bluuebunny/crossref_metadata_2025 huggingface dataset:
Microplate elsa for detection of antibodies to DNA in patients with systemic lupus erythematosus: Specificity and correlation with farr radioimmunoassay
Authors: Sutjita M. , Hohmann A. , Boey M. L. , Bradley J.
Published: March 2007

A 96‐well microplate ELISA for the detection of antibodies to DNA is described. A number of buffers and precoating treatments were used to evaluate the optimal method for coating the plate with DNA. These included pretreatment of the plates with poly‐L‐lysine or protamine sulfate, and posttreatment with glutaraldehyde, none of which improved the performance of the assay. Whereas bicarbonate and borate coating buffers gave equivalent and satisfactory results, TRIS buffer resulted in very high binding of immunoglobulin to wells not coated with antigen.

Sera from groups of patients with autoimmune disease as well as normal sera were tested against plates optimally coated with native E. coli DNA, calf thymus DNA, and heat‐denatured DNA. Using native E. coli DNA, virtually none of 35 normal sera had any detectable antibody. With this antigen, as well as with native calf thymus DNA, significant levels of DNA antibody were found only in SLE patients. Most patients with SLE or drug‐induced lupus, as well as some patients with rheumatoid arthritis and normal individuals had antibodies that bound to heat‐denatured (single‐stranded) DNA. Using either native E. coli or calf thymus DNA, a good correlation was found between the amount of DNA antibody detected by ELISA and the Farr‐type radioimmunoassay.
Evaluation of cancer patient leukocyte responses in the presence of physiologic and pharmacologic pyridoxine and pyridoxal levels
Authors: Gridley D. S. , Stickney D. R. , Shultz T. D.
Published: March 2007

Peripheral blood samples from six cancer patients (five colon cancer, one lung cancer) and six healthy volunteers were tested in vitro for oxygen radical production by phagocytic cells and in assays of mitogen‐induced lymphoblastogenesis at physiologic and pharmacologic concentrations of pyridoxine (PN, 1.8‐96 nmol/ml) or pyridoxal (PL, 0.08‐90 nmol/ml). Plasma levels of pyridoxal‐5′‐phosphate (PLP), 4‐pyridoxic acid (4PA), pyridoxamine phosphate (PMP), and PL were also determined. Phagocytic cells from three patients showed significantly increased capacity for oxygen radical production when incubated in PL‐, but not PN‐supplemented media. Oxygen burst capacity of cells from healthy subjects was significantly enhanced by PN‐, but not PL‐enriched media. Lymphocyte responsiveness to phytohemagglutinin or pokeweed mitogen (PWM) stimulation showed a modest increase in cell activation in three patients as the concentration of PN was increased; with concanavalin A, two showed enhanced responsiveness. On the other hand, PL‐supplementation resulted in greater cell proliferation only with PWM. The cancer patients had significantly lower plasma PLP, 4PA, and PMP levels when compared with the healthy volunteers. These data indicate that in the cancer patients and in a majority of the healthy volunteers, vitamin 6‐6 status was marginal or deficient and suggest that increasing PN or PL in vivo levels may augment functions related to cell‐mediated immunity.
Nucleic acid analysis by sandwich hybridization
Authors: Nicholls Peter J. , Malcolm Alan D. B.
Published: March 2007

One of the most significant achievements of the biochemist during the past two decades is the use to which immunologically based assays have been put in clinical diagnosis (Hood et al.: Immunology, 1984). The problem faced and surmounted by immunologists in effecting the transition from research tool to routine clinical assay bears a remarkable similarity to that confronting the molecular biologist today; i.e., how can nucleic acid hybridization, a technique of obvious potential (Meinkoth and Wahl: Anal Biochem 138:267‐284, 1984; Syvanen: Med Biol 64:313‐324, 1986; Matthews and Kricka: Anal Biochem 169:l‐25, 1988), be modified in order to fulfill all necessary parameters of a routine diagnostic assay? There are several such requirements, and the importance placed on each depends on the objectives of the assay: the technique must be sensitive, specific, and reproducible. Other advantages would be cost‐effectiveness, ease of manipulation, and amenability to automation. Ideally, the signal detection should be based on a non‐radioactive system, because of the instability of probes labelled with isotopes like 32p, and the potential hazards involved in their handling and disposal.

The sandwich hybridization for the analysis of nucleic acid sequences was first used in 1977 (Dunn and Hassell: Cell 12:23‐36, 1977), but its potential as a diagnostic assay was not realized until 1983, when it was applied to the detection of adenovirus DNA in nasopharyngeal aspirates from children with acute respiratory infection (Ranki et al: Gene 21:77‐85, 1983). It has since been modified and used not only for the detection of microbial infection (Virtanen et al.: Lancet i:381‐383, 1983; Ranki et al.: Cur Top Microbiol Immunol 104:307‐318, 1983; Lehtomaki et al.: J Clin Microbiol 24:108‐111, 1986; Virtanen et al.: J Clin Microbiol 20:1083‐1088, 1984; Palva and Ranki: Clin Lab Med 5475‐490, 1985; Polsky‐Cynkin et al.: Clin Chem 31:1438‐1443, 1985; Parkkinen et al.: J Med Virol 20:279‐288, 1986; Palva: FEMS Microbiol Lett 28%‐91, 1985; Palva et al: FEMS Microbiol Lett2333‐89,1984; Zolg et al.: Mol Biochem Parasitol 22:145‐151, 1987; Palva: J Clin Microbiol 18:92‐100, 1983), but also for the analysis of nucleotide sequence variations (Langdale and Malcolm: Gene 36:201‐210, 1985). We will discuss the development of the sandwich technique and the advantages it conveys over the more conventional nucleic acid hybridization formats, together with new developments which will ensure that it earns a place alongside immunoassay in the diagnostic laboratory.
Immunoblot reactivity of a non‐hiv protein with human sera
Authors: Norman Gary L. , Barker Shu Su , Rasheed Suraiya , The Transfusion Safety Study Group
Published: March 2007

