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The growing importance of the clinical laboratory in patient healthcare is evident from the fact that about 70% of medical decisions are now based on laboratory test results. (1) The [...]

'Are you going to tell her before she goes on holiday?' asked my consultant anaesthetist colleague as I was closing the wound. 'Of course, I will,' I said. 'If I [...]

DanShen is a traditional Chinese medicine indicated for cardiovascular diseases. The potential interference of DanShen with serum digoxin measurement was investigated using a new enzyme-linked chemiluminescent immunosorbent (ECLIA) digoxin assay. Aliquots of drug-free serum were supplemented with ethyl acetate extract of DanShen (4 different brands studied), and apparent digoxin concentrations were measured by the ECLIA as well as fluorescence polarization immunoassay (FPIA) and a turbidimetric assay for comparison. Mice were also fed 4 DanShen preparations and apparent digoxin concentrations were subsequently measured. In another experiment, serum pools containing digoxin were further supplemented with DanShen extracts and digoxin concentrations were measured again by all 3 assays. No apparent digoxin concentration was observed when aliquots of drug-free serum pools were supplemented with DanShen and digoxin concentrations were measured by the ECLIA or the turbidimetric assay. In contrast, significant apparent digoxin concentrations were observed using FPIA, and the highest apparent digoxin concentration was observed with brand 4 of DanShen extract. Similarly, when mice were fed with this herb, significant apparent digoxin concentrations were also observed using FPIA, but neither ECLIA nor turbidimetric assay showed any apparent digoxin concentration. When aliquots of digoxin pool were further supplemented with various DanShen extract, the apparent digoxin concentrations were significantly increased when FPIA was used. In contrast, digoxin concentrations in the presence of DanShen extract compared well with the digoxin concentration of the original pool when ECLIA or turbidimetric assay was used. We conclude that DanShen does not interfere with serum digoxin measurement using a more recently released ECLIA digoxin assay.

A recent report indicates that hydroxyzine and its active metabolite cetirizine interfere with the PENTINA carbamazepine assay. The potential interference of hydroxyzine and cetirizine with the fluorescence polarization immunoassay (FPIA) and CEDIA assay of carbamazepine as well as with the fluorescence polarization immunoassay of tricyclic antidepressants (TCA) was studied. Aliquots of drug-free serum pools were supplemented with various concentrations of hydroxyzine and cetirizine representing therapeutic, mild to moderate toxic as well as very toxic concentrations. Then apparent carbamazepine and TCA concentrations were measured by immunoassays. Although no interference of hydroxyzine and cetirizine was observed with carbamazepine assays (FPIA and CEDIA), significant apparent TCA concentrations were observed when aliquots of drug-free serum were supplemented with hydroxyzine or cetirizine. Mathematical formula was devised to predict hydroxyzine and/or cetirizine concentration in serum based on observed apparent TCA levels. Hydroxyzine and cetirizine also falsely increased total TCA values when aliquots of serum pool prepared from patients receiving TCA were further supplemented with these drugs. In conclusion, hydroxyzine and cetirizine do not interfere with the FPIA and CEDIA carbamazepine assays but interfere with the measurement of total TCA using the FPIA.

Despite known toxicity of oleander, this product is used in herbal preparations. Oleander interferes with various digoxin immunoassays. It is possible that a person taking digoxin also may take oleander-containing herbal products, and digoxin immunoassays interfering with oleander cannot be used for therapeutic monitoring of digoxin. Recently, Bayer Diagnostics introduced a new enzyme-linked chemiluminescent immunosorbent digoxin assay for application on the ADVIA IMS®System (ECLIA-digoxin). We studied potential interference of oleander with this new digoxin assay and found that this assay is virtually free from oleander interference. When aliquots of drug-free serum pools were supplemented with ethyl alcohol extract of oleander leaf or pure oleandrin standard, we observed significant apparent digoxin concentration when measured by the fluorescence polarization immunoassay (FPIA) but minimal digoxin-like immunoreactivity using the ECLIA digoxin assay. Because cross-reactivity should be studied in the presence of primary analyte, we prepared 2 serum pools using sera from patients receiving digoxin. Then aliquots of first digoxin pool were supplemented with oleandrin standard and aliquots of second digoxin pool with oleander extract. We observed significant increases in apparent digoxin concentration in the presence of both oleandrin and oleander extract using the FPIA. However, we observed no statistically significant change in digoxin concentration when ECLIA digoxin assay was used, indicating that this assay is virtually free from oleander interference.

