Showing papers in "Journal of Clinical Laboratory Analysis in 1993"

Expression of cell adhesion molecules in the salivary and lacrimal glands of sjogren's syndrome

Abstract: We attempted to determine whether cell adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin (endothelial-leukocyte adhesion molecule-1; ELAM-1), are involved in the lymphoid cell infiltration of the salivary and lacrimal glands in Sjogren's syndrome (SS) patients. Both immunohistochemical analysis and the reverse-transcripts polymerase chain reaction (RT-PCR) were used to analyze the expression of VCAM-1, ICAM-1, ELAM-1, very late antigen 4 (VLA-4 [alpha 4,beta 1]), lymphocyte function-associated antigen-1 (LFA-1), interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), and interleukin-1 beta (IL-1 beta). Immunohistochemical analysis of salivary gland biopsies from SS patients showed a marked expression of VCAM-1 and ICAM-1 in the venules surrounded by infiltrated CD4+ CD45RO+ T cells. E-selectin was expressed on vascular endothelium with weak intensity. Increased levels of VCAM-1, ICAM-1, IFN-gamma, and IL-1 beta mRNA were demonstrated by RT-PCR, whereas E-selectin mRNA were weakly expressed in SS lacrimal and salivary gland tissues. This is in contrast with strong expression of ELAM-1 in IL-1 beta-stimulated human umbilical vascular endothelial cells (HUVEC) in vitro. Cytokine-mediated up-regulation of VCAM-1 and ICAM-1 that facilitates the recruitment of VLA-4 and LFA-1 expressing T cells might contribute to lymphoid cell infiltration in the salivary and lacrimal glands in SS.

Glycoconjugates of Trypanosoma cruzi: a 74 kD antigen of trypomastigotes specifically reacts with lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas disease.

Abstract: Protective, lytic antibodies are believed to be correlated with active Trypanosoma cruzi infection. In patients with chronic infection, antibodies lysing trypomastigote forms recognize chiefly alpha-galactosyl structures at the parasite surface. The target molecules on cell-derived trypomastigotes that react with anti-alpha-galactosyl antibodies (anti-Gal) from patients with chronic Chagas disease were investigated. Glycoconjugates were isolated from trypomastigotes and shown to absorb purified Chagasic (Ch) anti-Gel effectively as well as lytic antibodies from Ch sera. Active fractions were F2 (74 kD and 95.6 kD) and F3 (120-200 kD). A differential reactivity with antibodies from untreated Ch patients (trypanolytic) and from treated, presumably cured, individuals (not trypanolytic) was evident using F2 and F3 antigenic fractions. No cross-reactivity with heterologous sera (other infections) was observed. The F2 glycoconjugate (mostly 74 kD) can be used in the diagnosis of active Chagas infection, replacing the quantitative determination of complement-mediated lysis. With the present sample of patients' sera and normal human sera, it showed 100% sensitivity and specificity.

Advanced glycosylation end products: a new disease marker for diabetes and aging.

Abstract: Advanced glycosylation end products (AGEs) are a potentially useful marker for monitoring glycemic control, predicting the risk of diabetes- and aging-associated clinical complications, and monitoring the treatment of patients with micro- and macrovascular diseases, including retinopathy, atherosclerosis, nephropathy, and neuropathy. AGEs or AGE-proteins are derived from nonenzymatically glycated proteins (Amadori products) after further cross-linking with other proteins and additional rearrangement. AGE-proteins can be assayed by either radioreceptor or immunoassays in blood and tissues. No commercial kit is available at this time.

