bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzyme‐amplified receptor assay screening test for chlorpromazine, trifluoperazine, and phencyclidine
Authors: Hallowell Susan F. , Echnitz Garry A. R
Published: March 2007

A new receptor based assay is described for the determination of classes of drugs which have high affinities for the acetylcholine receptor. The method is based upon the inhibition of the enzyme activity of an enzyme‐drug conjugate by the binding to the receptor protein, and competition between free drugs and the enzyme‐drug conjugate for a limited number of receptor sites. The activity of the enzyme marker system, glucose‐6‐phosphate dehy‐drogenase covalently conjugated to desipra‐mine, is monitored by colorimetric detection of the rate of NADH formation at 340 nM. The procedure proposed is designed to provide a simple drug screen which can be done in a minimally equipped laboratory while achieving the required sensitivity. The technique is illustrated for three acetylcholine channel binding compounds: the hallucinogen phencyclidine (PCP) and the antipsychotic agents chlorpromazine and trifluoperazine. This procedure yields calibration curves with detection limits at nanomolar levels of drug, with binding responses dependent on the amounts of receptor and enzyme‐labeled drug used. Aspecific binding responses of unlabeled enzyme to drug or receptor to compounds with low affinity for the receptor are shown to have minimal effect on the assay.
Detection of antibodies against avian antigens in bronchoalveolar lavage from patients with pigeon breeder's disease: Usefulness of enzyme‐linked immunosorbent assay and enzyme immunotransfer blotting
Authors: Sandoval J. , Bañales J. L. , Cortés J. J. , Reyes P. A. , Mendoza F. , Selman M.
Published: March 2007

The study reported here evaluated the usefulness of the enzyme‐linked immunosorbent assay (ELISA) in the detection of antibodies against pigeon antigens in the serum and bronchoalveolar lavage (BAL) of patients with clinical, radiological, and functional evidence of interstitial lung disease (ILD) with and without pigeon breeder's disease (PBD). The results were compared with those obtained by the simultaneous use of counterimmuno‐electrophoresis (CIE) in the same patients. In PBD, ELISA detected antibodies against pigeon's sera in both serum and BAL in 100% of patients, while CIE failed to detect the anti‐bodies in the serum of one patient and in most of the samples of BAL. In addition, we used enzyme immunotransfer blotting to determine the number of epitopes in pigeon serum recognized by antibodies present in serum and BAL. There was a heterogeneous response in both fluids, but the reaction pattern demonstrated that patients' sera recognize to—25 different pigeon epitopes. We conclude that ELISA is a highly sensitive and specific method for the detection of antibodies against pigeon antigens in the serum and BAL of patients with PBD and that the host response involves a great number of avian antigens.
Soluble interleukin‐2 receptor: Elevated levels in serum and synovial fluid of patients with rheumatoid arthritis
Authors: Carpenter A. B. , Eisenbeis, Jr. C. H. , Carrabis S. , Brown M. C. , Ip S. H.
Published: March 2007

Soluble interleukin‐2 receptor (sIL‐2R) levels were quantitated in the seum and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and degenerative joint disease (DJD). A sandwich immunoassay, employing two monoclonal antiodies against distinct epitopes on the IL‐2R, was utilized for measurement. We found a striking elevation of sIL‐2R in RA SF as compared with DJD SF (RA, 1319 ± 135; DJD, 416 ± 59; p < 0.001). RA serum sIL‐2R levels were also significantly elevated over DJD levels. There was no interaction between rheumatoid factor (RF) and sIL‐2R. RA patients with elevated sIL‐2R levels had significantly longer disease duration, higher c‐reactive protein (CRP) levels in serum and SF, and higher RF levels in serum and SF. The groups were similar in regard to other laboratory variables. The presence of elevated levels of sIL‐2R in RA serum and SF confirms the presence of a heightened immune reactivity and in vivo activation of lymphocytes in RA.
Oligoclonal immunoglobulins in serum of patients with chronic viral hepatitis
Authors: Nicholas M Rea , Papadopoulos A. , Tsianos Epaminondas V. , Costello Rene
Published: March 2007

Zone electrophoresis on agarose gel was performed in serum samples from patients with chronic viral hepatitis. On the basis of the clinical, serological, and biochemical evaluation, the patients were divided into three categories: chronic type B hepatitis; chronic type delta hepatitis; and chronic non‐A‐non‐B (NANB) hepatitis. The results of the electrophoretic analysis showed a high incidence of oligoclonal immunoglobulin bands (OIBs) in patients with chronic B hepatitis and chronic delta hepatitis and a low incidence of OIBs in patients with NANB. Since other biochemical and serological characteristics are variable and do not correlate with clinical symptoms of disease, we suggest that oligoclonal immunoglobulins could be used as a marker to aid in studies of the natural history and pathologic course of the various types of chronic hepatitis.
Novel and sensitive enzyme immunoassay (immune‐complex‐transfer enzyme immunoassay) for alpha‐fetoprotein from hepatocellular carcinoma
Authors: Kohno Takeyuki , Ishikawa Eiji , Kitamura Tooru , Tsuda Kazunori
Published: March 2007