Phenytoin is an anticonvulsant that requires therapeutic drug monitoring. Recently, Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of phenytoin on the ADVIA 1650® analyzer. We evaluated the analytic performance of this assay by comparing values obtained in 52 patients receiving Phenytoin using this new assay with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA). The new turbidimetric immunoassay for phenytoin showed the following imprecision with the low, medium, and high controls: total CV of 5.2% (mean 4.81 μg/mL), 3.7% (mean 16.24 μg/mL), and 4.1% (mean 22.65 μg/mL), respectively. The detection limit of the assay was 0.79 μg/mL, and the assay was linear up to a phenytoin concentration of 46.1 μg/mL. The assay showed excellent dilution recovery and recovery of spiked samples (mean recovery 101.4% and 94.4%, respectively). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y = 1.06 x − 0.61, r = 0.98, n = 52). We also determined the cross-reactivity of 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), a major metabolite of phenytoin, and of oxaprozine, an analogue with a similar chemical structure to phenytoin, in both phenytoin assays. Both assays showed almost no cross-reactivity to oxaprozine and only small (5%-8%) cross-reactivity to HPPH. We also found that the turbidimetric assay was free from interference at least up to 1200 mg/dL of hemolysis, 30 mg/dL of free bilirubin, 34.5 mg/dL of conjugated bilirubin, and 750 mg/dL of triglyceride (Intralipid). When a drug-free serum was followed by a serum sample containing 38.5 μg/mL of phenytoin, no sample probe carryover effect was observed. We conclude that the new turbidimetric assay can be used for routine monitoring of phenytoin in clinical laboratories.

Endogenous digoxin-like immunoreactive factors (DLIF) may crossreact with antidigoxin antibody and falsely elevate immunoassay results. Recently, a new enzyme-linked immunosorbent chemiluminescent assay for digoxin has been available for use on the ADVIA IMS (Integrated Modular System) 800i analyzer (Bayer Diagnostics). We studied potential interference of DLIF with this new digoxin assay. We analyzed 30 serum specimens from patients who have pathologic conditions that may increase serum DLIF concentrations. These patients were never exposed to digoxin or other agents that may lead to a measurable digoxin concentration. We also analyzed 10 specimens from neonates, 10 cord blood specimens, and 10 amniotic fluid specimens. Apparent digoxin concentrations were measured using the new enzyme-linked immunosorbent digoxin assay (IMS-Digoxin), a fluorescence polarization immunoassay (FPIA), and also a chemiluminescent immunoassay (CLIA, run on ACS:180® system from Bayer Diagnostics). We observed measurable apparent digoxin levels with the FPIA in 4 uremic patients (range 0.21-0.36 ng/mL, digoxin equivalent), 7 patients with liver disease (range 0.21-0.72 ng/mL), and 3 patients in the third trimester of pregnancy (0.22-0.66 ng/mL). We also observed measurable DLIF concentrations with the FPIA in 2 neonates (0.22 and 0.36 ng/mL), 5 cord blood specimens (range 0.21-1.18 ng/mL), and 5 amniotic fluid specimens (0.21-0.50 ng/mL). None of these DLIF-positive specimens showed any measurable digoxin concentration using the IMS-Digoxin or the CLIA assay. When serum specimens containing elevated concentrations of DLIF but no digoxin (as measured by FPIA) were supplemented with known concentrations of digoxin, we observed falsely elevated digoxin concentrations, as expected, only by the FPIA. In contrast, we observed a good agreement between the target and observed concentrations when the new IMS-Digoxin or the CLIA assay was used. We conclude that the IMS-Digoxin assay is free from interference of DLIF.