Examination of serum for the presence of antibodies to the human immunodeficiency virus (HIV) by immunoblot analysis requires precise identification of reactivities with various HIV specific proteins. During a recent survey of approximately 2,000 sera, we identified 22 sera from non‐HIV‐reactive blood donors and 2 from individuals receiving blood products for congenital blood disorders, which consistently and exclusively reacted with a protein of a molecular weight slightly greater than 65,000 daltons (termed AT65). Since the HIV pol p65 protein reacts with specific antibodies at about the same position (i.e., 65,000 daltons), it was essential to determine the viral or nonviral origin of the AT65 reactivity. Our data indicate that the AT65 reaction is due to a protein present on normal or activated lymphocytes, which can co‐purify with HIV preparations used for immunoblot analysis. Recognition of HIV‐specific p65 and nonspecific AT65 reactions is important to those responsible for interpretation of HIV immunoblots and may aid in the evaluation of some “indeterminant” results.
More sensitive and simpler immune complex transfer enzyme immunoassay for antithyroglobulin igg in serum
Authors: Kohno Takeyuki , Mitsukawa Tomohiro , Matsukura Shigeru , Tsunetoshi Yoshizo , Ishikawa Eiji
Published: March 2007

A more sensitive and simpler immune complex transfer enzyme immunoassay for anti‐thyroglobulin IgG in serum and its use for the measurement of anti‐thyroglobulin IgG in healthy subjects and patients with thyroid diseases are described. Antithyroglobulin IgG in test serum was reacted simultaneously with dinitrophenyl thyroglobulin and thyroglobulin‐β‐D‐galactosidase conjugate. The complex formed of antithyroglobulin IgG, dinitrophenyl thyroglobulin, and thyroglobulin‐β‐D‐galactosidase conjugate was trapped onto two polystyrene balls coated with affinity‐purified rabbit (antidinitrophenyl bovine serum albumin) IgG. The polystyrene balls were washed to eliminate nonspecific IgG in the test serum, and the complex was eluted from the polystyrene balls with dinitrophenyl‐L‐lysine and transferred to two polystyrene balls coated with affinity‐purified rabbit (anti‐human IgG γ‐chain) IgG. β‐D‐Galactosidase activity bound to the polystyrene balls was assayed by fluorimetry using 4‐methylumbelliferyl‐β‐D‐galactoside as substrate. As compared with the previous immune complex transfer enzyme immunoassay, the procedure was simplified by reducing one incubation step and one washing step, and the detection limit of antithyroglobulin IgG in serum (0.1 μg/liter) was lowered 100‐fold. More careful and extensive examination than in a previous study revealed the presence of antithyroglobulin IgG in a large proportion of healthy subjects and in all patients with Graves' disease and chronic thyroiditis.
Competitive ELISA for detection of native ras gene‐related products in sera of cancer patients
Authors: Epelbaum R. , Bartal A. H. , Rivkin M. , Tugendhaft N. , Shalitin C.
Published: March 2007

A solid phase, enzyme‐linked immunosorbent assay (ELISA) competition kit was developed to detect circulating native ras gene‐related products in sera of 151 healthy volunteers and cancer patients. This assay uses monoclonal antibody (mAb) BST‐6A generated against a yeast‐derived, native ras‐related polypeptide Yp20. Only 2% (1 of 58) of normal control sera showed strong competition, as compared to 15% (5 of 34) of patients with early stage or no evidence of disease, and 44% (26 of 59) of patients with advanced disease. These differences were statistically significant (X2, P < 0.05–0.001). Eleven sera samples of cancer patients found to be strong competitors in the ELISA competition kit were tested for the presence of anti‐ras antibodies by ELISA. None showed higher ELISA values as compared with pooled normal human serum and control sera. It is thus suggested that our procedure detected circulating ras‐related oncoproteins in sera of cancer patients mainly with advanced disease.
Effect of dexamethasone on herpes simplex virus replication in mouse neuroblastoma cells (NB41 A3): Receptor characteristics
Authors: Dreyer Larry L. , Sydiskis Robert J. , Bashirelahi Nasir
Published: March 2007

Monolayers of NB41A3 (MNB) cells were exposed to pharmacologic doses of dexamethasone (DXM). After 24‐h, the cells were infected with herpes simplex virus type 1 (HSV‐1) at a multiplicity of infection (m.0.i.) = 0.1. After every 24‐h period, extracellular and intracellular virus aliquots were collected and frozen. The aliquots were titered in a standard plaque‐forming assay. It was shown that the hormone led to a statistically significant increase of extra‐ and intracellular virus titers above the titers exhibited by these cells without added hormone. The same experiment was repeated in Vero cells, but the hormone did not elevate the resultant HSV‐1 titers. A binding assay was performed on these two cell lines by use of 3H‐DXM to determine if a DXM receptor was present. Specific binding was seen, but only in the MNB cell line. The Bmax of this receptor was 480 fmol/mg protein and it had a Kd of 2.3 nM. The S value of the receptor ligand complex equalled 8.0. These results indicate that cells possessing hormone receptors allow for a more efficient replication of the virus and suggest that these hormones may play an important role in the exacerbation of herpes simplex virus infection in vivo.
Wb e34 monoclonal antibody: Further characterization and diagnostic use in bancroftian filariasis
Authors: Reddy M. V. R. , Parkhe K. A. , Harinath B. C. , Piessens W. F.
Published: March 2007

