Effect of Brazilian, Indian, Siberian, Asian, and North American ginseng on serum digoxin measurement by immunoassays and binding of digoxin-like immunoreactive components of ginseng with Fab fragment of antidigoxin antibody (Digibind)

We compared Brazilian, Indian, Siberian, Asian, and North American ginseng for potential interference with 3 digoxin immunoassays: fluorescence polarization (FPIA), microparticle enzyme (MEIA), and Tina-quant (Roche Diagnostics, Indianapolis, IN). We supplemented aliquots of a drug-free serum pool with ginseng extracts representing expected in vivo concentrations and overdose. We observed apparent digoxin-like immunoreactivity with FPIA, modest immunoreactivity with MEIA, and no apparent digoxin immunoreactivity with the Tina-quant with all ginsengs except Brazilian, which showed no immunoreactivity with any assay. When aliquots of serum pools prepared from patients receiving digoxin were supplemented with ginsengs, we observed falsely elevated digoxin values with FPIA, falsely lower digoxin values (negative interference) with MEIA, and no interference with the Tina-quant. Digoxin-like immunoreactive components of various ginsengs have moderate protein binding; monitoring free digoxin concentrations does not eliminate such interference. We also observed that Digibind (Burroughs Wellcome, Research Triangle Park, NC) can bind free digoxin-like immunoreactive components of ginsengs; such effects can be monitored by measuring apparent free digoxin concentrations. Indian, Asian, and North American ginsengs interfere with serum digoxin measurement by FPIA and MEIA; the Tina-quant is free of such interference. Digibind can bind free digoxin-like immunoreactive components of ginseng.     

Suppression of total digoxin concentrations by digoxin-like immunoreactive substances in the MEIA digoxin assay. Elimination of negative interference by monitoring free digoxin concentrations

Digoxin-like immunoreactive substances (DLIS) cross-react with antidigoxin antibody and falsely elevate immunoassay-measured total digoxin concentrations. The fluorescence polarization immunoassay (FPIA) for digoxin showed high cross-reactivity with DLIS, but a new microparticle enzyme immunoassay (MEIA) had low cross-reactivity. The concentration of digoxin in the presence of DLIS was falsely lowered (negative interference) when measured by MEIA. We prepared the following serum pools: 2 normal (no DLIS), 2 from patients with uremia, and 3 from patients with liver disease (high DLIS). No patients received digoxin or digitoxin. When normal pools were supplemented with known concentrations of digoxin, total and free concentrations measured by both assays were comparable, but when liver and uremic pools containing high DLIS were supplemented with digoxin, the measured total digoxin concentrations were lower by MEIA and higher by FPIA. However, by taking advantage of 25% protein binding of digoxin and high protein binding of DLIS, free digoxin levels were not affected by DLIS. In 2 patients receiving digoxin but without volume expansion, total and free digoxin concentrations measured by both assays were comparable; in the 2 volume-expanded patients, only free digoxin concentrations were comparable. Monitoring free digoxin concentration can eliminate negative interference of DLIS in the MEIA for digoxin.     

Factitious hypoglycemia. Diagnosis by measurement of serum C-peptide immunoreactivity and insulin-binding antibodies.

In seven patients with factitious hypoglycemia due to the surreptitious injection of insulin, we made the diagnosis by measurements of plasma insulin and C-peptide immunoreactivity (in seven patients), facilitated by the finding of circulating insulin-binding antibodies (in two patients). The simultaneous demonstration of low plasma glucose, high immunoreactive insulin and suppressed C-peptide immunoreactivity represents a triad of results pathognomonic of exogenous insulin administration. Determination of plasma free C-peptide and free insulin permitted patients with high titers of insulin antibodies, including those with a history of insulin-treated diabetes, to be studied and diagnosed in a way similar to that in subjects who had no circulating insulin antibodies.     