Carbamazepine, an anticonvulsant, requires therapeutic drug monitoring. Recently Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of carbamazepine on the ADVIA 1650 analyzer. We evaluated the analytic performance of this assay by comparing values obtained with this new assay in sera of 54 patients receiving carbamazepine with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA) and a chemiluminescent immunoassay (CLIA). The new turbidimetric immunoassay for carbamazepine showed excellent precision. The low control showed a total CV of 4.9% (mean 2.86, SD 0.14 μg/mL), the medium control demonstrated a total CV of 3.5% (mean 7.79, SD 0.27 μg/mL), and the high control showed a total CV of 4.8% (mean 16.15, SD 0.78 μg/mL). The assay was linear up to a carbamazepine concentration of 20 μg/mL. The assay showed excellent dilution recovery and recovery of samples supplemented with carbamazepine (mean recovery 102.2%). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y = 0.96 x − 0.46, r = 0.99, n = 54). We also observed excellent correlation between the values obtained by the CLIA (x-axis) and the turbidimetric (y-axis) assay (y = 1.10 × −0.32, r = 0.99, n = 54). However, the slope of 1.10 was higher than the slope of 0.96 observed with the regression equation obtained by using values obtained by the FPIA and the turbidimetric assay. The positive bias obtained with the new turbidimetric assay compared with the CLIA assay resulted from lower cross reactivity of carbamazepine 10,11-epoxide, the active metabolite of carbamazepine, with CLIA. On the other hand, the cross reactivity of the metabolite is similar between the new turbidimetric assay and the FPIA assay. We conclude that the new turbidimetric assay can be used for routine monitoring of carbamazepine in clinical laboratories.

Oleander is an ornamental shrub that grows in the United States, Australia, India, Sri Lanka, China, and other parts of the world. All parts of the plant are poisonous because the presence of cardiac glycoside oleandrin. Despite its toxicity, oleander extract is used in folk medicines. Because of its structural similarity, oleandrin cross-reacts with the fluorescence polarization immunoassay (FPIA) for digoxin. We studied the potential of detecting oleandrin in serum using 5 common digoxin immunoassays (FPIA, MEIA, both from Abbott; Beckman digoxin assay on Synchron LX, Chemiluminescent assay, CLIA from Bayer Diagnostics) and a recently FDA-approved turbidimetric assay on the ADVIA 1650 analyzer (Bayer). Aliquots of drug-free and digoxin-like immunoreactive substances (DLIS)-free serum pools were supplemented with ethanol extract of oleander leaves or oleandrin (Sigma Chemicals) in amounts expected in vivo after severe overdose. We observed significant apparent digoxin concentration with FPIA, Beckman, and the new turbidimetric assay (1 mL drug-free serum supplemented with 5.0 μL of oleander extract: apparent digoxin 2.36 ng/mL by the FPIA, 0.32 ng/mL by the MEIA, 0.93 ng/mL by the Beckman, 0.82 ng/mL by the new turbidimetric assay). The CLIA showed no cross-reactivity. Similar observations were made when serum pools were supplemented with oleandrin. Because cross reactivity should be tested in the presence of the primary analyte, we supplemented serum pools prepared from patients receiving digoxin with oleander extract or oleandrin. Thje measured digoxin concentrations were falsely elevated with the FPIA, Beckman, and turbidimetric assays, the highest false elevation being observed with the FPIA. Surprisingly, apparent digoxin concentrations were falsely lowered when MEIA was used. Digibind neutralizes free apparent digoxin concentration in vitro in serum pools supplemented with oleander extract, and this effect can be measured by the FPIA. We conclude that FPIA is most sensitive to detect the presence of oleander in serum. In contrast, the CLIA (no cross-reactivity) should be used for monitoring digoxin in a patient receiving digoxin and self-medicated with a herbal remedy containing oleander.

