA and protease inhibitors, and for the evaluation of the specificities of various anti‐PSA antibodies. We found multiple isoforms of free PSA on PAGE, which were all capable of forming complexes with protease inhibitors. The same isoform pattern can be produced from the original seminal fluid. The PSA isoforms differ from each other most likely in charge because they could be converted to one band on SDS‐PAGE and to a single peak by gel filtration chromatography. We found it difficult to form large quantities of PSA complex when mixing free PSA from seminal fluid with protease inhibitors, regardless of whether the free PSA or the protease inhibitors were in excess. Except for the PSA‐ACT complex, which was consistently detectable by both HPLC and PAGE‐immunoblotting techniques after incubation, these two procedures disagreed in their detection of PSA‐A2M and PSA‐AT complexes. The PSA‐A2M complex was usually observable by immunoblotting techniques but barely detectable on HPLC, whereas PSA‐AT was totally invisible by immunoblotting but appeared as a peak in the HPLC elution profile. Mixing free PSA with serum clearly resulted in both PSA‐ACT and PSA‐A2M complexes. However, more PSA‐ACT than PSA‐A2M was formed; the result was also confirmed by using 125I‐PSA for mixing. PSA could be separated into active and inactive PSA by DEAE Sepharose chromatography with a 14‐fold difference in protease activity. The difference in enzymatic activity apparently had no effect on complex formation. All the anti‐PSA antibodies examined in this study reacted with PSA isoforms, PSA‐ACT and PSA‐A2M complexes. We conclude that it would be almost impossible to establish an assay to measure all forms of PSA in the serum and to expect to produce precise and accurate PSA values.©1995 Wiley‐Liss, Inc.
Improvement of an eia system for basic fibroblast growth factor by use of biotinylated antibody prepared with nhs‐lc‐biotin
Authors: Takei Yoshifumi , Higashira Hanae , Hayashi Kyozo
Published: March 2007

We improved our previously devised enzyme immunoassay (FIA) system for basic fibroblast growth factor (bFGF) using biotinylated antibody prepared with sulfosuccinimidyl‐6‐(biotinamido)hexanoate (NHS‐LC‐Biotin, Pierce), a water‐soluble biotin analogue, with an extended spacer arm. The discriminatory detection limit of the improved EIA was found to be 5 pg/ml (0.5 pg/assay tube), sixfold more sensitive than that of the previous system. The reproducibility of within‐ and between‐assay series was 5.10–8.61% and 5.99–8.69%, respectively; and recovery of exogenous bFGF from serum was approximately 102%. Employing the improved EIA system, we investigated by chromatofocusing chromatography the isoelectric points of two immunoreactive bFGFs (high‐molecular‐weight bFGF‐like immunoreactive substance, designated as HMW‐bFGF‐LI and 16‐kd bFGF‐LI, having the same molecular weight as recombinant bFGF) detected in serum from a breast cancer patient. As a result, the pl value of HMW‐bFGF‐LI was estimated to be 7.13, and that of 16 kd bFGF‐LI, 9.58.©1995 wiley‐Liss, inc.
A new plastic evacuated tube with plasma separator
Authors: Landt Michael , Wilhite Timothy R. , Smith Carl H.
Published: March 2007

A new plastic‐walled, evacuated blood collection tube (Terumo Venoject II) with plasma separator was evaluated for characteristics related to the use of plastic in place of glass. Plastic tubes were highly resistant to breakage through handling mishaps or failure during centrifugation. Higher centrifugation speed (13,600g) was well tolerated, and centrifugation times as short as 3 minutes at 13,600g effectively cleared plasma of cellular components, at high plasma yield. Plastic tubes were nearly completely combustible under incineration conditions commonly used for medical waste, and plastic tubes effectively retained vacuum during the typical shelf life of evacuated blood collection tubes. Collection of carbamezepine specimens in plastic tubes decreased levels an average of 6.8% compared to a heparinized glass tube control under conditions approximating routine use; levels of other drugs (phenytoin, phenobarbital, valproic acid, theophylline, and cyclosporine) were less significantly affected. This modest decrease appeared to result from a combination of an immediate interaction with the plastic surface of the tubes and a time‐dependent interaction with the olefin‐based separator. Modest but clear benefits including reduction in specimen breakage, reduced centrifugation time and reduced solid waste after incineration derive from the use of plastic in place of glass in evacuated blood collection tubes.©1995 wiley‐Liss, inc.
Detection of autoantibodies to ss‐a/ro by indirect immunofluorescence using a transfected and overexpressed human 60 kd ro autoantigen in hep‐2 cells
Authors: Fritzler Marvin J. , Miller B. Joan
Published: March 2007

The objective of the study was to determine the sensitivity and specificity of an indirect immunofluorescence (IIF) assay using transfected HEp‐2 cells to detect anti‐SS‐A/Ro autoantibodies in human sera. Seventy‐three sera having SS‐A/Ro autoantibodies as determined by double immunodiffusion (ID) and immunoblotting (IB) were tested by IIF on a HEp‐2 cell substrate that had been transfected with a full‐length cDNA encoding a human 60 kD SS‐A/Ro autoantigen. Controls included 30 normal human sera and 50 sera with a variety of other antinuclear antibodies. Prototype human and rabbit sera directed against the 60 kD SS‐A/Ro antigen produced intense speckled nuclear and nucleolar staining of transfected cells. Sixty‐nine of 73 (95%) SS‐A/Ro positive sera also produced this characteristic staining pattern. The endpoint autoantibody titers on transfected cells was fivefold greater than on untransfected cells. The 30 normal human sera and the 50 sera with other antinuclear antibodies did not produce this characteristic staining. Six of 32 (19%) unselected sera that were sent for autoantibody testing had reactivity with transfectants by IIF. Four of the six sera were confirmed to have anti‐SS‐A/Ro antibodies by ID and 5/6 by IB. By contrast, only three of these sera were scored as having a staining pattern compatible with SS‐A/Ro antibodies by IIF on standard HEp‐2 substrates. We conclude that SS‐A/Ro autoantibodies can be detected by an IIF assay using a HEp‐2 cell substrate transfected with a SS‐A/Ro cDNA. This new substrate detects SS‐A/Ro antibodies that were not identified on standard HEp‐2 substrates and by other immunoassays.©1995 wiley‐Liss, inc.
Development and validation of an automated particle‐enhanced nephelometric immunoassay method for the measurement of human plasma c1q
Authors: Borque Luis , Olivan V. , Iguaz F.
Published: March 2007

We have developed a sensitive immunoassay based on latex particle agglutination for measuring C1q concentrations in human plasma. In this simple and fast particle‐enhanced immunoassay, we used carboxylated latex particles (diameter 210 nm) covalently coated with F(ab)2 fragments of anti‐C1q antibodies. These particles are incubated with diluted sample (400‐fold) for 6 min at room temperature, with the resulting agglutination quantified by measuring the change of light‐scatter produced. The assay has been automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hr. This assay generates a standard curve in the range of 24–775 mg/L, showing intraassay and interassay precision of <8% and <10%, respectively. Dilution linearity was validated throughout the dynamic range of the assay. There were no interferences from bilirubin, Intralipid, haemoglobin, and rheumatoid factor. Results obtained in 45 clinical samples correlated well with those obtained by a commercial radial immunodiffusion method (r=0.936), and with those obtained by the Behring immunoprecipitation nephelometric test (r=0.950). The mean concentration in plasma from healthy subjects was 180 mg/L and the reference interval was from 128 to 237 mg/L. This latex nephelometric procedure is a convenient method and an interesting alternative to other immunoassays for routine measurement of human C1q.