The Wb E34 monoclonal antibody raised against Wuchereria bancrofti microfilarial excretory secretory (mf ES) antigen was reported to be useful in detecting the filarial antigen in W. bancrofti and Brugia malayi infected sera. Further studies in this laboratory showed that this monoclonal antibody reacts with a stage–specific antigen of W. bancrofti filarial parasite. Wb E34 identified three antigenic components with molecular weights, 55, 57.5, and 63 kilodaltons (Kd) in western blot analysis. The target antigen of Wb E34 was found to be located in the cytoplasm of microfilariae by indirect immunofluorescence technique. The inhibitory antibodies to Wb E34 were detected in a higher percentage of microfilaraemic sera (81%) than in clinical filarial (33%) or tropical eosinophilia sera (35%). Thus the inhibition ELISA using W bancrofti mf ES antigen and Wb E34‐penicil‐linase may be useful in detecting the filarial antibody associated with the active stage of infection.
Properties of benzodiazepine binding sites in peripheral blood lymphocytes
Authors: Garau L. , Carai M. A. M. , Boi W. , Ennas M. G. , Manconi P. E. , Spano P. F. , Memo M. , Pizzi M. , Misale C.
Published: March 2007

Benzodiazepine (BDZ) binding sites were studied by using 3H‐diazepam and 3H‐Ro 5‐4864 in intact lymphocytes from peripheral blood (PBL), in comparison to kidney and cerebellum. Experiments with 3H‐diazepam performed at equilibrium and measuring kinetics revealed that BDZ binding sites are indeed present in rat PBL. The binding is saturable (Bmax 557 fmoles/106 cells), with high affinity (KD = 9.3 nM) and reversible. Specific binding sites are also observed by saturation experiments with 3H‐Ro 5‐4864 (Bmax 175 fmoles/106 cells, KD 2.2 nM). In addition, analysis of saturation isotherms obtained with 3H‐diazepam indicates that BDZ binding sites are also present in human PBL. Scatchard plot of binding isotherms revealed an apparent single population of sites in all cases. The pharmacological characterization of BDZ binding sites in PBL, as compared with those of kidney and cerebellum, showed that these sites belong to the so‐called “peripheral type”.
Pharmacological modulation of beta‐endorphin in rat peritoneal macrophages
Authors: And Paola Sacerdote , Panerai Alberto E.
Published: March 2007

The neuropeptide beta‐endorphin is present in cells of the immune system, i.e., lymphocytes and monocytes, and its expression can be induced by immunological stimuli. In the present study, we showed that the increase of the serotoninergic availability induces an increase of beta‐endorphin concentrations in rat peritoneal macrophages that is blunted by the administration of serotonin receptor antagonists. A significant increase of beta‐endorphin concentrations is also evident after blocking the dopaminegic receptors, whereas a dopaminergic agonist decreases the concentrations of the peptide. Our data are consistent with a similar modulation of beta‐endorphin concentrations in central nervous system and in immune cells, e.g., rat peritoneal macrophages.
Modified dot assay with increased sensitivity: Detection of small amounts of immunoglobulin molecules and the importance of different detection systems
Authors: Lavanchy Daniel , Stroun Jacques , Frei Philippe C.
Published: March 2007

The original method of the dot assay described by Hawkes et al. enabled the detection of 100 pg of immunoglobulin molecules. We compared different enzyme detection systems based on the avidin‐biotin complex, in conjunction with different substrates. Three commercial kits (Vectastain‐ABC, Streptavidin Bridge, and Streptavidin‐Peroxidase Complex) based on the same technique were also included in the comparison. In addition, we tested the Aureo‐Probe kit, which is based on gold‐ and silver‐enhanced staining. Various incubation times, blocking solutions (horse serum, bovine serum albumin, and milk), and commercial antihuman Ig antisera or monoclonal antibodies were tested. The greatest sensitivity was achieved by using the peroxidase‐avidin‐biotin complex together with 4‐chloro‐1‐naphtol as substrate, which detected 1 pg of immunoglobulin molecules. Equal sensitivity was also shown by the Aureo‐Probe system.
Comparison of the classical immunofluorescent antibody tests for epstein‐barr virus with commercially available test kits
Authors: Thiele Geoffrey M. , Johnson Ann M. , Okano Motohiko , Woods Gail L. , Purtilo David T.
Published: March 2007

Twenty reference sera with previously reported titers plus 10 normal seropositive and 10 seronegative sera were used to determine which assay for the detection of antibodies to Epstein‐Barr virus should be utilized in our laboratory. By three commercially available test systems and by slides prepared in our laboratory, all techniques detected the same number of samples positive for IgG antibodies to viral capsid antigen (VCA) and Epstein‐Barr virus‐determined nuclear antigen (EBNA), but the geometric mean titers (GMTs) varied. Major discrepancies arose in the sensitivity of detection of early antigen‐diffuse (EA‐D) and ‐diffuse and restricted (EA‐DR). Slides prepared in our laboratory gave the best results, detecting 12 of 12 (100%). Slides prepared according to Gull and Hillcrest detected 11 of 12 (92%), and those prepared according to Zeus detected 9 of 12 (75%). From these data, we conclude that all of these methods for detection of VCA and EBNA are adequate. However, in determining antibody levels to EA‐D and EA‐DR, great care should be taken as to the source of the slide and specificity and sensitivity.
Evaluation of the stick enzyme immunoassay in caranx sp. and seriola dumerili associated with ciguatera
Authors: Hokama Y. , Asahina A. Y. , Hong T. W. P. , Shang E. S. , Miyahara J. T.
Published: March 2007