Evaluation of vero cell lysate antigen for the ELISA of flaviviruses

Abstract: The vero cell lysate antigen for the enzyme-linked immunosorbent assay (ELISA) of flaviviruses was evaluated for sensitivity, specificity including cross-reactions, and background by comparing with the standard ELISA. Human sera, in serial dilutions, were taken from subjects 14, 35, and 210 days postvaccination with 17D antigen. Early after injection, high sensitivity (82.9%) was shown by the cell lysate antigen method. Late after infection, high sensitivity was achieved by the standard method (96.2% and 94%), with significant difference (P = 0.0001). However, sensitivity achieved by the cell lysate antigen method was also acceptable (91.7% & 88.9%). The cell lysate antigen method showed high specificity and low cross reactivity early after infection. At 35 days postvaccination, no difference in specificity was observed between the two methods, but higher cross-reactions were observed for the standard method. This pattern continued at 210 days postvaccination, with significantly higher cross-reactions with the standard ELISA. The optical density differences by the two methods did not show significant relationship with the serial dilutions of human sera. No difference was observed in early and late infections in the background values of the negative control (Western equine encephalitis) between the two methods. The ELISA by the cell lysate antigen, within the limits of the experiments done, was found to be a good replacement for the ELISA by the standard method.

Detection of antibody IgG to HIV-1 in urine by ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 as antigen for diagnosis of HIV-1 infection

Abstract: Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 gag protein (p24) of HIV-1 as antigen and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant p24 conjugate and recombinant p24-beta-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, eluted with epsilon N-2,4-dinitrophenyl-L-lysine, and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Bound beta-D-galactosidase activity was assayed by fluorometry. This assay was at least 3,000-fold more sensitive than conventional methods. The lowest signal among 49 asymptomatic carriers was 3.1-fold higher than the highest nonspecific signal among 100 seronegative subjects. The sensitivity and specificity were both 100%. The positivity could be confirmed by preincubation of urine samples with excess of the antigen. Thus, this assay would be a powerful tool for detecting IgG antibody to HIV-1 in urine.

Review of diabetes: identification of markers for early detection, glycemic control, and monitoring clinical complications.

Abstract: The hallmark of diabetes mellitus, whether type I or type II, is hyperglycemia. Clinical complications associated with diabetes are most likely the consequence of hyperglycemia via both altered metabolic pathways and nonenzymatic glycation of proteins. The nonenzymatic glycation of proteins is accelerated in diabetes due to elevated blood glucose concentration. The Amadori product of nonenzymatic glycation will further cross-link with other proteins to form advanced glycosylation end products (AGEs). The reaction of AGEs with long-lived proteins, such as collagen, and the uptake of AGEs by the receptors on macrophages, endothelial cells, and platelets are major reasons for the development of various clinical complications in diabetes. Several markers have been identified for the screening, diagnosis, and monitoring of the disease. Autoantibodies against beta cells are the best markers for mass screening and for early detection of type I diabetes. In addition to glycated hemoglobin, AGEs and blood glycated proteins of various half-lives could be used for monitoring glycemic control. Several abnormal metabolites have been identified as potential markers for monitoring the severity of various clinical complications. The most interesting findings in diabetic markers could be AGEs. The amount of AGEs found in the tissues could be related to the extent of micro- and macrovascular damage and might prove useful for monitoring the treatment of patients at early stages of either nephropathy, atherosclerosis, retinopathy, or neuropathy.

Detection of the extracellular domain of c-erbB-2 oncoprotein in sera from patients with various carcinomas: correlation with tumor markers.

Abstract: Using a serum enzyme immunoassay (EIA) kit from Triton diagnostics we detected c-erbB-2 oncoprotein activity in random sera containing highly elevated tumor markers and also in serial specimens from cancer patients expressing elevated oncoprotein activities. Elevated oncoprotein activity was found not only in sera of breast and ovarian carcinomas but also in sera from colorectal, pancreatic, and prostate carcinomas and even from primary hepatoma. Whenever oncoprotein was overexpressed in an individual patient, there was usually an excellent correlation between the oncoprotein activity and the level of dominant tumor marker in serial serum specimens. Based on the size exclusion S-200 column chromatography, we found only a single molecule containing c-erbB-2 oncoprotein activity in pooled sera from cancer patients whereas two oncoproteins slightly different in size were detected in breast tumor tissue cytosol. Using HPLC on a Superose 12 HR column, the serum portion of the oncoprotein was eluted at a position near IgG, suggesting that the extracellular domain of the oncoprotein exists as a dimer in the serum. © 1993 Wiley-Liss, Inc.