Alpha‐fetoprotein in sera from patients with hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin‐Sepharose 4B. One peak (the first peak), which passed through the column without adsorption, was found in both healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma. The second and third peaks were reactive with L. culinaris agglutinin and found only in patients with hepatocellular carcinoma. For α‐fetoprotein in the second and third peaks, a novel and sensitive enzyme immunoassay (immune‐complex‐transfer enzyme immunoassay) was developed. Alpha‐fetoprotein in test serum was reacted with dinitrophenyl affinity‐purified anti‐α‐fetoprotein IgG, and the complex formed was trapped onto affinity‐purified (antidinitrophenyl bovine serum albumin) IgG‐coated polystyrene balls. The polystyrene balls were washed to eliminate substance(s) other than a‐fetoprotein in the test serum, and the complex was eluted from the polystyrene balls with dinitrophenyl‐L‐lysine. The eluted complex containing α‐fetoprotein in the second and third peaks was trapped onto L. culinaris agglutinin‐coated polystyrene balls and reacted with affinity‐purified anti‐α‐fetoprotein Fab'‐β‐D‐galactosidase conjugate. Beta‐D‐galactosidase activity bound to the polystyrene balls was assayed by fluorimetry. The maximal volume of serum that could be used without interference was 20μl, which was 100‐fold larger than that in the previous enzyme immunoassay. As a result, the minimal detectable serum concentrations of α‐fetoprotein in the second and third peaks were 0.1 mg/L and 3.5 μg/L, respectively, which were 35‐ and 29‐fold lower than those obtained by the previous enzyme immunoassay. It was possible to confirm the presence of α‐fetoprotein in the second and third peaks by a significant difference between bound β‐D‐galactosidase activities in the absence and presence of α‐methyl‐D‐mannoside or D‐glucose. This assay may be more useful for diagnosis of hepatocellular carcinoma than was the previous enzyme immunoassay. However, the maximal serum concentration of a‐fetoprotein in the first peak that did not affect the detection limits of α‐fetoprotein in the second and third peaks was 0.35 mg/L, which was also 29‐fold lower than that obtained by the previous enzyme immunoassay. The detection limits of a‐fetoprotein in the second and third peaks remain to be lowered without lowering the maximal serum concentration of α‐fetoprotein in the first peak that does not affect the detection limit of serum α‐fetoprotein in the second and third peaks.
Novel and sensitive noncompetitive (two‐site) enzyme immunoassay for haptens with amino groups
Authors: Tanaka Koichiro , Kohno Takeyuki , Hashida Seiichi , Lshikawa Eiji
Published: March 2007

A novel and sensitive noncompetitive (two‐site) enzyme immunoassay for haptens with amino groups is described. L‐Thyroxine (T4) was used as a model hapten. T4 was indirectly biotinylated with glutathione as spacer between T4 and biotin molecules and trapped onto anti‐(T4‐bovine serum albumin) IgG‐coated polystyrene balls. After washing the polystyrene balls to eliminate unreacted biotin and other biotinylated substances, biotinylated T4 was eluted from the polystyrene balls with HCI and was reacted with anti‐(T4‐bovine serum albumin) Fab'‐horseradish peroxidase conjugate. The complex formed was trapped onto streptavidin‐coated polystyrene balls.

Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. The detection limit of T4 was 78 fg (0.1 fmol)/tube, which was 50‐fold lower than the limit by competitive enzyme immunoassay using the same antibody and T4‐peroxidase conjugate. This noncompetitive enzyme immunoassay was applied to the measurement of total T4 in serum. Serum levels of total T4 in 10 healthy subjects aged 25–40 yr were 94 ± 13 (SD) μg/L (range, 78–114). The principle of this noncompetitive enzyme immunoassay may allow it to measure other haptens with amino groups more sensitively than can competitive immunoassays.
Sensitive time‐resolved fluorimetric immune‐complex‐transfer immunoassay for antithyroglobulin igg in serum
Authors: Kohno Takeyuki , Ishikawa Eiji , Mitsukawa Tomohiro , Matsukura Shigeru
Published: March 2007

A sensitive time‐resolved fluorimetric immune‐complex‐transfer immunoassay for antithyroglobulin IgG in serum is described. Antithyroglobulin IgG in test serum was reacted with dinitrophenyl europium ion‐labeled thyroglobulin. The complex formed of antithyroglobulin IgG and dinitrophenyl europium ion‐labeled thyroglobulin 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 antihuman IgG y‐chain IgG. Europium ion bound to the polystyrene balls was measured by time‐resolved fluorimetry. Antithyroglobulin IgG was demonstrated in all patients with Graves' disease and all patients with chronic thyroiditis. This immunoassay was more sensitive than the conventional enzyme immunoassay and less time‐consuming than the previously described immune‐complex‐transfer enzyme immunoassays, although there were larger assay variations.
Cardiolipin liposomes: A novel flow reagent for detection of anticardiolipin antibodies
Authors: Pirruccello Samuel J , Pirruccello Samuel J , Brusnahan Susan K.
Published: March 2007

The association of autoantibodies with specificity for phospholipids and an increased risk for thromboembolic phenomena has received considerable recent clinical attention. These autoantibodies have been reported in patients with defined autoimmune disorders as well as in patients with no other obvious autoimmune disease symptoms other than isolated or recurrent thromboembolic disease. A significant component of this autoimmune response appears to be related to cardiolipin‐directed antibodies. Most studies reported to date have used either an enzyme immunoassay or a radioimmunoassay for detection and quantitation of antiphospholipid antibodies. We have developed a novel flow cytometric assay for detection of anticardiolipin antibodies. The assay, by analogy to polystyrene microsphere assay, utilizes cardiolipin liposomes as solid‐phase microspheres for antigen presentation. In comparison to enzyme‐linked immunosorbent assay, the flow assay shows similar sensitivity by serum titration, has immunoglobulin class specificity, and is semiquantitative as currently designed. The flow assay is relatively easy to perform and should allow detection of other antiphospholipid specificities with tailoring of the phospholipid makeup of the liposomes.
A rapid turbidimetric immunoassay for serum antistreptolysin‐o2
Authors: Otsuji Shogo , Kamada Tetsuro , Matsuura Tsuneaki , Seki Masashi , Tanaka Kiko , Shibata Hideaki , Honda Tetsuro
Published: March 2007