Positive and negative interference of the Chinese medicine Chan Su in serum digoxin measurement. Elimination of interference by using a monoclonal chemiluminescent digoxin assay or monitoring free digoxin concentration

An over-the-counter Chinese medicine, Chan Su, is used as a cardiotonic agent. We demonstrated significant digoxin-like immunoreactivity in various organic and aqueous extracts of Chan Su. For example, when a 20-microL aliquot of an aqueous extract of Chan Su powder (1 mg/mL) was added to a 2-mL aliquot of a drug-free serum, the observed digoxin-like immunoreactivity was 2.76 ng/mL (3.53 nmol/L) digoxin equivalent using the fluorescence polarization immunoassay (FPIA). The magnitude of interference was much lower (0.94 ng/mL [1.20 nmol/L]) with the microparticle enzyme immunoassay (MEIA), and no interference was observed with the chemiluminescent assay (CLIA). We also observed a significant positive interference of the extract with the serum digoxin measurement using FPIA. In contrast, we observed a negative interference (falsely lowered digoxin concentration) of the extract in the serum digoxin measurement with the MEIA. The extract had no effect on the serum digoxin measurement with the CLIA. By taking advantage of the high protein binding of Chan Su and only 25% protein binding of digoxin, we further demonstrated that positive interference of Chan Su in the FPIA and negative interference of Chan Su in the MEIA of digoxin could be eliminated by monitoring the free digoxin concentration.     

Rapid immunoassays for the measurement of immunoglobulin G subclass concentration in immunoglobulin preparations and human serum.

Enzyme immunoassays (EIA) capable of determining total IgG1, IgG2, IgG3 and IgG4 subclass concentrations in human serum preparations have been developed. Subclass-specific monoclonal antibodies (mAbs) are bound to polyacrylamide bead-conjugated anti-mouse immunoglobulin antibodies. Bound immunoglobulins are detected with a peroxidase-conjugated anti-IgG antibody or a biotin-conjugated anti-IgG antibody followed by peroxidase streptavidin. The standard curves were found to be linear in the regions 16.0-2.0 micrograms/ml for IgG1, 4.0-0.5 micrograms/ml for IgG2, 0.4-0.06 micrograms/ml for IgG3 and 0.25-0.05 micrograms/ml for IgG4. Coefficient of variation (CV) values range from 0.32-7.32% for IgG1, 0.66-4.85% for IgG2, 1.62-6.85% for IgG3 and 0.05-6.47% for IgG4 standard curves. The inter-assay variability for the control human serum samples was 9.6% for IgG1, 6.7% for IgG2, 9.5% for IgG3 and 6.8% for IgG4.     

Analysis of complement fixation and commercial enzyme immunoassays for detection of antibodies to Mycoplasma pneumoniae in human serum

The Meridian ImmunoCard (IC), GenBio ImmunoWELL-IgM, and Remel EIA commercial antibody tests are qualitative enzyme immunoassays that detect antibodies to Mycoplasma pneumoniae in serum. These tests were compared to an M. pneumoniae complement fixation (CF) assay, which uses a commercially available antigen component. The Meridian IC and the ImmunoWELL-IgM detect immunoglobulin M (IgM) only; the Remel EIA and the CF test detect both IgM and IgG antibodies. Detection of specific IgM antibody, which appears early in infection, can be, but is not always, indicative of a recent or current infection. Paired serum samples from 64 adult patients with probable M. pneumoniae infection were examined with each of the four tests. Thirty (47%) of the 64 acute-phase sera were IgM positive by Meridian IC, 26 (41%) were positive by Remel EIA, 24 (38%) were positive by CF, and 15 (23%) were positive by ImmunoWELL-IgM. When both the acute- and convalescent-phase serum samples from each patient were examined, 61 (95%) of the 64 patients were positive by CF, 60 patients (94%) were positive by Remel EIA, 52 patients (81%) were IgM positive by the Meridian IC, and 29 patients (45%) were IgM positive by the ImmunoWELL-IgM assay. The Meridian IC was more sensitive than the other tests for early detection of IgM antibodies. However, after examining paired serum samples, we concluded that the detection of IgM alone may not be useful for all cases of mycoplasma infection, especially in an adult population.     

人参花蕾皂甙对失血性休克犬心、肝、肺组织SOD及五种金属元素含量的影响

杂种犬１６只，按体重随机分为失血性休克（ｈｅｍｏｒｒｈａｇｉｃｓｈｏｃｋ，ＨＳ）组（８只），失血性休克（ＨＳ）人参花蕾皂甙（ｇｉｎｓｅｎｇｆｌｏｗｅｒｂｕｄｓａｐｏｎｉｎ）预治疗（ＨＳＧ）组（８只）。通过调整血容量使动脉血压维持于５．３～６．７ｋＰａ５ｈ，放血后５ｈ处死动物取心肌、肝、肺组织测微量元素铜（Ｃｕ）、锌（Ｚｎ）铁（Ｆｅ）及宏量元素钙（Ｃａ）、镁（Ｍｇ）及超氧化物歧化酶（ＳＯＤ）的含量。结果显示：ＨＳＧ与ＨＳ组比较肺组织Ｚｎ含量明显减少（Ｐ＜０．０１），心肌、肺和肝组织Ｃａ含量显著减少（Ｐ＜０．０５），而心肌、肺和肝组织ＳＯＤ含量明显增多（Ｐ＜０．００１，Ｐ＜０．００１，Ｐ＜０．０１）。作者对这些结果的意义进行了探讨。     