Production of hydrogen peroxide by neutrophilic polymorphonuclear leukocytes in patients with diabetic nephropathy

Abstract: The production of hydrogen peroxide (H2O2) by neutrophilic polymorphonuclear leukocytes (PMN) after stimulation with phorbol myristate acetate (PMA), n-formyl-I-methionyl-IIeucyl-I-phenylalanine (FMLP), aggregated human IgG, or Staphylococcus aureus was determined in 36 patients with non-insulin dependent diabetes mellitus (NIDDM). H2O2 production by PMN after stimulation was measured using flow cytometry. Thirty-six patients with NIDDM were divided into four stages as follows: 1) stage I: non-microalbuminuric stage;2) stage II: microalbuminuric stage; 3) stage III: proteinuric stage without impairment of renal function; and 4) stage IV: proteinuric stage with impairment of renal function. H2O2 production after PMA stimulation in all stages of NIDDM patients was higher than that in healthy controls. This increase of H2O2 production by PMN was particularly observed in stage IV of NIDDM patients after stimulation. Furthermore, H2O2 production in patients in stage IV was higher than that in patients with non-diabetic disease with impairment of renal function. It appears that reactive oxygen species produced by PMN after stimulation under some conditions may play an important role in the progression of diabetic nephropathy. © 1993 Wiley-Liss, Inc.

Autoantibodies in the sera of Trypanosoma cruzi-infected individuals with or without clinical Chagas disease

Abstract: Variations in the levels and the specificities of autoantibodies directed against a panel of antigens (cytoskeleton proteins, DNA, laminin) were analyzed in the sera from two groups of humans infected with Trypanosoma cruzi. One group was constituted of apparently healthy blood donors (BD) and the other of patients with clinically confirmed Chagas disease (CCH). In both infected groups, a high proportion but not all sera exhibited dramatic enhancement of IgM and IgG autoantibodies directed against all antigens tested. Sera positive for IgG autoantibodies were generally found more frequently in the CCH than in the BD group, except for anti-actin antibodies more often present in BD sera. Anti-laminin IgG antibodies were present in a similar number of individuals in both groups. Although the titers of anti-laminin IgG antibodies were in general higher in CCH, their dissociation constants were in the same range (7 × 10−8–10−7M) in both groups. IgG autoantibodies were demonstrated to be polyreactive with laminin and other self antigens as well. Circulating immune complexes were present in sera from both groups and the activity of the antibodies dissociated from these complexes was directed against all the antigens of the panel. Although the IgE concentration was significantly enhanced in several subjects from both groups, the incidence of positive sera was higher in the CCH (60%) than in the BD (39%) group. Our results demonstrate that autoantibodies with the characteristics of natural autoantibodies are found in both T. cruzi-infected apparently healthy individuals and patients. © 1993 Wiley-Liss, Inc.

Evaluation of the Hawaiian reef fishes with the solid phase immunobead assay.

Abstract: This study presents data on the evaluation of a laboratory ciguatera kit based on the solid phase immunobead assay (SPIA) for the detection of ciguatoxin in Hawaiian reef fish. The SPIA was performed on fish catches by volunteer fishermen throughout the State of Hawaii. A total of 1,067 fish of various species were tested for ciguatoxin (CTX) using the SPIA kit. Of the total 1,067 fish tested, 510 were from Oahu, 402 from Hawaii, and 75 from Maui. The number of fish from Molokai, Kauai, and Lanai were 23, 20, and 7 respectively. Twenty percent of the total fish tested were positive, 41% borderline, and 39% negative for ciguatoxin. The highest percentage of SPIA- positive fish were from Hawaii (27%) followed by Oahu (19%) and Kauai (15%). These results correlate with the reported incidents from the Department of Health (DOH) of actual ciguatera poisoning in the State of Hawaii. Fish in all three categories of the SPIA test values were eaten. No false negatives were noted with individuals eating SPIA negative fish. Of the 232 SPIA borderline values eaten, 3 species of fish caused ciguatera poisoning. These fish included 2 papio, 1 mullet, and 1 po'ou. Of the 17 SPIA positive fish eaten, 5 caused ciguatera poisoning: 2 papio, a kole, an uhu, and a weke. The SPIA ciguatera test did protect the public when only SPIA-negative fish were eaten. The borderline and positive SPIA fish were generally unsafe, especially the positive fish. The data indicated that the probability of getting ciguatera with a SPIA positive fish was 1 out of 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Principle and applications of ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for antibodies in body fluids