We designed a rapid, quantitative turbidimetric immunoassay for serum antistreptolysin‐O (ASO), based on the immunoprecipitating velocity with purified streptolysin‐O (SLO), which can be measured turbidimetrically at 340 nm. The assay system involves polyoxy‐ethylene mono‐p‐nonylphenyl ether to accelerate and enhance the immunoprecipitation reaction. No antibody excess phenomenon is observed, up to 1,700 U/ml. Grossly icteric sera can be assayed. Patient sera gave acceptable results. Free cholesterol offers no interference. The standard curve is linear for values up to 800 U/ml. Results measured by the proposed method and those by the conventional microtitration method correlated well. Analytical recovery averaged 90.1% (80.3%‐102.5%). Within‐run and day‐to‐day CVs ranged from 2.1% to 3.5% and from 2.5% to 6.9% respectively. Working SLO is stable for at least 15 days at 4°C. The proposed method has proven to be superior to other available methods for measuring serum ASO.
A dot‐elisa intended for the specific and simultaneous detection of antibodies directed to antigens derived from toxoplasma gondii, rubella virus, cytomegalovirus, and type 1 and type 2 herpesviruses
Authors: Leonardi Maria S. , Zummo Sebastiana , Mastroeni Pasquale , Fattal‐German Michéle , Bizzini Bernard
Published: March 2007

A procedure for the routine and simultaneous laboratory detection of IgG antibodies produced in humans in the course of various infectious diseases is described. The procedure, based on dot‐enzyme‐linked immunosorbent assay (ELISA), used single nitrocellulose strips onto which several antigens were dotted in close proximity. Optimal conditions were specified that allowed the unequivocal and simultaneous detection of IgG antibodies specifically directed against Toxopfasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus type 1 and type 2 antigens. This technique has proved to be simultaneously specific, sensitive, and reliable, and it has been applied to prenatal screening of sera from pregnant women. It is suggested that this technique should also be used for the screening of large numbers of sera under field trial conditions.
Comparative study with in situ hybridization and immunocytochemistry in detection of hiv‐1 in formalin‐fixed paraffin‐embedded cell cultures
Authors: Daugharty Harry , Long Earl G. , Swisher Billie L. , Warfield Donna T. , Feorino Paul M.
Published: March 2007

The sensitivities of three immunohistological techniques were compared in this study for detecting human immunodeficiency virus (HIV‐1) in infected cultured human lymphocytes that had been formalin‐fixed and paraffin‐embedded. The techniques included in situ hybridization (ISH) with HIV‐1 cDNA; immunocytochemistry with HIV‐1 p24 monoclonal antibody (ICC‐m); and immunocytochemistry with HIV‐1 polyclonal antibody from a patient with acquired immunodeficiency syndrome (AIDS) (ICC‐p). Procedures were optimized for enzyme digestion and for antibody reaction conditions. HIV‐1 ‐infected cells and noninfected control cells were tested. Noninfected controls were uniformally negative by all three methods. Infected cells had the highest positivity rate by the ISH method (p ≤ 0.0001), and the ICC‐p method was more positive than the ICC‐m (p ≤ 0.0001). Both the ICC‐p and the ICC‐m techniques were more positive with the cocultivated cell cultures than the ISH, which was more sensitive with the infected continuous cell line (P ≤ 0. 0001). The ICC‐p method had a lower standard deviation on positive results than either the ICC‐m or ISH method. The variability observed with these test procedures, reagents, and specimens suggests that these are important technological parameters in detecting p24, with implications for detecting other HIV‐1 markers in infected tissues.
Current status of clinical laboratory tests for the human immunodeficiency virus
Authors: Nakamura Robert M. , Bylund David J. , Rooney Kathleen E.
Published: March 2007

The predictive values of positive and negative test results for human immunodeficiency virus (HIV) antibody are extremely high in laboratories that have good quality control and high performance standards and use licensed FDA‐approved enzyme immunoassay (EIA) and Western blot standardized tests. With a carefully designed protocol, the false‐positive rate of combined EIA and Western blot has been reported to be as low as 1 in 105. When results of HIV confirmatory antibody tests are indeterminate, other tests such as culture and nucleotide probe methods for HIV DNA or RNA may help resolve false‐reactive screening EIA tests. Improvements are constantly in progress for HIV laboratory tests with the use of recombinant DNA‐derived antigens and synthetic polypeptides. With the use of new‐generation synthetic polypeptide antigens, specific assays to identify HIV‐1 and HIV‐2 have been developed. Recently, assays for the HIV regulatory gene products have been studied for their predictive potential. Antibodies to nef protein, a regulator of HIV‐1 replication, may be an early indicator of HIV infection.
Screening antisera for subtype specificity based on immunoblotting of lectin‐affinity electrophoresis: Application toward alpha‐fetoprotein subfractions
Authors: Jone Cyrenius M. , Wu Alan H. B. , Spillman Thomas , Fritsche Herbert A.
Published: March 2007