Effect of serum content and diluent selection on assay sensitivity and signal intensity in multiplex bead-based immunoassays

INTRODUCTION: In the Luminex multiplexed immunoassay system, complex samples such as human serum are diluted to minimize disturbing matrix effects with a specific diluent. This diluent has to imitate the sample matrix to allow interpolation and has to provide optimal cytokine-antibody binding for all cytokines. Because diluents influence multiplex immunoassay results, this paper explores several methods to determine the quality of a chosen diluent. MATERIALS AND METHODS: Two commercially available diluents, DY997 and RD6 from R&D Systems, were compared in a 19-plex immunoassay setup from Luminex. RESULTS: Using diluent DY997, multiplex signal intensity was reduced by 55% when spiked samples (chemokines and cytokines at 100 pg/mL) contained 50% v/v human serum, compared to samples containing 25% v/v. When using diluent RD6, signal intensity was reduced by 20% when samples contained 50% v/v human serum, compared to 25% v/v human serum. Diluent DY997 showed decreasing multiplex assay sensitivity with increasing protein concentrations, but not as low as in the presence of 50% v/v human serum. CONCLUSIONS: In a 19-plex setup, this paper describes signal intensity, assay sensitivity and background signal levels in relation to the total volume-fraction of serum and protein concentration. For the determination of cytokines in serum samples with the multiplexed system Luminex the diluent RD6 seems more appropriate than the diluent DY997.     

Relevance of anti-nucleosome antibodies detected by enzyme-based immunoassays in lupus diagnosis. Comparative analysis of four commercial kits

Among the biological assays used for the diagnosis of systemic lupus erythematosus (SLE), the detection of anti-double strand DNA antibodies (dsDNA Ab) is regarded as highly specific. However this biological parameter is negative among 20 to 40% of patients. Recent studies have revealed potential interest of the anti-nucleosome antibodies in the diagnosis of the lupus, in particular when any anti-dsDNA antibody activity could be detected. We selected 80 sera in order to evaluate four commercial anti-nucleosome enzyme-based immunoassays (EIA) kits. Their sensitivity and specificity values were compared with those obtained by the detection of anti-dsDNA Ab, carried out with both a Farr assay and two EIA kits. No anti-nucleosome EIA kits reached performances of the Farr assay for the diagnosis of lupus. On the other hand, our results show an higher diagnostic value for some anti-nucleosome EIA kits compared with 2 anti-dsDNA EIA kits. Apart from SLE, anti-nucleosome antibodies can be observed in others auto-immune diseases, in particular Sj?gren's syndromes, the primary antiphospholipid syndrome, the systemic sclerosis and the mixed connective tissue disease. Compared results of the four anti-nucleosome EIA kits highlight many discordances. These variations, testifying to the absence of standardization for this new parameter, must encourage with a careful interpretation of results, according to the clinical context.     

Rotavirus-specific antibodies in fetal bovine serum and commercial preparations of serum albumin.

Rotavirus-specific antibodies were detected in fetal bovine serum, bovine serum albumin, and human serum albumin by radioimmunoprecipitation with the NCDV strain of bovine rotavirus as the detecting antigen. Fetal bovine sera neutralized bovine rotavirus in a plaque reduction neutralization test to titers of 1:20 or greater. Immunoglobulins purified from fetal bovine serum by protein A-agarose affinity chromatography precipitated rotavirus antigens but did not neutralize bovine rotavirus. Rotavirus antibodies in fetal bovine serum and in purified serum albumin preparations may interfere with diagnostic assays for the detection of rotavirus antigens or antibodies.     

Evaluation of five immunoassays for detection of Chlamydia psittaci in cloacal and conjunctival specimens from turkeys.