Abstract: The sensitivity and specificity of enzyme immunoassay for antibodies in body fluids have been improved considerably by transferring the complex of labelled antigen and antibody to be detected from one solid phase to another to eliminate interfering substance(s) in the samples (immune complex transfer enzyme immunoassay). Usefulness of the new method has been tested for antibodies in serum as well as in urine. Anti-thyroglobulin IgG could be measured not only in serum of all patients with autoimmune thyroid diseases and almost all healthy subjects but also in the urine of most of the patients. Anti-HTLV-I IgG was unequivocally demonstrated in some of sera, which were indeterminate or negative by Western blotting, and diagnosis of HIV infection by detecting anti-HIV IgG in urine and saliva would be possible with higher reliability than by conventional methods. © 1993 Wiley-Liss, Inc.

Protein 1: its purification and application in clinical medicine.

Abstract: Protein 1 (P1) is a low-molecular-weight protein recently isolated from the urine of patients with chronic renal failure. Its molecular weight is 14 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and pI 4.7 on isoelectric focusing. We purified this protein, characterized its physicochemical properties, and analyzed its amino acid sequences to show that it is probably identical to human lung Clara cell 10 kDa protein. Its monoclonal antibody was prepared, and a reliable enzyme-linked immunosorbent assay employing the sandwich method was developed and used to investigate distribution and variation in concentration of P1 in various body fluids under an array of physiologic and pathologic conditions. Clinical studies indicated that, as is the case with other proteins of low molecular weight, the main catabolic site of P1 of plasma origin is the kidney: P1 is filtered by the renal glomeruli and reabsorbed by the renal tubules. Unabsorbed P1 is thus excreted into the urine. This protein is also synthesized in the genital tissues of males, however, from which it is also excreted into the urine. Clinical data obtained in our study of this protein is summarized here, and an attempt is made to determine the potential value of this protein in laboratory medicine.

Antibodies to Sm, RNP and SSB detected by solid-phase ELISAs using recombinant antigens: A comparison study with counter immunoeloctrophoresis and immunoblotting

Abstract: Sera from 64 patients with systemic lupus erythematosus (SLE) were tested for the presence of anti-Sm, anti-RNP, and anti-SSB antibodies using commercially available solid-phase enzyme-linked immunosorbent assays (ELISAs) and recombinant nuclear proteins as substrates. The results were compared to those obtained with counterimmunoelectrophoresis (CIE) and immunoblotting (IBT) using a rabbit thymus extract (RTE) as the substrate. The ELISAs detected antibodies to Sm, RNP, and SSB in, respectively, 25%, 36%, and 15% of the SLE sera. Neither IBT-positive/ELISA-negative nor CIE-positive/ELISA-negative sera were found, regardless of the specificity considered, suggesting that ELISAs using recombinant nuclear antigens are highly sensitive. Discrepancies were observed between the results obtained with these different techniques. In addition to sera positive by both IBT and ELISA but negative by CIE, a substantial number of sera had ELISA-detectable anti-Sm, anti-RNP, and anti-SSB antibodies which failed to react with the corresponding polypeptides by IBT. The reasons for ELISA/IBT discrepancies were explored; however, no single explanation was found. Instead, a higher sensitivity of the ELISA to detect antibodies directed against certain polypeptides, the possible inability of IBT using RTE as the substrate to detect antibodies reacting with conformational antigenic determinants, and false-positive reactions in the ELISAs were suggested. Thus, it is still advisable to perform both IBT and ELISAs simultaneously in well-defined autoimmune diseases to further analyze the potential advantages of ELISAs using recombinant antigens.

Primary structure of the variable regions of a monoclonal antibody MUSE11 recognizing the tandem repeat domain of a mucin core protein, MUC1

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

In situ hybridization for epidermal growth factor receptor (EGFR) external domain transcripts in prostatic adenocarcinoma.