We adapted a method for evaluating monoclonal antibody specificity toward isoforms and examined 29 alpha‐fetoprotein (AFP) antibodies from Hybritech. These antibodies were separated by affinity electrophoresis on agarose‐containing erythroagglutinating phytohemagglutinin (PHA‐E) followed by immunoblotting onto nitrocellulose. Under these conditions, AFP separates into four subfractions: NR (nonreactive with PHA‐E), WR (reactive weakly), RS2 (strongly reactive), and RS1 (very strongly reactive). When polyclonal goat anti‐AFP control was used as the test antibody, the distribution of amniotic fluid AFP was approximately 50°h, 31%, 12%, and 7% for NR, WR, RS2, and RS1, respectively. The distribution for these same bands from the serum of a patient with hepatocellular carcinoma was 39%, 31 %, 30%, and 0%; from the serum of a patient with embryonal carcinoma, it was 29%, 38%, 0%, and 33%, respectively. Twenty‐six monoclonal antibodies, including the two used in the Hybritech Tandem‐E assay for AFP, showed similar distribution, while the remaining three antibodies showed no reactivity to any of the AFP antigens in this system. Although we were not able to demonstrate any specificity of these anti‐sera toward any single AFP isoform, use of this affinity electrophoresis system provides a model for studying isoform specificity of other AFP antibodies and other antibody‐antigen systems.
Report of the symposium on the use of intravenous gammaglobulin in adults infected with the human immunodeficiency virus
Authors: De Simone C. , Di Fabio S. , Antonaci S. , Chirigos M. , Delia S. , Good R. A. , Hadden J. W. , Lockey R. , Jirillo E. , Milazzo F. , Scalise G. , Tinelli M.
Published: March 2007

On July 27, 1989, the International Conference on Molecular Aspects of Immune Response and Infectious Diseases devoted a symposium to the subject of the use of intravenous gamma globulin (IVIG) in acquired immunodeficiency syndrome (AIDS). The information presented confirmed that IVIG benefits human immunodeficiency virus (HIV)‐infected children with recurrent infections and that much remains to be learned about the influence of IVIG in adult AIDS. The symposium participants recognized the urgent need to develop randomized clinical trials using a control group to assess the efficacy of a treatment with IVIG in PGL (persistent generalized lymphadenopathy), ARC (AIDS‐related complex), and AIDS. To prepare this report, a committee was established, including individuals with expertise in immunology, immunopharmacology, microbiology, virology, infectious diseases, general medicine, and pediatrics and representing research experience in academia and hospitals. After an introduction to the report with a summary of immunotherapeutic agents under evaluation to treat HIV infection, section 1 lays out the present understanding of the disease pathogenesis. Section 2 then outlines the treatment of HIV‐seropositive individuals, discussing the uncertainties that any treatment entails. Section 3 discusses the rationale for treating HIV‐infected individuals with WIG, and Section 4 examines the major differences between IVIG and hyperimmuneglobulins for the treatment of HIV infection. Section 5 looks at IVIG as a mean to delay the emergence of opportunistic infections and restore immunocompetence in AIDS and related illnesses, and Sections 6 and 7 suggest a pilot protocol on the use of WIG in association with low‐dose or standard‐dose zidovudine (AZT). Limited time precluded the committee's addressing certain important aspects, and it must be remembered that, since this report represents the committee evaluations as of July 1989, the use of WIG in adults infected with HIV should be studied on a continuous basis according to more up‐todate knowledge of HIV infection.
Optimal conditions for protease use in the assay of serum mitochondrial aspartate aminotransferase
Authors: Okabe Hiroaki , Uji Yoshinori , Sugiuchi Hiroyuki , Watazu Yoshifumi , Shirahase Yasushi , Kaneda Nobuaki
Published: March 2007

The optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase (c‐AST) to determine mitochondrial aspartate aminotransferase (m‐AST) in serum were studied. Protease 401 was found to be effective over a pH range of 6.0‐10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of c‐AST and preservation of m‐AST, lactic dehydrogenase (LDH), and malic dehydrogenase (MDH) in the assay procedure. The presence of serum endogenous protein inhibitors such as aα1‐antitrypsin and α2‐macroglobin did not inhibit protease 401.
Human α1‐microglobulin: Its measurement and clinical significance
Authors: Itoh Yoshihisa , Kawai Tadashi
Published: March 2007

α1‐Microglobulin (α1‐M), also called protein HC, is a low‐molecular‐weight (LMW) glycoprotein (about 30 kDa) with unique physicochemical properties. Using purified urinary α1‐M as a standard and specific antibody against α1‐M, an assay system for α1‐M was developed, and the clinical significance of this protein was investigated by measuring total levels of α1‐M under physiological and pathological conditions. α1‐M is distributed in various body fluids: in serum, it consists mainly of free LMW α1‐M and monomeric IgA–α1‐M complex. The total α1‐M level in serum and urine usually reflects LMW α1‐M variation sensitively, and its determination is quite useful as an indicator of renal glomerulotubular dysfunction and hepatic dysfunction. Serum levels can vary, depending on IgA‐α1‐M complex level, in parallel with the IgA concentration. The heterogeneity of α1‐M purified from different sources of urine by different procedures and underestimation of IgA‐α1‐M complex by solid‐phase antibody assays can be important causes for the discrepancy of serum levels between assays.
Alloalbumin in cerebrospinal fluid: Report of a case and remarks on physiopathology
Authors: Banfi Giuseppe , Marinelli Marcello , Comi Giancarlo , Martinelli Vittorio , Bonini Pierangelo
Published: March 2007