The stick enzyme immunoassay (S‐EIA) using monoclonal antibody to ciguatoxin (MAb‐CTX) was used to examine clinically implicated fish and to pre‐screen two species of fish, Caranx sp. (ulua or jack) and Seriola dumerili (kahala or amberjack), supplied by sports fishermen. All of the clinically implicated fish from the Department of Health gave S‐EIA values ≥ 1.3. The Caranx sp. and Seriola dumerili considered safe (≤1.2 value) and consumed after testing gave no false‐negative results. The S‐EIA procedure using MAb‐CTX proved to be specific, sensitive, and simple to use in the laboratory. It also proved to be useful in screening two large carnivorous fish for ciguatoxin and related polyethers prior to consumption.
Estimation of serum sex hormone–binding globulin by five direct and two indirect methods
Authors: Brotherton Janet
Published: March 2007

Serum sex hormone‐binding globulin (SHBG) has been estimated over the range 2–190 nM by six commercially available methods. The Farmos immunoradiometric assay (IRMA) and the Pharmacia‐LKB DELFIA fluoroimmunoassay, which use the same monoclonal antiserum, produced statistically identical but widely spread results. The Techland polyclonal radioimmunoassay produced similar results but was considerably less sensitive, and agreement with the other two direct methods was poor at low concentrations. These three direct methods gave slightly higher results than those obtained with the two indirect methods: the BioMerieux concanavalin‐A–binding method and, especially, the Serono ammonium sulphate precipitation method. The Diagnostic Products monoclonal IRMA gave results that were up to double the values obtained with the other five methods and is considered unsatisfactory. Monoclonal antibodies are now available for the direct estimation of SHBG, although an amino acid sequence has not yet been published for this protein, and there is still some controversy about the size of its structural units.
Isolation of human herpesvirus‐6 from clinical specimens using human fibroblast cultures
Authors: Luka Janos , Okano Motohiko , Thiele Geoffrey
Published: March 2007

The isolation and characterization of human herpesvirus‐6 (HHV‐6) has been hindered by the lack of cell lines useful for its rapid propagation. Recently, we have reported that the MRC‐5 cell line (human diploid lung fibroblasts) was susceptible for HHV‐6 infection. In this study, we report on the isolation of HHV‐6 from the peripheral blood or buffy coat of three chronic fatique syndrome patients, one post‐liver transplant patient, and one severe chronic active Epstein‐Barr virus syndrome patient using the MRC‐5 cell line. Additionally, it was observed by Southern blot hybridization studies that four of five isolates had different restriction enzyme fragment patterns than the isolate obtained from the National Institutes of Health with Eco RI. These data suggest the usefulness of the MRC‐5 cell line in the isolation and characterization of HHV‐6 from various patients.
Differentiation of myeloperoxidase and glandular peroxidase in biological fluids: Application to human saliva
Authors: Venezie Ronald D. , Jenzano Joyce W. , Lundblad Roger L.
Published: March 2007

A modification of the assay system for peroxidase activity in human mixed saliva using 2,2′‐azinobis‐(‐ethylbenzthiazoline‐6‐sulphonic acid) (ABTS) as the substrate is reported. The modifications permit differentiation between glandular peroxidase (SPX) and myeloperoxidase (MPX) activity. In addition, factors endogenous to saliva such as thiocyanate did not adversely affect the modified assay system. The basic peroxidase assay utilized 1.0 mM ABTS and 0.1 mM H2O2 in 0.1 M sodium acetate, pH 4.7. The addition of 100 mM NaCl to the assay inhibited 90% of the activity of purified MPX but only 40% of the SPX activity in parotid saliva presumed to be free of MPX. Addition of Cl− to the assay had varied effects on the mixed saliva SPX activity (44–68% inhibition), suggesting that MPX is present in varying amounts in mixed saliva. Assay of mixed saliva from patients with different states of periodontal health indicated that the amount of Cl− inhibition is dependent on amount of inflammation and thus amount of MPX present. The results suggest that the modified assay yields a reliable estimate of relative SPX activity in mixed saliva.
Acetylcholine receptors from normal human muscle: Concentration, purification, and use in radioreceptor assays for autoantibodies
Authors: Wu James T. , Knight Joseph A. , Miya Terry G. , Bringhurst Cory
Published: March 2007

We measured the concentration of acetylcholine receptor (AChR) in various normal human muscles, obtained at autopsy, in order to find a more reliable and convenient source than the amputated leg muscle from diabetic patients for the isolation of AChR. We found that human calf muscle contains the highest concentration of AChR, approaching the concentration of receptor required for the radioreceptor assay for autoantibodies in myasthenia gravis (MG). Because the amount of contaminant proteins in the receptor preparations affect the sensitivity and precision of the assay, various chromatographic techniques were tested to improve the purity of the receptor preparation. We found that both G‐100 Sephadex and DEAE Sephacel chromatographies were effective in removing contaminant proteins. DEAE Sephacel chromatography is particularly useful because the procedure provides higher recovery of receptor. Against the same pool of autoantibodies from patients with MG, AChRs from diabetic and normal leg muscle exhibit similar affinities. We conclude that AChR from normal human calf muscle can be used in radioreceptor assays for measuring autoantibodies in patients with myasthenia gravis.
Correlation of histoplasma capsulatum polysaccharide antigen with the severity of infection in murine histoplasmosis
Authors: Williams Brian J. , Connolly‐Stringfield Patricia , Bartlett Marilyn , Durkin Michelle , Blair Robinette , Connolly Kathleen , Wheat L. Joseph , Garringer Todd , Tewari Ram P.
Published: March 2007