Abstract: We examined prostatic adenocarcinomas from 19 formalin fixed radical prostatectomy specimens for EGFR by in situ hybridization employing a 24 base synthetic biotin-labeled oligonucleotide probe complementary to the 5' end of EGFR mRNA. All slides were examined by light microscopy using a 25 × objective. Each field was given three values: 1) Gleason grade (1-5), 2) Nuclear grade [small ( 10 μ)], and 3) EGFR staining intensity score (0, absent; 1, weak; 2 +, moderate to strong). A total 851 25 × fields of prostatic adenocarcinoma were studied. All cancers demonstrated at least some degree of cytoplasmic EGFR message. The EGFR intensity score correlated best with tumor nuclear size. No correlation with Gleason grade was observed. Cytoplasmic staining was also identified in the basal cell layer of benign glands, high grade prostatic intraepithelial neoplasia, stromal nodules, transitional epithelium, periurethral glands, and ganglion cells. Competitive hybridization experiments using an unlabeled EGFR probe showed markedly diminished hybridization signal, while in situ hybridization with a biotin-labeled EGFR sense probe was negative. Immunohistochemistry on 13 of the tumors with 2 monoclonal antibodies against EGFR showed staining in only 1/13 and 10/13 tumors. EGFR expression appears to be most prominent in tumors of high nuclear grade. Further studies will be necessary to explore this growth factor as a prognostic variable in this tumor. © 1993 Wiley-Liss, Inc.

Immunologic significance of increased soluble cd8/cd4 molecules in patients with active systemic lupus erythematosus

Abstract: This study attempted to estimate soluble CD4(sCD4)/CD8(sCD8) molecules in active systemic lupus erythematosus (SLE) patients. Measurements were made by solid-phase enzyme-linked immunosorbent assay. sCD8 or sCD4 molecules were significantly increased in the patients as compared to control subjects. sCD8 correlated with the erythrocyte sedimentation rate. sCD4 correlated with the anti DNA antibody titer, the IgG concentration, and negatively with the complement titer. An association of these molecules with immunologic abnormalities and disease activity exists in SLE patients. © 1993 Wiley-Liss, Inc.

Detection of antineutrophil autoantibodies by flow cytometry : use of unfixed neutrophils as antigenic targets

Abstract: Antineutrophil antibodies may be found in the sera of patients with chronic neutropenia as well as in the sera of a variety of patients with neutropenia and associated autoimmune or infectious disorders. We evaluated an immunofluorescent flow cytometric technique for the measurement of antineutrophil antibodies in serum. Sera from patients with suspected immune neutropenia were studied and compared with a group of sera from normal healthy individuals, as well as with sera from patients with rheumatoid arthritis and systemic lupus erythematosus. Of 159 patients with suspected immune neutropenia and a variety of associated clinical disorders, 59 (37%) were found to have evidence for enhanced binding of IgG to normal target neutrophils, interpreted as positive for antineutrophil antibodies. Whereas 0/37 nonneutropenic patients with typical RA had positive results, 51/244 (21%) of sera from nonneutropenic patients with SLE or other collagen vascular disorders showed enhanced IgG binding to neutrophils. Living neutrophils were used to study the effects of cellular activation, and increased antibody binding was observed with certain sera that contained IgG directed against activation-dependent antigens. We found that, under controlled conditions, flow cytometry can be reliably used to detect antineutrophil autoantibodies, with unfixed, living neutrophils as antigenic targets. © 1993 Wiley-Liss, Inc.

Detection of hepatitis C virus infection with recombinant immunoblot assay, synthetic immunoblot assay, and polymerase chain reaction

Abstract: The newly developed immunoblot assay, RIBA SIA (recombinant and synthetic polypeptide immunoblot assay), Chiron, Calif., was compared with the commercially available second generation recombinant immunoblot assay (RIBA-2) for the detection of antibody to hepatitis C virus (anti-HCV). The two immunoblot tests were also compared with the polymerase chain reaction (PCR) for the detection of HCV RNA. Ninety-one percent of samples reactive by RIBA-2 were positive for anti-HCV by RIBA SIA. A total of 31% of RIBA-2 indeterminate samples became reactive by RIBA SIA, 24% became non-reactive, and 45% remained the same. Samples reactive by RIBA-2 or SIA from different risk groups, were mostly positive (67-100%) by PCR for HCV RNA. All indeterminate samples from hemophiliacs and intravenous drug users were PCR positive. RIBA SIA is more sensitive and specific than RIBA-2 and correlates well with PCR results.