Alloalbumin Tagliacozzo(313Lys→ Asn) was detected in the cerebrospinal fluid of a 27‐yr‐old woman. We studied this rare finding by means of high‐resolution electrophoresis, isoelectric focusing, and immunofixation. We herein discuss the importance of molecular mass versus superficial electrical charge in the transfer of plasma proteins through the blood‐brain barrier.
Effect of azidothymidine on soluble cd4 levels in patients with aids or aids‐related complex
Authors: Reddy Mohan M. , Grieco Michael H. , Vodian Morton
Published: March 2007

Circulating soluble CD4 (sCD4) levels were measured in 20 patients (11 with the acquired immunodeficiency syndrome [AIDS] and 9 with AIDS‐related complex [ARC]) treated with azidothymidine (AZT) and in 12 patients (nine with AIDS and three with ARC) who were in the placebo group. The mean CD4 level in the AZT treatment group at baseline was 41 ± 12 (SEM) U/ml. After 4 wk of treatment, the mean level decreased to 23 ± 5; it was29 ± 10,31 ± 14, and 21 ± 5 at 8,12, and 16 wk of therapy, respectively. No significant changes were observed in the placebo group. These results suggest that AZT administration may be associated with reduced sCD4 levels in these patients.
Experimental approach to the study of immune function in children with possible human immunodeficiency virus infection
Authors: Cunningham‐Rundles S. , Yeger‐Arbitman R. , Edelson P. , Sanders D. , Giardina P. V. , Hilgartner M. W.
Published: March 2007

The clinical immunology laboratory is often called on to assess risk in pediatric patients with clinical evidence of immunodeficiency and possible human immunodeficiency virus (HIV) exposure while antibody tests are being considered or are underway. Since non–HIV‐related conditions including neoplasia, certain viruses, and primary immunodeficiency can potentially produce lymphocyte subset imbalance and functional impairment, there is need for a laboratory approach to differential analysis of pediatric immunodeficiency. In addition, laboratory methods may also influence the results obtained. In order to investigate these issues, we screened pediatric patients with and without HIV exposure. Altered lymphocyte subset expression and function were found among non‐HIV‐infected pediatric patients. The use of percentage and absolute lymphocyte numbers was found to affect the results obtained in a significant manner. Some patients who were chronic blood transfusion recipients were found to have blocking factors, presumably alloantibodies, in serum, which affected detection of lymphocyte surface antigens. In this population, age at seroconversion was a factor influencing subsequent levels of CD4 + T lymphocytes. Significant differences in CD4+ T lymphocyte percentages were also observed in children congenitally exposed to HIV compared with controls, even among those children with CD4/CD8 ratios greater than 1.0, who therefore had possibly escaped infection. Immune changes in children should be interpreted with caution.
Diagnostic value of measuring pancreatic isoamylase with a double–monoclonal antibody immunoassay in serum of hospitalized hyperamylasemic patients
Authors: Panteghini Mauro , Pagani Franca
Published: March 2007

We evaluated the diagnostic utility of measuring pancreatic isoamylase (P‐AMY) with a double‐monoclonal antibody technique in a population of 43 consecutive hospitalized hyperamylasemic patients in comparison with serum pancreatic lipase (LPS) activity. In 27 cases (62.8%), the final diagnosis was acute pancreatitis. Predictive values were calculated for P‐AMY and LPS activities, and a P‐AMY percentage was calculated for selected decision levels. The maximal diagnostic efficiency was 0.930, 0.814, and 0.767 for LPS, P‐AMY activity, and P‐AMY percentage, respectively, indicating that serum LPS measurement was clinically superior to P‐AMY for distinguishing patients with or without pancreatitis. Measurement of both P‐AMY activity and percentage in serum did not significantly improve diagnostic accuracy.
Identical igm antibodies recognizing a glycine‐alanine epitope are induced during acute infection with epstein‐barr virus and cytomegalovirus
Authors: Rhodes Gary , Rubin Ruth E. , Vaughan John , Smith Richard S. , Horwitz Charles A.
Published: March 2007

We studied antibody production in serial serum samples from patients with acute Epstein‐Barr virus (EBV) and cytomegalovirus (CMV) infections. Sera were analyzed both by enzyme‐linked immunosorbent assay (ELISA) using a synthetic peptide (P62) derived from the glycine‐alanine repeating region of the Epstein‐Barr nuclear antigen (EBNA‐1) and by immunoblotting. In prior studies, we have shown that patients with acute EBV infection make IgM antibodies that react with this peptide, that recognize a viral‐specific protein (EBNA‐l), and that bind with a number of proteins present in uninfected cells; however, antibody binding to these autoantigens was inhibited by the peptide. IgG antibodies reactive with the peptide did not appear until months after the disease and were specific for the EBNA‐1 protein. We now find that patients with acute CMV infection but not those with acute infections from a variety of other nonherpes organisms also produce IgM antibodies that recognize the EBV‐derived peptide P62. These antibodies also appear to recognize the same cellular proteins as the EBV‐induced IgM antibodies. The IgM antibodies appeared in all acutely infected CMV patients studied and occurred both in patients with previous EBV infections and in one patient studied who had not previously been exposed to EBV. It appears that infection with EBV or CMV can induce the synthesis of a very similar or identical set of IgM antibodies.
Semi‐Automated fluorometric assay for urinary total and b n‐acetyl‐β‐d‐glucosaminidase: Analytical investigation
Authors: Kristz William , Samaan Ghassan Joseph , Leberre Corinne , Demelier Jean‐François , Biou Daniel
Published: March 2007