Endogenous digoxin-like immunoreactive factors (DLIF) cross-react with antidigoxin antibody and falsely elevate or lower measured serum digoxin concentrations, depending on the assay design. Recently, Bayer Diagnostics released a turbidimetric assay for digoxin on the ADVIA 1650 analyzer. We studied potential interference of DLIF with this new digoxin assay. We analyzed 40 serum specimens from patients who have pathologic conditions that may increase serum DLIF concentrations. These patients were never exposed to digoxin or other agents that may lead to a measurable digoxin concentration. We also analyzed five specimens from autopsy and five specimens from neonates. Apparent digoxin concentrations were measured using the new turbidimetric digoxin assay, the fluorescence polarization immunoassay (FPIA, Abbott Laboratories, Abbott Park, IL), and also the chemiluminescent immunoassay (CLIA, Bayer Diagnostics). We observed measurable apparent digoxin levels with the FPIA in 5 uremic patients (range 0.24–0.86 ng/mL), 6 patients with liver disease (range 0.21–0.72 ng/mL), in 3 patients in the third trimester of pregnancy (0.21–26 ng/mL), and in 3 neonates (range 0.21–0.46 ng/mL). Four out of 5 autopsy specimens showed measurable apparent digoxin concentrations (0.23–0.81 ng/mL). In contrast, only 1 specimen (a uremic patient) showed an apparent digoxin concentration of 0.26 ng/mL with the turbidimetric digoxin immunoassay (FPIA value 0.86 ng/mL, CLIA value 0.32 ng/mL). Because DLIF is absent in the protein-free ultrafiltrate, we also measured free digoxin concentrations in DLIF-positive patients to ensure that the apparent digoxin concentrations were caused by DLIF. We observed no apparent digoxin concentrations in the protein-free ultrafiltrate in any DLIF-positive specimens. When serum specimens containing elevated concentrations of DLIF but no digoxin were supplemented with a known concentration of digoxin, we observed falsely elevated digoxin concentrations by the FPIA, as expected. In contrast, we observed a good agreement between the target and observed concentrations when the new turbidimetric assay was used. We conclude that DLIF has minimal effect on serum digoxin measurements by the new turbidimetric assay.

Spironolactone and potassium canrenoate aldosterone antagonist diuretics are often used with digoxin in clinical practice. It has been well documented in the literature that spironolactone, potassium canrenoate, and their common metabolite canrenone crossreact with the fluorescence polarization immunoassay FPIA for digoxin and falsely elevate measured serum digoxin concentrations. Recently a new turbidometric assay for digoxin became commercially available from Bayer Diagnostic for application on the ADVIA 1650 Chemistry analyzer. We studied the potential interference of these compounds in this new digoxin assay. Aliquots of drugfree serum were supplemented with therapeutic and abovetherapeutic concentrations of spironolactone, canrenone, and potassium canrenoate, and apparent digoxin concentrations were measured. We observed apparent digoxin concentrations with the FPIA digoxin assay as expected but observed no apparent digoxin levels with the new turbidometric immunoassay. When serum pools prepared from patients receiving digoxin were supplemented with these compounds in concentrations expected in serum in patients receiving these medications, we observed falsely elevated digoxin levels with the FPIA digoxin assay, but no statistically significant change was observed with the new turbidometric assay. We conclude that the new turbidometric assay for digoxin is free from interference by spironolactone, potassium canrenoate, and their common metabolite canrenone.

Asian and Siberian ginsengs contain glycosides with structural similarities to digoxin. We studied potential interference of ginseng in 5 digoxin immunoassays in 3 Asian (2 liquid extracts, 1 capsule) and 3 Siberian ginseng preparations (1 liquid extract, 2 capsules). With the fluorescence polarization immunoassay (FPIA), we observed apparent digoxin activity in 1 Asian liquid preparation and in the liquid extract and 1 capsule form of Siberian ginseng. In mice fed ginseng, we observed digoxin activities in the serum (Asian, 0.48-0.68 ng/mL [0.6-0.9 nmol/L]; Siberian, 0.20-0.47 ng/mL [0.3-0.6 nmol/L]), indicating that such interferences also occur in vivo. Serum pools prepared from samples from patients receiving digoxin and then supplemented with Asian or Siberian ginseng showed falsely increased digoxin values using the FPIA (e.g., for Asian ginseng, 1.54 ng/mL [2.0 nmol/L] vs control value, 1.10 ng/mL [1.4 nmol/L]) and falsely decreased values using the microparticle enzyme immunoassay (MEIA; 0.73 ng/mL [0.9 nmol/L] vs control value, 1.04 ng/mL [1.3 nmol/L]). Digoxin-like immunoreactive substances (DLISs) showed synergistic effects with ginsengs in interfering with the FPIA and MEIA for digoxin. No interference was observed with 3 other digoxin assays, even in the presence of elevated DLISs.