Immunoquantification of lipoprotein(a): comparison of nephelometry with electroimmunodiffusion.

Abstract: A new fully automated nephelometric immunoassay for Iipoprotein(a) quantification in human serum was evaluated using the Behring Nephelometer Analyzer. The assay exhibited a good linearity in the concentration range of 110-1,770 mg/l; at higher concentrations, samples were automatically diluted by a factor of 4. The method is simple, robust, and shows an excellent stability of the calibration curve over several weeks. Intraassay and day-to-day coefficients of variation were 2% and 4.5%, respectively. The method correlated well with electroimmunodiffusion (r = 0.977; n = 123; P = 0.0001). Unspecific turbidity as expressed by an elevated blank value occurred in 3% of all freshly measured samples (n = 392). Storage of the samples for 1 week at 4°C had no significant influence on the results. Frozen sera, on the other hand, cannot be assayed by this method. We believe that this assay is well suited for use in clinical routine work. © 1993 Wiley-Liss, Inc.

Double immunochemical screening test (hemoglobin and albumin) for the detection of occult intestinal bleeding.

Abstract: The authors have developed a combined electrophoretic-immunoprecipitation technique suitable for the simultaneous demonstration of two blood proteins, i.e., hemoglobin and albumin, in the feces. The analytical sensitivity of the method is 2 μg hemoglobin and albumin/ml. The technique is simple, inexpensive, and suitable for the reliable demonstration of occult intestinal bleeding for either inpatients or mass screening. This technique is an aid not only to the fact that bleeding can be detected, but to its intensity, as well. This new technique improves the diagnostic accuracy of early detection of carcinomas and polyps, presumably without raising significantly the number of “false-positive” reactions. © 1993 Wiley-Liss, Inc.

Characterization of marine toxin(s) in Myripristis sp. by immunological, mouse toxicity, and guinea pig assays.

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

Prostate-specific antigen and prostate volume: a meta-analysis of prostate cancer screening criteria.

Abstract: Objective: To establish the value of serum prostate-specific antigen (PSA) and prostatespecific antigen per unit volume of prostate gland (PSAD) in detecting prostate carcinoma (CaP) in a hypothetical screening algorithm, a meta-analysis of the sensitivities, specificities, predictive values and likelihood ratios were combined from the published data. Data Sources: Journal articles identified by a MEDLINE database search from 1988 to October 1992, using prostate-specific antigen as a key word were used to calculate the distribution of PSA in healthy men, men with benign prostatic hyperplasia (BPH) and men with prostate carcinoma (CaP) Study Selection: Only studies that contained the specified serum PSA values and patient outcomes were included. Data Extraction: The distributions of the serum PSA were plotted versus serum PSA for healthy men (2567), men with BPH (798) and men with CaP (835) from the abstracted data. Prostate volume distributions were estimated from the published transrectal ultrasound (TRUS) calculations. Data Synthesis: Hypothetical cohorts of 1,000 men between the ages of 60 and 70 years were screened using three different screening decision algorithms. Using a serum PSA cutoff of 3.0 ng/ml for referral for transrectal biopsy, 59 of 80 (74%) CaP would be detected and 21 (26%) would be missed. 209 transrectal biopsies would be performed, and 150 (72%) of them would be negative for CaP. Using a serum PSA cutoff of 4.0 ng/ml, 52 of 80 (65%) CaP would be detected and 28 (35%) would be missed. 146 transrectal biopsies would be performed, and 94 (64%) of them would be unnecessary. Using a cutoff of 2.0 ng/ml for serum PSA and 0.1 ng/ml/cc for PSAD, 55 of 80 (69%) of the cancers would be detected and 25 (31%) would be missed. Only 84 transrectal biopsies would be performed, and 29 (35%) of them would be negative for cancer. Conclusion: This algorithm maximizes the number of cancers detected (true-positive cases) and at the same time reduces the number of false-positive cases, minimizing the number of patients who would have to receive an unnecessary transrectal biopsy, compared to using a serum PSA cutoff of 3.0 or 4.0 ng/ml. © 1993 Wiley-Liss, Inc.