Total N‐acetyl‐β‐D‐glucosaminidase (NAG) and NAG‐B isoenzyme determination are determined in order to investigate renal tubular function. Here, we propose a semi‐automated adaptation of Sandman's manual method for the Monarch centrifugal analyzer. Fluorescence of the released 4‐methylumbelliferone is automatically read and results are directly expressed in nanokatal/l. Mean (1 SD) values expressed in nanokatal/millimole creatinine in urine from healthy female (n = 30) and male (n = 30) subjects were 8.2 (4.0) and 7.8 (2.9) for total NAG and 1.9 (1.4) and 1.5 (0.7) for the NAG‐B isoenzyme activity. The assay of six patients with various renal disorders shows definite increase in total NAG and NAG‐B isoenzyme activities.
Human melanoma metastasis culture supernatant contains chemotactic factors for phagocytes
Authors: Ventura M. T. , Cerri A. , Jirillo E. , Antonaci S. , Bonomo L.
Published: March 2007

Several findings point out that melanoma culture supernatants release soluble factors which modulate mononuclear cell chemotactic responsiveness. In this regard, either an enhancement or an impairment of chemotactic capacity has been found. Here, we provide evidence that supernatant recovered from human melanoma cell metastasis culture is able to trigger monocyte and polymorphonuclear cell chemotaxis. This activity is not affected by the presence of anti‐IL‐1 antibody during the assay. Further studies are in progress to isolate and characterize soluble factors involved in this activity.
State‐Of‐The‐Art of zidovudine monitoring
Authors: Stretcher Brian N.
Published: March 2007

Zidovudine, the only drug approved by the FDA for the treatment of AIDS, has well‐documented efficacy, but numerous toxic side effects. Despite considerable knowledge of the pharmacology of the drug, however, a therapeutic concentration range has not been established. This article reviews methodologies used for measurement of zidovudine in serum. Procedures for several methods are described and comparisons offered. In addition to methodologies, other issues of concern in establishing or maintaining a zidovudine monitoring service are also discussed, including the clinical relevance of routine zidovudine monitoring, method‐related quality control, and laboratory safety. Methodologies currently being developed to measure the active intracellular metabolites of zidovudine are also described. These methods are believed to be the key to fully understanding the relationships between zidovudine pharmacology, toxicity, and efficacy.
Phagocytic cell chemiluminescence using different zymosan preparations
Authors: Gridley Daila S. , Lau Benjamin H. S. , Tosk Jeffrey M.
Published: March 2007

This study compared the effectiveness of opsonized and unopsonized zymosan prepared in our laboratories with a commercially available opsonized preparation used for induction of luminol‐dependent oxidative burst in phagocytic cells. The production of chemiluminescence (CL) by human whole blood, isolated human neutrophils, normal BALB/c mouse splenocytes, and an immortal BALB/c mouse macrophage cell line (J774A. 1) was tested in an automated luminometer. Recombinant murine or human interferon‐γ(IFN‐γ) and lipopolysaccharide (LPS) were used as priming agents in some of the experiments. With human leukocytes and normal mouse spleen cells the laboratory‐prepared zymosans (regardless of opsonization) induced equal or significantly greater CL than did the commercially prepared zymosan. In addition, greatly increased CL was evident with IFN‐γ‐ and LPS‐primed neutrophils tested with our zymosans compared with the commercial preparation. These results suggest that effective zymosans capable of inducing strong, reproducible CL responses from several different phagocytic cell populations can be readily made in the laboratory.
Altered steady‐state levels of mrna coding for extracellular matrices in renal tissues of ddy mice, an animal model for iga nephropathy
Authors: Tomino Yasuhiko , Nakamura Tsukasa , Ebihara Isao , Funabiki Kazuhiko , Yaguchi Yutaka , Shimizu Masahiko , Shirato Isao , Koide Hikaru
Published: March 2007

Correlations between the steady‐state mRNA levels of extracellular matrices using specific cDNA probes for the α1 chain of type IV collagen (α1 (IV) chain); laminin A, B1, and B2 chains; and heparan sulfate proteoglycan (HSPG); and glomerular injuries in ddY mice were evaluated. Eight‐, sixteen‐ and forty‐week‐old ddY mice were used in this study. ICR mice of the same age served as control. Extracted total RNA of pooled kidneys was fixed on a filter and then hybridized with the cDNA probes. Renal cryostat sections were incubated with rabbit anti‐mouse type IV collagen, laminin, and HSPG antisera and then stained with FITC‐labeled goat anti‐rabbit IgG antiserum. The sections were also stained with FITC‐labeled goat anti‐mouse IgA, IgM, IgG, and C3 antisera. In light microscopy, the average number of glomerular cells was calculated at each age. Increased expression of extracellular matrices genes for the α1(IV) chain; laminin A, B1, and B2 chains; and HSPG was found in renal tissues of ddY mice. Staining of type IV collagen, laminin, and HSPG was observed in renal tissues of ddY mice at each age. Increased proteinuria in 40‐week‐old ddY mice might be related to the decrease in glomerular basement membrane HSPG which acts as the anionic sites in such areas. Marked proliferation and or expansion of glomerular cells and mesangial matrices were observed in 40 week‐old‐ddY mice. The intensity of IgA and C3 deposits in the glomeruli was parallel to the levels of mRNA for such components. It appears that increased mRNA levels for such matrices coincided with the development of renal injuries in ddY mice. It is concluded that evaluation of steady‐state mRNA levels of the extracellular matrices in renal tissues of ddY mice is useful in determining progressive mechanisms in patients with IgA nephropathy.
Molecular forms of soluble human t lymphocyte receptor for sheep erythrocytes in serum and saliva
Authors: Itano Eiko N. , Ono Mario A. , Sumigawa Mari , Longo Ieda M. , Moura Nayla C. , Mendes Nelson F.
Published: March 2007

Using a specific serum anti‐soluble T lymphocytes receptor for sheep erythrocytes (E) and SDS‐PAGE, we detected radioactive bands of molecular weight 58,000 in immunoprecipitates of supernatant of heated human lymphocytes (SHL), in the supernatant of PHA stimulated lymphocyte cultures (SLC), normal human serum (NHS), and serum from cancer and uremia patients, labelled with 131I. By Sephadex G‐200 chromatography, in addition to this fraction, we detected molecules of molecular weight higher than 150,000 which interact with the anti‐soluble receptor serum (anti‐Rs), in serum from cancer and uremia patients. These molecules were detected in NHS or SHL after concentration or by prolonged exposure of SDS‐PAGE with some labelled and immunoprecipitated SHL samples.