Chan Su is a Chinese medicine prepared from the skin gland of a Chinese toad and is used in treating arrhythmia and other heart diseases. Danshen is prepared from the Chinese medicinal plant Salvia miltiorrhizaand is used for various cardiovascular diseases including angina pectoris. The authors studied the potential interference of such medicines with the widely used EMIT 2000 (Dade Behring; Deerpark, IL) digoxin assay and the recently marketed Randox digoxin assay (Randox Laboratories Ltd, Antrim, United Kingdom) (both run on the Bayer ADVIA 1650 analyzer) (Bayer Diagnostics, Tarrytown, NY) and compared their results with an FPIA (Abbott Laboratories) and a chemiluminescent immunoassay (CLIA; Bayer Diagnostics) for digoxin. Aliquots of drug-free serum were supplemented with 1 L ethyl acetate extract of Danshen or aqueous extract of Chan Su, and apparent digoxin concentrations were measured by all four digoxin immunoassays (FPIA, EMIT, Randox, CLIA). The authors also supplemented aliquots of several different serum pools prepared from patients taking digoxin with very small amounts of Chan Su or Danshen extract and compared digoxin values with the control digoxin values (serum pool containing no Chinese medicine). The authors observed no interference of Danshen in either EMIT, Randox, or CLIA assay but observed an interference with the FPIA assay. On the other hand, the authors observed high interference of Chan Su in the FPIA assay but moderate interference with the EMIT 2000 and Randox digoxin assays. CLIA assay was again free from any interference. The authors also observed a wide variation in digoxin-like immunoreactivity and magnitude of interference in digoxin immunoassay in different brands of Chan Su and Danshen, indicating poor quality control in manufacturing of these Chinese medicines. Taking advantage of the high protein binding of digoxin-like immunoreactive components of Chan Su, the authors further demonstrated that interference of Chan Su in EMIT 2000 and Randox assays can be mostly eliminated by monitoring free digoxin.

Digoxin fab antibody (Digibind; Burroughs Wellcome, Research Triangle Park, NC, USA) is used in the treatment of digoxin overdose. The effect of digibind on the measurement of total and free digoxin has been extensively studied. However, the effect of digibind on digitoxin measurements has not been studied thoroughly. The authors studied the effect of digibind on the measurement of total and free digitoxin in vitrousing the fluorescence polarization immunoassay and a new chemiluminescent immunoassay. We also studied the capability of digibind to bind digitoxigenin, the major aglycon metabolite of digitoxin. Digibind neutralized both digitoxin and digitoxigenin in vitro, as evidenced by significant reductions in free digitoxin and digitoxigenin (measured as digitoxin equivalent) concentrations. Digibind caused negative interference in the measurement of total digitoxin concentrations by both fluorescence polarization and chemiluminescent assays. However, the magnitude of negative interference was significantly higher with the chemiluminescent assay. For example, in a serum pool supplemented with 80 ng/mL of digitoxin, the concentrations of total and free digitoxin measured by the fluorescence polarization immunoassay were 82.1 ng/mL and 3.3 ng/mL respectively. In the presence of 5 µg/mL of Digibind, the corresponding total and free digitoxin concentrations were 73.9 ng/mL and none detected, respectively. In another serum pool supplemented with 70 ng/mL of digitoxin, the concentrations of total and free digitoxin as measured by the chemiluminescent assay were 69.1 ng/mL and 3.8 ng/mL, respectively. In the presence of 5 µg/mL of Digibind, the corresponding total and free digitoxin concentrations were 29.0 ng/mL and none detected, respectively. Because this effect may also occur in vivo, the progress of Digibind therapy in treating a patient with digitoxin overdose may be monitored by measuring the free digitoxin concentrations.