Five commercially available immunoassays were evaluated for the detection of Chlamydia psittaci in cloacal and conjunctival swabs from industrially raised turkeys: IMAGEN (DAKO Diagnostics, Ely, Cambridgeshire, United Kingdom), Chlamydia CEL-VET IF (Cellabs, Brookvale, Australia), IDEIA (DAKO Diagnostics), CELISA (Cellabs), and CLEARVIEW (Unipath, Bedford, United Kingdom). Results were compared with isolation in Buffalo Green Monkey cells as a reference method. For the conjunctival samples, the sensitivities of the IMAGEN test, the Chlamydia CEL-VET IF test, the IDEIA, the CELISA, and the CLEARVIEW test were found to be 100, 66, 0, 0, and 0%, respectively, as compared to the reference test. Also for the conjunctival samples, the specificities of the IMAGEN test, the Chlamydia CEL-VET IF test, and the IDEIA were found to be 100, 11, and 92.8%, respectively. For the cloacal specimens, the sensitivities of the IMAGEN test, the Chlamydia CEL-VET IF test, the IDEIA, the CELISA, and the CLEARVIEW test were found to be 100, 93.3, 26.6, 0, and 53.3%, respectively. Also for the cloacal specimens, the specificities of the IMAGEN test, the Chlamydia CEL-VET IF test, the IDEIA, and the CLEARVIEW test were found to be 92, 12, 100, and 88%, respectively. The IMAGEN test was the most sensitive and specific direct chlamydia antigen detection test for cloacal and conjunctival samples from turkeys.     

Comparison of five commercial enzyme-linked immunosorbent assays and Western immunoblotting for human immunodeficiency virus antibody detection in serum samples from Central Africa.

Detection by five different enzyme-linked immunosorbent assays (ELISAs) of antibody to human immunodeficiency virus (HIV) in sera from three Zairian populations consisting of 1,998 individuals with various risks for HIV infection was evaluated. Sera that were reactive by at least one assay and 10% of the nonreactive serum samples were analyzed by Western blot (immunoblot) by using U.S. Public Health Service interpretation criteria. Sera which were positive by ELISA for detection of antibody to HIV-1 and HIV-2 and negative or indeterminate by HIV-1 Western blot were also analyzed by HIV-2 Western blot. Overall, 443 (22.2%) serum specimens were HIV-1 Western blot positive, 390 (19.5%) had indeterminate HIV-1 Western blot patterns, and no samples were HIV-2 Western blot positive. The sensitivity of the ELISAs ranged from 97.5 to 99.8%, and the specificity ranged from 51.7 to 98.4%. By population group, the negative predictive value ranged from 97.1 to 100%, in contrast to the positive predictive value, which varied from 6.6 to 100%. Follow-up results for sera which were indeterminate for antibody to HIV-1 documented only four seroconversions (6.0%) among 67 individuals at high risk for HIV-1 infection and no seroconversions among 202 individuals at relatively low risk for HIV-1 infection. This study demonstrates the importance of evaluating commercial ELISAs with sera from appropriate geographical regions in order to select the most cost-effective and practical assay for use in that region. Furthermore, the high frequency of indeterminate Western blots for African sera emphasizes the continual need for improved confirmatory assays and interpretation criteria.     

Detection of rotavirus by hybridization with a nonradioactive synthetic DNA probe and comparison with commercial enzyme immunoassays and silver-stained polyacrylamide gels.

Three enzyme-linked immunosorbent assays (ELISAs) (rotavirus enzyme immunoassay [EIA; International Diagnostic Laboratories], Pathfinder [Kallestad Laboratories], and Rotaclone [Cambridge Bioscience, Inc.]) and hybridization of viral RNA with a nonradioactive, synthetic oligonucleotide DNA probe (SNAP; Molecular Biosystems, Inc.) were compared with silver-stained polyacrylamide gel electrophoresis (PAGE) of viral RNA for the detection of rotavirus in fecal specimens. Hybridization was performed following column purification of the viral RNA. Of 286 specimens analyzed by PAGE, SNAP, rotavirus EIA, Pathfinder, and Rotaclone, 88 were positive by PAGE. All 88 specimens were also positive by the other four assays. Nine specimens that were positive by one or more of the assays were positive by blocking ELISAs but were negative by PAGE. If these nine specimens were considered to be true positives, the final sensitivities and specificities were as follows: PAGE, 91 and 100%; SNAP, 94 and 97%; rotavirus EIA, 96 and 97%; Pathfinder, 100 and 94%; and Rotaclone, 96 and 97%, respectively.     