Specificity of an immunochemical reagent for quantifying the isoforms of creatine kinase-MB.

Abstract: Fractionation of the creatine kinase-MB isoforms is promising for use in diagnosing acute myocardial infarction and for monitoring myocardial perfusion status after thrombolytic therapy. An immunochemical reagent intended for use in fractionating the MB1 and MB2 isoforms of creatine kinase-MB was examined before and after immunoextraction, qualitatively by visually examining electrophoresis separation of various MB1 and MB2 mixtures, and quantitatively by comparing the observed and predicted enzymatic activity of various MB1 and MB2 mixtures. Qualitatively the reagent showed greater reactivity for MB1 than for MB2, as demonstrated by a marked decrease in the MB1 electrophoretic region following immunoextraction. Quantitatively, the reagent consistently eliminated about 75% of MB1 activity; however, the assay also eliminated about 40% of MB2 activity from isoform mixtures. Although the performance of the immunochemical reagent was not ideal, the greater reactivity for MB1 may have clinical use.

A survey of ciguatera: Assessment of Puako, Hawaii, associated with ciguatera toxin epidemics in humans

Abstract: A survey for the assessment of the ciguatera problem has been determined in Puako, South Kohala, on the Island of Hawaii. This is in the area of persistent ciguateric outbreaks during the months of January through March, caused by a specific species of fish (Cheilinus rhodochrous, red rose wrasse, or po'ou). Analyses of algae, Gambierdiscus toxicus, and various species of fish, including herbivores and carnivores, gave positive indications of Puako as a potential ciguateric area. Algae associated with Gambierdiscus toxicus blooms and the dinoflagellate itself were found in transects A and D. Transects A and D showed 291 G. toxicus per gram of Tolycarpidia glomurata and 9 G. toxicus per gram of Turbinaria sp. with epiphytic Jania sp., respectively. No G. toxicus was found in transects B and C. This may be attributed to the low salinity from intrusion of freshwater in this vicinity. Examinations of the fish, kole, manini, Hawaiian kole, roi, and po'ou by the solid-phase immunoassay showed 89% of fish in the borderline and positive categories from all transects. Extracts of viscera and flesh showed high levels of toxicity in mouse (13 of 23 deaths), particularly in the viscera (gut) of both herbivores and carnivores. The guinea pig atrial analysis generally showed a few ciguatoxin-like, but most were nonciguateric type responses. The data presented in this Puako survey showed evidence of toxic fish associated with ciguatoxin-like and most probably other toxins, either polyethers or non-polyethers as yet unidentified.

Stability studies of the components of a prototype penicillinase (beta-lactamase)-linked ELISA kit.

Abstract: Penicillinase (beta-lactamase) enzyme-linked immunosorbent assay (ELISA) for various reproductive hormones developed in the laboratory were found to have wide applicability in the fertility check clinic of the Institute. A need was thought to transform these assays into ready-to-use kit forms. Therefore, prototype ELISA kits for these hormones were developed and stability of the individual component was ascertained at various temperatures (room temperature, 37 degrees C and 2-8 degrees C). Stability studies were conducted on previously validated assay for pregnanediol-3 alpha-glucuronide (PdG). The studies showed that immunosorbents (antibody coated plates) are stable at room temperature for a period of 2 weeks, at 37 degrees C for 1 week and at 2-8 degrees C for a period of 9 months when preserved after treatment with glycerol solution. The lyophilised conjugate, standard and immunoassay buffer, colour reagent, and its substrate were stable at 37 degrees C up to 1 week and at room temperature up to 2 weeks and at 2-8 degrees C for a period of 6 months, during which the stability was studied.