Endogeneous digoxin-like immunoreactive factors (DLIF) can interfere with some digoxin immunoassays. We looked for similar interference, called digitoxin-like immunoreactive factors (DTLIF) in two digitoxin immunoassays: A new chemiluminescent assay (CLIA), processed on the automated random access immunoassay system ACS:180, and a fluorescent polarization assay (FPIA), processed on the semiautomated TDx batch analyzer. One hundred thirty-seven samples of sera were tested from nondigitalized pregnant women, patients with liver or kidney diseases, and cord blood. The CLIA digitoxin assay uses a murine monoclonal antibody and requires no sample pretreatment; the FPIA digitoxin assay uses a polyclonal rabbit antibody and requires sample precipitation. Both assays have a similar dynamic range and sensitivity and give comparable results with commercial controls and external quality control survey samples. Although the CLIA detected no digitoxin in any sample tested, the FPIA showed apparent digitoxin concentrations of more than 2.0 ng/ml for 100% and 44% among cord blood and liver disease specimens, respectively. The highest DTLIF concentration was found in serum from a patient with liver disease (18.1 ng/ml). When spiked with 32 ng/ml digitoxin, six of the samples containing DTLIF generated FPIA digitoxin values of 6% to 27.5% more than the expected digitoxin levels. Two specimens with no detectable DTLIF activity were run as controls, and when spiked with digitoxin, showed target digitoxin concentrations in the FPIA. The CLIA recovered near the target digitoxin values (32 ng/ml) in all spiked samples. It was concluded that the polyclonal FPIA digitoxin assay may give discordant digitoxin concentrations in some patient groups because of interference from digitoxin-like immunoreactive factors. The CLIA digitoxin assay is not affected by DTLIF interference.

The presence of hydrophobic sites in the lectin-I molecule was indicated by hydrophobic probes like 1-anilinonapthalene-8-sulfonic acid (ANS), 2-p-toluidinyl napthalene-6-sulfonic acid (TNS), N-phenyl-1-napthylamine (NA) and rose bengal (RB). This was further confirmed by amino acid modifications in the hydrophobic region of the lectin-I molecule. The binding of ANS, TNS, NA and RB to lectin-I was affected in the presence of NaCl. The involvement of hydrophobic interactions in rice-bean lectin-I-endogenous lectin receptor (ELR) complex were indicated by alterations in the circular dichroism and fluorescence emission spectra. The percentage of β-conformation (55–63%) of lectin-I was decreased by addition of ELR. ELR on reacting with lectin-I reduced the fluorescence emissions of the hydrophobic probes while fluorescence emission of ANS, TNS, NA and RB were greatly enhanced in presence of lectin-I alone. N-aceyl-galactosamine did not change the fluorescence emissions of any of the hydrophobic probes in presence or in absence of lectin-I. This demonstrates that carbohydrate and hydrophobic sites may be different and non-interacting. It is proposed that the ELR in reacting with lectin-I, induced conformational changes in the lectin-I molecule and thereby affected its erythroagglutinating activity with human blood group “A” erythrocytes.

The importance of high specificity in immunoassays used in therapeutic monitoring is highlighted by a case study in which therapeutic-to-toxic borderline digoxin levels were measured by a digoxin immunoassay in the serum sample from a patient administered digitoxin rather than digoxin. The sample, mistakenly sent to the laboratory for digoxin analysis, gave discordant results in three digoxin immunoassays: 1.99 and 0.79 ng/ml in assays using polyclonal antibodies (fluorescence-polarization immunoassay and microparticle enzyme immunoassay, respectively), and <0.1 ng/ml in a chemiluminescent immunoassay using more specific monoclonal antibody. The presence of digitoxin(∼40 ng/ml) in the sample was confirmed by three different digitoxin immunoassays. Based on these results, the interference of different levels of digitoxin was studied in the presence of 0, 0.85, 1.9, and 4.7 ng/ml digoxin in all three digoxin assays. The chemiluminescent assay showed no significant interference. The fluorescence-polarization immunoassay showed positive interference in all cases; however, the microparticle enzyme immunoassay showed a bidirectional interference: a positive interference observed at digoxin level <1.8 ng/ml, changing to a negative interference at higher digoxin concentrations. The authors conclude that in countries such as Germany, where both digoxin and digitoxin may be prescribed, caution should be used to interpret digoxin immunoassay results. Digoxin assays, with cross-reactivity to digitoxin <0.1% should be used.