Effect of low serum total protein on sodium and potassium measurement by ion-selective electrodes in critically ill patients

Hypoproteinaemia may lead to spuriously high electrolyte values using indirect ion-selective electrodes (ISE) compared to direct ISEs. This study evaluates the impact on electrolyte status assessment of direct compared to indirect ISE sodium and potassium measurements in samples from critically ill patients who have a high prevalence of hypoproteinaemia. Serum sodium and potassium measurements were compared using indirect and direct ISE in 190 samples received from critical care units over a three-week period. Serum sodium and potassium measurements were higher (P < 0.0001) using indirect ISE (140.0 +/- 5.0 and 4.5 +/- 0.6, respectively) compared to direct ISE (136.5 +/- 5.2 and 4.5 +/- 0.6, respectively). The calculated difference between indirect and direct ISE values for sodium increased as total protein concentration decreased (Y = 7.2-0.07X, 95% CI slope -0.1 to -0.05, P<0.0001, r2 = 0.14). Hypoproteinaemia was present in 85% of samples. Indirect ISE, compared to direct ISE, misclassified 28% of samples as pseudonormonatraemia (19%), pseudohypernatraemia (8%), pseudonormokalaemia (0.8%) and pseudohyperkalaemia (0.4%). Hypoproteinaemia is common in critically ill patients and this may lead to spuriously high indirect ISE electrolyte measurements, resulting in significant misclassification of electrolyte (particularly sodium) status. In such patients, direct ISE (as employed in point-of-care testing) offers more accurate and consistent electrolyte results than does indirect ISE (commonly used in major laboratory analysers).     

Evaluation of end-point titration, single dilution and capture enzyme immunoassays for measurement of antirotaviral IgA and IgM in infantile secretions and serum

In order to facilitate measurement of antirotaviral IgA in large collections of faeces and secretions, adaptations of enzyme immunoassay methods for estimating antirotaviral IgA and IgM in duodenal fluid, saliva, faeces and serum were studied. To quantitate specific IgA, a single dilution of each sample was assayed. Results were expressed as antirotaviral IgA units derived from a standard curve. Units were calculated by log-logit analysis on computer. There was strong correlation between antirotaviral IgA units and end-point titres in 257 faecal samples (correlation coefficient r = 0.92) and in 182 duodenal fluids and salivary samples (correlation coefficient r = 0.74). The assay was validated using acute and convalescent faeces from children with or without rotavirus infection. Immune conversions in IgA were detected in 33 (75%) of the children by units and 34 (77%) by titres. None of nine children with gastroenteritis due to other infectious agents showed immune conversions to rotavirus. A monoclonal capture IgM assay showed similar end-point titres and numbers of immune conversions when compared with a direct assay for antirotaviral IgM in serum and secretions. Use of the capture method eliminated false-positive reactions with the cell control. The assay for antirotaviral IgA units in secretions is simple, rapid, reproducible and reliable, and has proven of value in longitudinal epidemiological studies of rotavirus coproIgA profiles. Both the capture IgM technique and the single dilution IgA method permit analysis of large numbers of specimens and are appropriate for examination of immune responses to natural rotavirus infection or during vaccine trials.     

Effect of temperature on bacterial killing by serum and by polymorphonuclear leukocytes.

Bacterial killing by serum alone and by polymorphonuclear )PMN) leukocytes was studied at 37 degrees C and compared with killing at 39 and 41 degrees C. The test organisms for serum killing were Staphylococcus aureus 502A (serum resistant) and Escherichia coli O14 (serum sensitive). The organisms used in PMN killing tests were Streptococcus pneumoniae type 29 and E. coli O86.S aureus was not killed by serum alone at any temperature. Changes in temperature did not affect the rate of serum killing of E. coli O14 for the first 60 min, but by 90 and 120 min there was a discrepancy with continued killing at 37 degrees C, but no further killing at 39 and 41 degrees C. PMN phagocytic killing of the pneumococcus was enhanced at 39 degrees C compared with 37 degrees C, and phagocytic killing of E. coli O86 was decreased at 41 degrees C when compared with 37 degrees C. Therefore, it appears that under certain circumstances fever may aid the host PMNs in destroying organisms, whereas under other circumstances it may interfere with such destruction.