Reticulocyte count with maturation fractions in pancytopenic evaluation by a fully automated counter

Abstract: Using a fully automated reticulocyte counter, the roles of the reticulocyte count with maturation in pancytopenia were evaluated. Different groups of pancytopenia including aplastic anemia, infiltrative marrow disorder, hypersplenism, and megaloblastic anemia were recruited. All patients had bone marrow examinations for morphological diagnosis and reticulocyte evaluation using an automated counter. The roles of these parameters were then analyzed statistically in the differential evaluation among these conditions. The following subjects were studied: 292 normal subjects, 67 cases of aplastic anemia, 69 cases of marrow infiltration by different malignancies, 35 cases of hypersplenism, and 13 cases of megaloblastic anemia. The results showed that the absolute reticulocyte counts were lowest in the groups of aplastic anemia and megaloblastic anemia and highest in hypersplenism. Both showed significant differences from the infiltrative groups. The maturation fractions were most immature in the group of marrow infiltration and are significantly different from the other groups. It was concluded that the highest absolute reticulocyte count (> 0.09 10(12)/L) obtained in pancytopenic patients suggests it to be a case of hypersplenism. The lowest counts ( 30%) with a nearly normal reticulocyte count favor the group of marrow infiltration.

Measurement of antimicrobial agents in cerebrospinal fluid using the Abbott TDx analyzer.

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

Enzyme immunoassay and characterization of pancreatic stone proteins in human urine

Abstract: An enzyme immunoassay of pancreatic stone protein (PSP) in human urine was developed. Mean analytical recovery of pure PSP-S2-5 added to urine was 102.3% (SD 5.9%), and the precision of the assay was 2.0-2.7% within an assay and 2.5-2.9% between assays. In healthy volunteers (age 20-55 years), the mean value of the PSP concentration, expressed as ratios to urine creatinine, was 129 ± 88 (mean ± SD) μg/g without any differences for sex. Urine PSP correlated with urine N-acetylglucosaminidase (NAG) (r = 0.354). The molecular forms of immunoreactive PSP in urine were characterized by using cation exchange chromatography (Mono S), SDS-PAGE, N-terminal sequence, and enzyme immunoassay analysis. The urine PSP, eluted at the position corresponding to PSP-S2-5 on cation exchange chromatography, was converted to PSP-S1 by trypsin digestion. The difference in mobility on SDSPAGE between urine PSP and PSP-S2-5 seems to be due to a glycosylated undecapeptide (N-terminal 1-11). The proposed method offers a sensitive, specific, and reproducible tool for laboratory analysis of human urine PSP levels. © 1993 Wiley-Liss, Inc.

Automated latex nephelometric immunoassay for the measurement of serum lipoprotein (a)

Abstract: A sensitive immunoassay based on latex particle agglutination has been developed for measuring lipoprotein Lp(a) concentrations in serum or plasma. Carboxylated latex particles (diameter 240 nm) covalently coated with F(ab')2 fragments of anti-lipoprotein Lp(a) antibodies are incubated with diluted sample (400-fold) for 12 min at room temperature, with the resulting agglutination quantified by measuring the change of light-scatter produced. The assay has been automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. This assay generates a standard curve in the range of 27 to 1750 mg/L, showing inter-assay precision of less than 8%. There were no interferences from plasminogen, bilirubin, Intralipid™, haemoglobin, rheumatoid factor, and apolipoprotein B. No significant differences were observed when fresh and frozen samples were compared. Sample pretreatment with “Lipoclean” clearing agent and sample lyophillization decreased the agglutinating reaction. In two separate studies using 77 and 112 patient sera the Lp(a) values, determined by the latex nephelometric method, the Terumo Macra™ Lp(a) ELISA test, and the Pharmacia Apo(a) radioimmunoassay method, gave correlation coefficients of 0.948 and 0.974, respectively. Physiological lipoprotein (a) values were determined in a blood donor group, with the distribution of serum Lp(a) highly skewed, with a mean (SD) and median values of 213(236) mg/L and 116 mg/L, respectively. Concentrations of Lp(a) were found to be age-and sex-independent. This latex nephelometric procedure is a convenient method and an interesting alternative to other immunoassays for routine measurement of human lipoprotein (a). © 1993 Wiley-Liss, Inc.