Intoxication caused by digitalis-like substances after ingestion of cooked toad soup has been reported. Bufalin, a cardioactive compound, is found in toad. Bufalin is also found in many Chinese medicines. Earlier reports demonstrated cross reactivity of bufalin with fluorescence polarization immunoassay for digoxin. In this report, the authors demonstrated a significantly higher cross reactivity of bufalin with the fluorescence polarization assay for digitoxin. They supplemented aliquots of normal plasma that had various concentrations of bufalin (1 to 50 ng/ml) from a local blood bank and measured apparent digitoxin concentrations using fluorescence polarization immunoassay and chemiluminescent assays (ACS digitoxin) for digitoxin. They measured apparent digoxin and digitoxin concentrations using fluorescence polarization, microparticle enzyme immunoassay, and chemiluminescent assays for digitoxin. They observed apparent digitoxin or digoxin concentrations in sera supplemented with bufalin only with the fluorescence polarization assays. For example, the apparent digitoxin concentration observed in a serum supplemented with 25 ng/ml of bufalin was 24.3 ng/ml of digitoxin equivalent. The apparent digoxin concentration observed in the same specimen was 1.33 ng/ml digoxin equivalent. Bufalin caused positive interference in serum digoxin or digitoxin measurements in specimens containing digoxin or digitoxin when concentrations were measured by fluorescence polarization assays. In contrast, bufalin lowered the measured digoxin concentrations in serum pools containing digoxin when digoxin concentrations were measured by the microparticle enzyme immunoassay. The authors conclude that bufalin toxicity can be rapidly detected by the fluorescence polarization assay for digitoxin.

A lectin from early long pod var. of Vicia faba seed has been purified to homogeneity on chitin. The purified lectin is shown to be homogeneous in nature by Bio Gel P - 150 gel filtration, fast protein liquid chromatography and polyacrylamide gel electrophoresis. The lectin is a glycoprotein with molecular weight of 51,000. The lectin molecule is possibly composed of two types of subunits devoid of any covalent linking through sulfhydryl groups, with molecular weights 9,000 and 15,000 respectively in the ratio 2:2. The purified lectin shows a high affinity for N-acetyl-D-glucosamine (GlcNAc).Amino acid analyses show that cysteine and methionine are absent, and a high proportion of aspartic acid and glutamic acid are present in the protein molecule. The extinction coefficient of the purified lectin is 7.22. The lectin behaves as a, cold agglutinin displaying stronger agglutination than the naturally occurring ABO agglutinin in the cold.

The authors, as a beta testing site, evaluated the ACS:180-phenytoin chemiluminescent assay (Ciba-Corning Diagnostics Corp., Medfield, MA, U.S.A.) by comparing its performance with a widely used fluorescence assay for phenytoin (Abbott Laboratories, Abbott Park, IL, U.S.A.). The ACS:180-phenytoin assays were run on a ACS-180 analyzer and fluorescence polarization assays on a TDx analyzer. The within-run precision for ACS-phenytoin assay was determined using controls obtained from Ciba-Corning. The CVs were 2.9% for low control (X = 5.5, SD = 0.16 µg/ml, n = 10), 2.8% for the medium control (X = 13.4, SD = 0.37 µg/ml, n = 10), and 2.7% for the high control (X = 24.6, SD = 0.66 µg/ml, n = 10). The corresponding between run precisions were 4.1% for the low control (X = 5.4, SD = 0.22 mg/ml, n = 10), 3.1% for the medium control (X = 13.8, SD = 0.43 mg/ml, n = 10), and 2.9% (X = 24.5, SD = 0.70 mg/ml, n = 10) for the high control. The assay was linear from 0.5 to 40 µg/ml of serum phenytoin concentrations with a detection limit of 0.24 µg/ml. The recoveries were 93-97% for concentrations of phenytoin of 5-30 µg/ml. They also compared 111 serum specimens collected from patients receiving phenytoin. The concentrations of phenytoin ranged from none detected to 32.4 µg/ml. Using fluorescence polarization assay as x-axis (reference method) and ACS:180-phenytoin assay as y-axis, they obtained the following regression line: y= 1.0x- 0.26, r= 0.993. They conclude that the ACS-phenytoin assay has a good precision and that the results correlate well with the fluorescence polarization assay.