The soluble receptors of molecular weights 58,000 (Rs1) and more than 150,000 (Rs2) were fully identical when analyzed by immunodiffusion with anti‐Rs serum. When submitted to immunoelectrophoresis, Rs1 showed electrophoretic migration similar to that of albumin, while Rs2 showed a pattern close to that of alpha2‐globulin. However, Rs2 did not show antigenic relationship with IgM and was not an immune complex with IgG. Even though the presence of Rs in human saliva has not yet been reported, molecules that interact with anti‐Rs serum have been detected in human saliva and are fully identical to molecules found in supernatant of heated human T lymphocytes and NHS. The Rs molecules present in human saliva have a molecular weight and electrophoretic migration similar to those of Rs 1 from SLC and from human serum and have no antigenic relationship with human albumin.
Use of inactive β‐d‐galactosidase for elimination of interference by anti‐β‐d‐galactosidase antibodies in immune complex transfer enzyme immunoassay for anti‐thyroglobulin igg in serum using β‐d‐galactosidase from escherichia coli as label
Authors: Kohno Takeyuki , Ishikawa Eiji , Katsumaru Hiroyuki , Nakamoto Hidetaka , Yasuda Taeko , Mitsukawa Tomohiro , Matsukura Shigeru , Tsunetoshi Yoshizo
Published: March 2007

A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti‐thyroglobulin IgG using β‐D‐galactosidase from Escherichia coli label was reported previously. This immunoassay was highly sensitive in demonstrating anti‐thyroglobulin IgG not only in all patients with Graves' disease and chronic thyroiditis but also in a large proportion of healthy subjects. However, the detection of anti‐thyroglobulin IgG at low levels in some serum samples was difficult, probably due to the presence of anti‐β‐D‐galactosidase antibodies. In the present study, the use of inactive β‐D‐galactosidase was tested for elimination of interference by anti‐β‐D‐galactosidase antibodies. Preincubation of serum samples with excess of inactive β‐D‐galactosidase resulted in sufficiently low backgrounds to detect low levels of antithyroglobulin IgG with little effect on the dose‐response of anti‐thyroglobulin IgG. As a result, it was revealed that anti‐thyroglobulin IgG was present in almost all healthy subjects as well as all patients with Graves' disease and chronic thyroiditis.
Importance of detection of ss‐a/ro autoantibody in screening immunofluorescence tests for autoantibodies to nuclear antigens
Authors: Bylund David J. , Nakamura Robert M.
Published: March 2007

SS‐A/Ro autoantibodies are related to Sjögren's syndrome and to several clinical subsets of lupus erythematosus. The most widely used laboratory procedure for ANA screening is the indirect immunofluorescence test (IF‐ANA); the laboratory detection of anti‐SS‐A/Ro requires implementation and adherence to several technical and quality assurance recommendations. Using appropriate substrate cells containing the SS‐A/Ro antigen, many so‐called “ANA‐negative” lupus erythematosus patients will demonstrate a positive IF‐ANA.
Validation of an immunoradiometric assay to measure plasma levels of active renin
Authors: Wathen Lynne K. , Nuorala Kenneth W. , Wathen Michael W.
Published: March 2007

An immunoradiometric assay (IRMA) for active renin in human plasma was analytically and clinically validated. Analytical validation established (1) precision, (2) recovery, (3) linearity, (4) cross‐reactivity, (5) sample stability, and (6) the validity and specificity of the 125l‐labeled anti‐renin monoclonal in the Diagnostics Pasteur immunoradiometric renin kit. Clinical validation included (1) establishing a normal reference range for renin, (2) comparing plasma renin activity (PRA) results to immunoreactive renin levels in subjects on Upjohn research protocols, and (3) comparing the renin responsiveness of sodium replete subjects to that of sodium deplete subjects prior to, during, and after infusion with Upjohn renin inhibitory peptide, ditekiren. This study was undertaken to demonstrate the research validity of an assay tool for the differentiation of enzymatically active renin from inactive renin or a form of prorenin.
Immune complex transfer enzyme immunoassay for (anti‐human t‐cell leukemia virus type i) igg in serum using a synthetic peptide, cys‐gag p19(100–130), as antigen
Authors: Kohno Takeyuki , Ishikawa Eiji , Sakoda Iwane , Suzuki Mika , Izumi Atushi
Published: March 2007