We sought to determine if Histoplasma capsulatum polysaccharide antigen (HPA) levels correlate with the extent of infection in murine of histoplasmosis. Separate groups of mice were inoculated intratracheally with varying numbers of H. capsulatum yeast cells. After 1 week, HPA levels and fungal burden (quantitative culture of lung and spleen and histopathologic stain of lung) were determined in lung and spleen, and HPA levels in serum. HPA levels, cultures and histopathological stain results of lung and spleen tissue showed a direct correlation with increasing inoculum size. HPA levels in serum also correlated with the size of inoculum. H. capsulatum antigen in lung correlated with silver stain scores of lung tissue, (R = 0.948, P <0.001) and with quantitative culture scores of lung, (R = 0.929, P <0.001). HPA levels in spleen tissue also correlated with spleen culture scores, (R = 0.724, P <0.001). These results indicate that determination of HPA level in serum and tissue may be a useful test in evaluating the severity of diseases as well as efficacy of antifungal therapy in histoplasmosis.
A novel igm‐indirect hemagglutination test for the serodiagnosis of acute toxoplasmosis
Authors: Yamamoto Yoshimi Imoto , Hoshino‐Shimizu Sumie , Camargo Mário E.
Published: March 2007

A new indirect hemagglutination (M‐Toxo‐IHA) test was standardized for the serodiagnosis of acute toxoplasmosis. The reagent for this test consisted of stabilized human red cells coated with a Toxoplasma gondii heatstable alkaline‐solubilized extract which reacted predominantly with IgM antibodies found in serum samples from patients with a recent infection. Almost no reactivity was verified with serum samples from chronic or ancient toxoplasmosis. The reagent preparation was simple and obviated prior fractionation of parasite components reactive with IgM antibodies. Rheumatoid factor or high levels of IgG antibodies did not interfere in the M‐Toxo‐IHA test. A total of 358 serum samples was assessed for the evaluation of this test, comprising sera from patients with recent and ancient toxoplasmosis, from nonrelated diseases, as well as from clinically healthy individuals with no previous T. gondii infections. Serodiagnostic performance of the M‐Toxo‐IHA test in terms of sensitivity, specificity, efficiency, and positive and negative predictive values was verified, and the values obtained were 1.000, 0.985, 0.989, 0.952, and 1.000, respectively, in relation to IgM‐immunofluorescence or IgM‐capture ELISA. Six different batches of reagent, successively produced for the current study, provided reproducible results and were stable for at least 22 months at 4°C.
Immunoreactivity of nerve lipid antigens in leprosy
Authors: Vemuri Nalini , Mukherjee Rama
Published: March 2007

Neural lipid antigens, namely, galactocerebroside and ganglioside, have been implicated in demyelinating diseases. We were interested in finding the role of these antigens in leprosy neuritis. The humoral immune response to these lipid antigens was quantitated by enzyme‐linked immunosorbent assay in sera from 91 leprosy patients and 18 normal individuals. Our data revealed the presence of antibodies to total nerve lipids (TNL) and galactocerebroside (GalC) and a significantly low level to ganglioside (Gg) in all the categories of leprosy. No antilipid antibodies were detected in normals. Anti‐TNL and anti‐GalC antibodies were highest in tuberculoid leprosy patients. Statistically significant positive correlation was observed between anti‐TNL and anti‐GalC antibodies in lepromatous borderline, tuberculoid, and neuritic patients.
Rapid and specific cyclosporine assay for the du pont aca® discrete clinical analyzer performed directly on whole blood
Authors: Hansen Joel B. , Lau H. Phillip , Janes Christopher J. , Miller Warren K. , Wiebe Donald A.
Published: March 2007

A rapid, sensitive, and specific enzyme immunometric assay for cyclosporine in whole blood has been developed for the aca® analyzer, using a monoclonal antibody from Sandoz, Ltd. Between‐run CVs ranged from 6.5 to 7.6% for samples containing between 60 and 400 ng/mL cyclosporine. Sensitivity was better than 25 ng/mL in the assay, which has an effective upper range of greater than 600 ng/mL. Two correlation studies compared cyclosporine values from the Du Pont method to those determined by HPLC procedures in three hospital laboratories. The results from a total of 120 whole blood samples with CsA between 20 and 800 ng/mL showed excellent correlation between the methodologies. HPLC and Du Pont CsA values from 10 day serial studies also correlated well for samples from a kidney, kidney‐pancreas, heart, and two liver transplant patients. We conclude that the Du Pont CSA assay provides accurate and reproducible results in a convenient format in less than 30 minutes.
The phadiatop test allows adequate screening for atopy with a marked reduction in cost
Authors: Salkie Mark L.
Published: March 2007

Sera from 35 patients aged 5 years or younger and sera from 95 patients 6 years and older were assayed for both total lgE level, allergen‐specific lgE by RAST and either the Phadiatop Paediatric or Phadiatop procedures, depending on age. Good agreement was found allowing exclusion of specimens anticipated to be RAST negative with an expected reduction in RAST testing of up to 80% of the present workload, without significant exclusion of RAST positive specimens.
Measurement of human ventricular myosin light chain‐1 by monoclonal solid‐phase enzyme immunoassay in patients with acute myocardial infarction
Authors: Uji Yoshinori , Sugiuchi Hiroyuki , Okabe Hiroaki
Published: March 2007

A monoclonal solid‐phase enzyme immunoassay has been developed for the detection of human serum ventricular myosin light chain‐1. Cross‐reactivity of this with human skeletal muscle myosin was observed, but the enzyme immunoassay with the sera of patients with acute myocardial infarction gave similar results with radioimmunoassay. The human ventricular myosin light chain‐1 levels in the healthy subjects were 0.2–6.6 ng/ml in males and 0.2–4.1 ng/ml in females. Withinrun and betweenrun precision (CVs) of the assays was on the order of 2.3–4.7% and 4.3–8.7, respectively. Sensitivity of the assay was 1.0 ng/ml, and working range was 5–100 ng/ml. In all patients with define acute myocardial infarction, serum ventricular myosin light chain‐1 levels increased three‐ to tenfold the upper reference range within 6 hr after the onset of chest pains. Two types of subtrend were discovered: its levels remain elevated for 3–4 days and its levels increased and then decreased 1–2 days after the initial rise but became elevated again for the next 4–7 days after the onset of chest pain, which is in contrast to the case with all conventionally used biochemical cardiac markers.
Evaluation of serum sialic acid and carcinoembryonic antigen for the detection of early‐stage colorectal cancer
Authors: Tautu Carmen , Alhadeff Jack A. , Pee David , Dunsmore Marlene , Prorok Joseph J.
Published: March 2007