High concentrations of digoxin-like immunoreactive substances (DLIS) artificially increase serum digoxin concentrations. However, DLIS are absent in the protein-free ultrafiltrate because of their strong binding with serum macromolecules, whereas 75% of digoxin can be found in the ultrafiltrate. Using regression analysis, we devised equations by which total digoxin concentration can be calculated from free digoxin and albumin concentrations in serum. We used two different assays, fluorescence polarization and chemiluminescence, for measuring total and free-digoxin concentrations in sera. Both equations were very similar. Because measured concentrations of digoxin in the serum exhibit DLIS interferences, the measured concentrations were sometimes higher in volume-expanded patients than the calculated digoxin concentrations. We also estimated the extent of interferences from DLIS by subtracting the calculated digoxin concentration from the measured digoxin concentration in volume-expanded patients.

On binding toVicia fabalectin, the fluorescence of 4-methylumbelliferyl-α-D-glucoPyranoside was quantitatively quenched showing that the interaction of 4-methylumbelliferyl-α-D-glucoPyranoside took Place in a binding environment. The binding of the fluorescent sugar was saccharide sPecific as evidenced by the reversal of 4-methylumbelliferyl-α-D-glucoPyranoside fluorescence quenching by D-fructose. The association constant,Ka, values for the 4-methylumbelliferyl-α-D-glucoPyranoside was determined by comPetition study emPloying reversal of fluorescence quenching of 4-methylumbelliferyl-α-D-glucoPyranoside by D-fructose. TheKavalue obtained for D-fructose was 1.07 ±0.03 X 104M-1and for 4-methylumbelliferyl-α-D-glucoPyranoside was 1.60 ±0.05 X 104M-1at 15°C. TheKavalues of 2.51 ±0.06 X 104M-1, l.26 ±0.02 X 104M-1and 0.56 ±0.01 X 104M-1, resPectively at 10°, 20° and 30°C were obtained from the ChiPman equation. The relative fluorescence quenching, ΔFa, at infinite concentration of the free saccharide sites ofVicia fabalectin [P′] was 93.5% at 30°C and the binding constant for 4-methylumbelliferyl-α-D-glucoPyranoside lectin interaction as derived by Yank and Hanaguchi equation was 0.63 ±0.01 X 104M-1.

Digitoxin is very strongly bound to serum albumin. Although free digitoxin is pharmacologically active, it is not monitored because of the lack of a sufficiently sensitive technique. The concentration of free digitoxin in the protein-free ultrafiltrate is usually below the detection limit of digitoxin immunoassays. A modified technique is described by which free digitoxin can be routinely monitored using commercially available immunoassays. The fluorescence polarization immunoassay for determining total digitoxin concentration requires that 100 L of serum be treated with 300 L of methanol to precipitate proteins. It is demonstrated that free digitoxin can easily be measured by adding 100 L of methanol to 300 L of ultrafiltrate, thus improving the sensitivity of the assay three-fold. The free digitoxin concentration can easily be calculated by dividing the observed value by 3. An attempt to use only ultrafiltrate (no methanol added) caused significant bias in the result, probably as a result of a matrix problem. The chemiluminescent assay for digitoxin does not require any specimen pretreatment and requires only 10 L of serum. The program was modified and used 50 L of ultrafiltrate to improve the sensitivity of the free digitoxin assay. If the chemiluminescent assay is used to measure free digitoxin, the true free digitoxin concentration can be calculated by dividing the observed value by 4.3. The free digitoxin concentrations were comparable in eight patients receiving digitoxin as measured by both methods. To show an application of this technique, two serum pools were prepared from patients receiving digitoxin and supplemented with various concentrations of phenytoin. A significant increase in free digitoxin concentration was observed because of the displacement of digitoxin from protein binding sites by phenytoin.