An 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‐gag p19(100–130), is described. Anti‐HTLV‐IIgG 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‐gag p19(100–130) conjugate and cys‐gag p19(100–130)‐β‐D‐galactosidase conjugate. The complex formed 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 100‐fold more sensitive than the conventional enyme immunoassay, in which a cys‐gag p19(100–130)‐bovine serum albumin‐coated polystyrene ball was incubated with test serum and, after washing, with (anti‐human IgG γ‐chain) Fab'‐peroxidase conjugate. The degree of inhibition by preincubation of test sera with excess of cys‐gag p19(100–130) in combination with an appropriate cut‐off value for the fluorescence intensity of bound β‐D‐galactosidase activity discriminated almost all seropositive samples from seronegative ones.
Novel and sensitive noncompetitive enzyme immunoassay (hetero‐two‐site enzyme immunoassay) for α‐human atrial natriuretic peptide in plasma
Authors: Hashida Seiichi , Yamamoto Naoko , Ishikawa Eiji
Published: March 2007

A novel and sensitive noncompetitive enzyme immunoassay (hetero‐two‐site enzyme immunoassay) for α‐human atrial natriuretic peptide (α‐hANP) in plasma, which uses only one monoclonal IgG for the ring structure of α‐hANP, is described. Plasma was filtered through polysaccharide membrane to separate peptides from proteins. The plasma filtrate was incubated with N‐hydroxysuccini‐midobiotin to biotinylate α‐hANP and subsequently with a polystyrene ball coated with monoclonal IgG for the ring structure of α‐hANP to trap biotinylated α‐hANP. The polystyrene ball was washed to eliminate unreacted N‐hydroxysuccinimidobiotin and other biotinylated substances, and biotinylated α‐hANP was eluted from the polystyrene ball with HCI. The eluate was neutralized and incubated with horseradish peroxidase‐labeled antibody IgG for the ring structure of α‐hANP and subsequently with two streptavidin‐coated polystyrene balls. Peroxidase activity bound to the streptavidin‐coated polystyrene balls was assayed by fluorometry. The detection limit of α‐hANP was 20 amol, and the assay range of plasma α‐hANP was 0.8–1,200 ng/L using 100 μl of plasma filtrates corresponding to 75 μl of plasma. Plasma levels of hANP in healthy subjects were 9.8–21.5 ng/L. These values were significantly lower than those measured by a two‐site enzyme immunoassay probably due to the presence of α‐hANPs lacking some N‐terminal amino acids, which were as reactive as α‐hANP [1–28] in the two‐site enzyme immunoassay but less reactive in the hetero‐two‐site enzyme immunoassay.
Production of a murine monoclonal antibody with anti‐hiv‐1 neutralizing properties
Authors: Hinkula Jorma , Gilljam Gustav , Wahren Britta
Published: March 2007

A mouse monoclonal hybridoma cell line producing IgG 1k to human immunodeficiency virus (HIV‐1) gp 120 envelope protein was cultured in several systems. A small‐scale flask culture was essential for characterizing the culture variables of the hybridoma. A dialysis tubing culture appeared to be an excellent alternative to in vivo cultures of ascitic fluid, and gave high mouse monoclonal antibody (Mab) concentrations. Two continuous culture systems were both very effective in producing large amounts of Mabs. The hollow fiber system has the advantage of giving a concentrated product in the harvest. The ceramic core system, on the other hand, allows excellent monitoring of the cellular growth and production phases and gave the highest HIV antigen reactivity/μg of the produced IgG.

Twelve grams of HIV‐1 neutralizing Mabs were produced. The Mab was purified with a yield of 61%. The neutralizing capacity of the Mab was studied in vitro and shown to be excellent with 50% neutralizing titers using 5 ng Mab. The biological half‐life of the Mab given intraveneously to an HIV‐infected individual was shown to be around 30 h.
Dna ploidy and cell‐cycle analysis: Tools for assessment of cancer prognosis
Authors: Aziz Douglas C. , Peter James B.
Published: March 2007

DNA ploidy and cell cycle analysis as measured by flow cytometry (FC) and image analysis (IA) have moved out of the realm of the research laboratory to become valid clinical tests used in the assessment of prognosis of the cancer patient. Although much information on the relationship of DNA ploidy/ %S‐phase analysis to patient prognosis is available in the literature, the data are not presented in such a way as to be helpful in clinical decision making. Because predictive values and confidence intervals, which measure the likelihood that a given clinical test will rule in or rule out a clinical outcome, were not calculated in previous reviews, conclusions about the clinical utility of these analyses were not possible. Using the available raw data on DNA ploidy and %S‐phase analysis from previously published papers, predictive values and confidence limits were calculated for specific clinical presentations. In several such clinical situations (tumor type, stage, etc.), predictive value of greater than 90% was derived. We conclude that in these situations DNA ploidy and %S‐phase analysis can be used to predict clinical outcome, to design treatment, and to guide patient management. The evaluation of the clinical utility of these tests must ultimately rest on prospective trials which show that randomized arms respond to treatment regimens dependent upon the DNA ploidy and %S‐phase status.
An enzyme immunoassay screening test for the detection of total antinuclear antibodies
Authors: Moncé Napoleon M. , Cappel N. Niels , Bogusky Ronald T.
Published: March 2007

The enzyme immunoassay antinuclear antibody (EIA ANA) screening test method is a new assay format for the qualitative determination of antinuclear antibodies (ANAs) in human serum or plasma. This assay method collectively detects ANAs against double stranded DNA (dsDNA), SS‐A/Ro, SS‐B/La, Scl‐70, Sm, and Sm/RNP antigens, along with serum positive for peripheral, homogeneous, speckled, nucleolar, and centromere patterns. This assay correlated well with data obtained with hemagglutination tests for specific ANA antigens, with the indirect immunofluorescence (IFA) ANA HEp‐2 test, and with the Crithidia luciliae IFA test for antidsDNA. This new test procedure is both highly specific and sensitive and substantially decreases the time involved when screening large numbers of patient samples.