Various expressions of elevated serum sialic acid (total sialic acid, TSA: lipid‐associated sialic acid, LASA; LASA/TSA; TSA normalized to total protein, TSA/TP) have been evaluated and compared with increased serum carcinoembryonic antigen (CEA) levels for the detection of early‐stage colorectal cancer. This evaluation was done blindly on a coded panel of 320 sera from staged colorectal cancer patients and controls provided by the Mayo Clinic‐National Cancer Institute Diagnostic Bank. Unlike the findings of a previous preliminary study (Tautu et al., JNCI 80:1333–1337, 1988), the ratio of LASA/TSA was not useful for detecting early‐stage (Dukes A and B) colorectal cancer. However, TSA and TSA/TP values were significantly elevated in each colorectal cancer subgroup compared with normal controls. TSA and TSA/TP values displayed a marginally better discriminatory power than CEA values in the case of Dukes A subgroup with respect to normal controls. CEA still appears to be the best single overall marker for discriminating between colorectal cancers and controls. However, multiple marker analysis using CEA and TSA (and related markers) appears to be more sensitive than CEA alone for detecting colorectal cancer.
Direct enzyme‐linked immunosorbent assay: A simple immunoassay using leishmania donovani promastigote for diagnosis of kala‐azar
Authors: Mukerji Krishna , Pal Abhijit , Naskar Kshudiram , Ghosh Dilip K. , Basu Debashish , Mallick K. K.
Published: March 2007

For immunodiagnosis of kala‐azar enzyme‐linked immunosorbent assay (ELISA) and immunofluorescence testing (IFAT) are commonly used. In IFAT, whole parasite antigen and in ELISA the soluble antigen have been used. Preparation of ELISA antigen has certain inherent difficulties. We have developed a simple, specific, and quantitative immunoassay, “direct ELISA” for diagnosis of kala‐azar. Intact formalinized promastigote suspension has been used to combine with the antibodies of the patient sera. The colour developed in the supernatant by the enzyme conjugate combined on the parasite surface was measured with a spectrophotometer. The test was able to detect kala‐azar‐specific antibodies at very high serum dilution and could discriminate between kala‐azar and the common diseases prevalent in Asia. The optical densities of the sera of different control groups were significantly low. The method has potential for use as a diagnostic tool in less well equipped laboratories.
New method for the determination of pancreatic amylase evaluated
Authors: Lorentz Klaus , Seiler Dieter , Nagel Dietmar , Naegele Ulrich , Koller Peter , Bais Renze , Bayer Peter M. , Keller Herbert , Puukka Matti , Ruostesuo Juhani , Puukka Raija , Rosalki Sidney B. , Foo Ying
Published: March 2007

We have carried out a multicenter evaluation of a new reagent carrier for Reflotron®, specific for pancreatic amylase where the salivary isoenzyme is inhibited by two specific monoclonal antibodies. This new procedure combines easy handling with low imprecision (median CV < 3%) in control material, serum, heparinized blood, and plasma and close correlation (r = 0.991 to 0.999) with established manual and automated methods. The same close correlation was found with values obtained from either venous or capillary finger‐stick blood. Salivary amylase up to 54 kU/L (37°C) was inhibited to about 97%. Endogenous interference by hemoglobin, bilirubin, triglycerides, cholesterol, or hematocrit was found to be negligible within a wide range of interferent concentrations. Out of a panel of 28 commonly used drugs it was shown that only two (ascorbic acid and paracetamol), and then only at toxic concentrations, caused a deviation in amylase activity of greater than 10%. From the results of this study we conclude that this new method is suitable for highly precise and accurate measurements of pancreatic amylase in emergency and routine laboratories.
Leukocyte membrane markers on cell populations defined by six flow cytometric bitmaps
Authors: Uz̆arević Branka , Batinić Drago , Petrovec̆ki Mladen , Marus̆ić Matko
Published: March 2007

Peripheral blood samples from healthy adults, patients with transplanted bone marrow, and healthy children were analyzed for the proportions of cells positive for CD2, CD4, CD8, CD19, CD56, CDw65, and KiM8 cell membrane markers. Six cell gates were defined on granularity vs. cell size (L90°LSx‐FALS) flow cytometric display for each blood sample: large lymhocytic, small lymphocytic, monocytic mononuclear, granulocytic, and all‐cell gate, and the listed markers were determined in each of them. Considerable differences in marker‐positive cell proportions were found between healthy adults and children, and particularly between healthy adults and patients with transplanted bone marrow. Marker‐positive cell frequencies corresponded to biologic distribution of three leukocyte populations within the defined gates, in a general agreement with known specificities of the antibodies used. A considerable degree fo variations in the positions of gates drawn for different samples and the numbers of cells counted in them was observed. Still it appeared that all six gates, otherwise commonly used in flow cytometric analysis, could be precisely defined and yielded reproducible data. Large and small lymphocytic gates yielded very similar marker frequencies, revealing that in cases where three leukocyte populations were not clearly delineated on (90°LSxFALS) display, a smaller lymphocytic gate could be safely drawn in order to avoid contamination with monocytes.
Low frequency and low level of elevation of serum CA 72‐4 in human carcinomas in comparison with established tumor markers
Authors: Wu James T. , Carlisle Pam
Published: March 2007

We evaluated a new circulating tumor marker, CA 72‐4, by comparing its frequency of appearance and level of elevation with other established tumor markers in serial serum specimens from patients with various carcinomas. We found that CA 72‐4, though highly expressed and widely found in various tumor tissues, is present at low concentration and frequency in the serum. In breast, colon, ovarian, and pancreatic carcinomas, only 21%, 30.9%, 16%, and 26.5% of specimens, respectively, showed elevated CA 72‐4. When elevated, the level of elevation was also low, much lower than that of the dominant markers. Poor response of CA 72‐4 to therapy was especially noticeable in serial specimens. In most cases, the CA 72‐4 remained normal for the entire series while other markers remained at elevated levels. However, changes of the level of CA 72‐4 usually paralleled those of other markers but at a much lower concentration. Simultaneous measurement of CA 72‐4 and CA 19‐9 appears useful to differentiate colorectal from pancreatic carcinomas when they all contained elevated levels of CA 19‐9. There was a much higher ratio of CA 72‐4 to CA 19‐9 with colon than with pancreatic and other carcinomas (247 ± 524 vs. 4.7 ± 6.8).
Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti‐human T‐cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys‐Env gp46(188‐224), as antigen
Authors: Kohno Takeyuki , Sakoda Iwane , Ishikawa Eiji
Published: March 2007

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti‐human T‐cell leukemia virus type I) IgG (anti‐HTLV‐I IgG) in serum using a synthetic peptide, Cys‐env gp46(188‐224) of HTLV‐I, is described. Anti‐HTLV‐I IgG in test serum, which had been incubated with excess of inactive β‐D‐galactosidase to eliminate interference by anti‐β‐D‐galactosidase antibodies, was reacted simultaneously with 2,4‐dinitrophenyl‐bovine serum albumin‐Cys‐env gp46 (188‐224) conjugate and Cys‐env gp46 (188‐224)‐β‐D‐galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the β‐D‐galactosidase conjugate, the complex was eluted from the polystyrene balls with ϵN‐2,4‐dinitrophenyl‐L‐lysine and transferred to polystyrene balls coated with affinity‐purified (anti‐human IgG‐γ‐chain) IgG. β‐D‐Galactosidase activity bound to the (anti‐human IgG‐γ‐chain) IgG‐coated polystyrene balls was assayed by fluorometry. This assay was more sensitive and useful than the immune complex transfer enzyme immunoassay using Cys‐Arg‐env gp46(188‐209) and other methods using HTLV‐I as antigen.
Tumor markers CA 19‐9 and CA 195 are also useful as markers for cystic fibrosis
Authors: Wu James T. , Olson June , Walker Kay
Published: March 2007

When monoclonal kits are used we can no longer detect highly elevated serum concentrations of carcinoembryonic antigen in cystic fibrosis (CF) patients as we could earlier (Pediatr Res 10:235–236, 1975). Instead, we find increased concentrations of CA 19‐9 or CA 195 in the CF sera. The serum levels of CA 19‐9 not only reflect the pulmonary condition of CF patients but also respond well to antibiotic therapy. Several lines of evidence suggest that the elevated serum concentration of CA 19‐9 is derived from sputum and corresponds with the amount of sputum in the lung. Correlations between CA 19‐9 and CA 195 in random and serial specimens from both patients with CF and patients with pancreatic carcinoma suggest that all sera contain heterogeneous, Lewis blood group‐related epitopes and the proportions of various epitopes are different among individual patients. When monitored on multiple tumor markers, the pattern of CF is different from that of pancreatic carcinoma although both usually show elevated CA 19‐9. Our study indicates that both CA 19‐9 and CA 195 can be used as sensitive markers for the early detection of exacerbation in CF patients. © 1992 Wiley‐Liss, Inc.
Novel and ultrasensitive noncompetitive enzyme immunoassay (hetero‐two‐site complex transfer enzyme immunoassay) for α‐human atrial natriuretic peptide
Authors: Hashida Seiichi , Ishikawa Eiji
Published: March 2007

A novel and ultrasensitive noncompetitive enzyme immunoassay (hetero‐two‐site complex transfer enzyme immunoassay) for α‐human atrial natriuretic peptide (α‐hANP) in plasma is described. α‐hANP was biotinylated using N‐hydroxysuccinimidobiotin and trapped onto an anti‐α‐hANP [6–28] IgG‐coated polystyrene ball. After washing, biotinylated α‐hANP was eluted from the polystyrene ball with HCI and was reacted with 2,4‐dinitrophenyl‐fluorescein‐bovine serum albumin‐disulfide‐rabbit anti‐α‐hANP [6–28] IgG conjugate. The complex formed was trapped onto (anti‐2,4‐dinitrophenyl group) IgG‐coated polystyrene balls and, after washing, reacted with avidin‐β‐D‐galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with ϵN‐2, 4‐dinitrophenyl‐L‐lysine and transferred to anti‐fluorescein IgG‐coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2‐mercaptoethylamine and transferred to (anti‐rabbit IgG) IgG‐coated polystyrene balls. β‐D‐Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of α‐hANP [1–28] was 3 fg (1 amol)/tube. Interference by plasma proteins was eliminated by separation of peptides from proteins using a molecular sieve. The assay range of plasma α‐hANP [1–28] was 0.04–120 ng/L, and plasma levels of hANP in healthy subjects (11–56 ng/L) were measured without concentration. © 1992 Wiley‐Liss, Inc.
Enzyme‐linked immunoadsorbent assay for the detection of cytomegalovirus‐IgM: Comparison between eight commercial kits, immunofluorescence, and immunoblotting
Authors: Lazzarotto T. , Casa B. Dalla , Campisi B. , Landini M. P.
Published: March 2007

Eight commercially available enzyme‐linked immunoadsorbent assays (ELISA) for the detection of cytomegalovirus (CMV)‐specific IgM were used in parallel to determine the presence of CMB‐IgM in 123 serum samples from pregnant women. The results obtained with the eight kits were compared. Based on concordance of six or more of the eight kits, we assessed sensitivity, specificity, and overall agreement, as well as incidence of false‐positive and ‐negative results for each kit. The results obtained by ELISA were then compared with those obtained by immunofluorescence (IF) and immunoblotting (IB).

Our study did not single out one outstanding ELISA kit among the eight evaluated, nor did it suggest that IF or IB are better than ELISA. Furthermore our results indicate that IB might be useful in several cases as, beside its good sensitivity, most IB‐false‐positive sera are easily recognized as reacting exclusively with pp 150, the unique reactivity to pp 150 not being among the IB profiles of IB‐true‐positive sera. Nevertheless 14.6% of sera remained CMV‐IgM‐indeterminate. © 1992 Wiley‐Liss, Inc.
User‐defined serum aspartate and alanine aminotransferase, cholesterol, triglycerides, urea, and uric acid for the Beckman synchron CX 4/5 using Ames Sera‐Pak reagents
Authors: Lolekha Porntip H. , Nitipaichit Pornpen
Published: March 2007

Beckman aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol, triglycerides, urea, and uric acid liquid Reagents for Synchron CX 4/5 (48, 48, 25, 60, 26, and 30 cents US/test, respectively) are expensive. We have established our own methods for serum AST, ALT, cholesterol, triglycerides, urea, and uric acid (6, 6, 5, 12, 13, and 6 cents US/test, respectively) using Ames Sera‐Pak reagents.

Linearity of our AST, ALT, cholesterol, triglycerides, urea, and uric acid methods were either similar to or higher than the Beckman mehtods. The within run and day‐to‐day run precisions were acceptable. Recovery of our AST, ALT, cholesterol, triglycerides, urea, and uric acid wee excellent. Our results for AST, ALT, cholesterol, triglycerides, urea, and uric acid correlated well with the Beckman results. Bilirubin (340.8 μmol/L) did not significantly interfere on our AST, ALT, cholesterol, triglycerides, and urea, while its concentrations of 165.8 μmol/L started giving negative interference on uric acid. Turbidity (2+) did not interfere significantly on our AST and ALT but started giving positive interference on cholesterol, triglycerides, urea, and uric acid. Hemolysis (2+) gave positive interference on our cholesterol, triglycerides, urea, and uric acid. Stability of Ames Sera‐Pak working reagents was at least 30 days for AST, ALT, urea, and uric acid and 40 days for cholesterol and triglycerides. © 1992 Wiley‐Liss, Inc.
Rapid screening method for polymorphism of group A apolipoproteins
Authors: Harake Bouchra , Caines Patrick S. M. , Thibert Roger J.
Published: March 2007

Polymorphism of apolipoproteins Al and All (ap Al and apo All) can be easily investigated in plasma by a simple method involving a 30‐min incubation of EDTA plasma in the presence of urea, dithiothreitol, and Nonidet P‐40 followed by subsequent isoelectric focusing (IEF). The sample (2 μL) was applied to an ultrathin flat acrylamide gel of pH range 4–6, and focused using a Bio‐Rad Mini IEF Cell for 1.5 h at a maximum of 500 V. Coomassie Blue R‐250 was used to visualize the apolipoproteins. To verify the identity of the different apolipoproteins aftr IEF, the gel was immunofixed directly with anti‐apo Al, or immunoblotted on polyvinylidene difluoride (PVDF) membrane using monospecific antibodies to apo Al and ap All and an antiimmunoglobulin‐alkaline phosphatase conjugate. High‐density lipoprotein (HDL) was used as a standard for Apo Al variants. Employing these techniques, human plasma apo Al was resolved into one major band (apo Al0 pl 5.54), and four minor bands identified as apo Al+2 (pl 5.75), apo Al+1 (pl 5.66), apo Al−1 (pl 5.45), and apo Al−2 (pl 5.34). Apo All was resolved into one major isoprotein designated as apo All+1 and apo All−1 which focused at pls of 5.18 and 4.58, respectively.

The results showed that these methods can be used to identify apo Al and All isoforms without prior ultracentrifugation to isolate the HDL. The entire procedure, including IEF, Fixation (chemical or immunofixaiton). and staining, can be accomplished in 5 h compared to 2 days using previously reported technique. The identification and characterization of human apolipoprotein Al and All isoforms is important in clinical practice, e.g., diagnosis of tangier disease, and may be useful in studying structure‐function relationships of these apoproteins. © 1992 Wiley‐Liss, Inc.
Serum magnesium, copper, and zinc concentrations in acute myocardial infarction
Authors: Tan I. K. , Chua K. S. , Toh A. K.
Published: March 2007

This study was carried out to assess the serum levels of magnesium, copper, and zinc after an acute episode of myocardial infarction. Determination of the metal concentrations were carried out on 41 patients with myocardial infarction and 41 healthy controls matched for age and sex. A slight decrease in the mean level of magnesium (P<.05) was observed in patients (2.0 mg/dl) compared with the controls (2.1 mg/dl). The mean serum copper concentratin was significantly higher (P<.001) in patients (138 μg/dl) than in controls (98 μg/dl), while the mean serum zinc concentration was significantly lower (P<.001) in patients (75 μg/dl) than in controls (100 μg/dl). The differences in serum copper and zinc levels between patients and controls were magnified considerably when the copper/zinc ratios were calculated for both groups (P<.001). The mean copper/zinc value obtained for patients (1.91) wa almost double that for the controls (1.02). © 1992 Wiley‐Liss, Inc.