Discovering Cross-Reactivity in Urine Drug Screening Immunoassays through Large-Scale Analysis of Electronic Health Records.
ABSTRACT: BACKGROUND:Exposure to drugs of abuse is frequently assessed using urine drug screening (UDS) immunoassays. Although fast and relatively inexpensive, UDS assays often cross-react with unrelated compounds, which can lead to false-positive results and impair patient care. The current process of identifying cross-reactivity relies largely on case reports, making it sporadic and inefficient, and rendering knowledge of cross-reactivity incomplete. Here, we present a systematic approach to discover cross-reactive substances using data from electronic health records (EHRs). METHODS:Using our institution's EHR data, we assembled a data set of 698651 UDS results across 10 assays and linked each UDS result to the corresponding individual's previous medication exposures. We hypothesized that exposure to a cross-reactive ingredient would increase the odds of a false-positive screen. For 2201 assay-ingredient pairs, we quantified potential cross-reactivity as an odds ratio from logistic regression. We then evaluated cross-reactivity experimentally by spiking the ingredient or its metabolite into drug-free urine and testing the spiked samples on each assay. RESULTS:Our approach recovered multiple known cross-reactivities. After accounting for concurrent exposures to multiple ingredients, we selected 18 compounds (13 parent drugs and 5 metabolites) to evaluate experimentally. We validated 12 of 13 tested assay-ingredient pairs expected to show cross-reactivity by our analysis, discovering previously unknown cross-reactivities affecting assays for amphetamines, buprenorphine, cannabinoids, and methadone. CONCLUSIONS:Our findings can help laboratorians and providers interpret presumptive positive UDS results. Our data-driven approach can serve as a model for high-throughput discovery of substances that interfere with laboratory tests.
Project description:Hantavirus pulmonary syndrome (HPS) is a human disease caused by a newly identified hantavirus, which we will refer to as Four Corners virus (FCV). FCV is related most closely to Puumala virus (PUU) and to Prospect Hill virus (PHV). Twenty-five acute HPS serum samples were tested for immunoglobulin G (IgG) and IgM antibody reactivities to FCV-encoded recombinant proteins in Western blot (immunoblot) assays. All HPS serum samples contained both IgG and IgM antibodies to the FCV nucleocapsid (N) protein. FCV N antibodies cross-reacted with PUU N and PHV N proteins. A dominant FCV N epitope was mapped to the segment between amino acids 17 and 59 (QLVTARQKLKDAERAVELDPDDVNKSTLQSRRAAVSALETKLG). All HPS serum samples contained IgG antibodies to the FCV glycoprotein-1 (G1) protein, and 21 of 25 serum samples contained FCV G1 IgM antibodies. The FCV G1 antibodies did not cross-react with PUU G1 and PHV G1 proteins. The FCV G1 type-specific antibody reactivity mapped to a segment between amino acids 59 and 89 (LKIESSCNFDLHVPATTTQKYNQVDWTKKSS). One hundred twenty-eight control serum samples were tested for IgG reactivities to the FCV N and G1 proteins. Nine (7.0%) contained FCV N reactivities, 3 (2.3%) contained FCV G1 reactivities, and one (0.8%) contained both FCV N and FCV G1 reactivities. The epitopes recognized by antibodies present in control serum samples were different from the epitopes recognized by HPS antibodies, suggesting that the control antibody reactivities were unrelated to FCV infections. These reagents constitute a type-specific assay for FCV antibodies.
Project description:The accurate diagnosis and seroprevalence investigations of Zika virus (ZKV) infections remain complex due to cross reactivity with other flaviviruses. Two assay formats, both using labelled Zika virus NS1 antigen as a revealing agent (a double antigen binding assay, DABA, and an immunoglobulin Ig capture assay, G capture) were initially developed and compared with the indirect EuroimmunZ assay for the detection of anti-Zika antibody. Of 147 pre-Zika period serum samples, 39 (27%) were reactive in the EuroimmunZ or the DABA assays, 28 sera concordantly so. Such false reactivity was influenced by the serotype of Dengue virus (DV) to which individuals had been exposed to. Thus, of sera from patients undergoing secondary Dengue virus infection of known serotype, 91%, 45% and 28% of Dengue virus serotype 2, 3 and 4 respectively were reactive in one or more of the three assays. A novel method of quenching false sero-reactivity was therefore developed for the DABA and G capture assays. Initial addition of a single homologous Dengue virus serotype 3 NS1Ag quench significantly ablated false reactivities in the pre-Zika period sera. An equipotent quadrivalent quench comprising homologous Dengue virus serotypes 1 to 4 NS1Ag was shown to be optimum yet retained sensitivity for the detection of specific anti-Zika antibody. Comparing DABA and G capture assays using quenched and unquenched conjugates in comparison with EuroimmunZ early in the course of PCR-confirmed infection indicated that a significant component of the apparent early anti-ZIKA antibody response is likely to be due to a Zika virus-driven anamnestic anti-Dengue virus response. The increased specificity provided by homologous antigen quenching is likely to provide a significant improvement in sero-diagnostics and to be of clinical value.
Project description:Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures.
Project description:Enterotoxigenic Escherichia coli (ETEC) is a leading diarrheagenic bacterial pathogen among travelers and children in resource-limited regions. Adherence to host intestinal cells mediated by ETEC fimbriae is believed to be a critical first step in ETEC pathogenesis. These fimbriae are categorized into related classes based on sequence similarity, with members of the class 5 fimbrial family being the best characterized. The eight related members of the ETEC class 5 fimbrial family are subdivided into three subclasses (5a, 5b, and 5c) that share similar structural arrangements, including a fimbrial tip adhesin. However, sequence variability among the class 5 adhesins may hinder the generation of cross-protective antibodies. To better understand functional epitopes of the class 5 adhesins and their ability to induce intraclass antibody responses, we produced 28 antiadhesin monoclonal antibodies (MAbs) to representative adhesins CfaE, CsbD, and CotD, respectively. We determined the MAb cross-reactivities, localized the epitopes, and measured functional activities as potency in inhibition of hemagglutination induced by class 5 fimbria-bearing ETEC. The MAbs' reactivities to a panel of class 5 adhesins in enzyme-linked immunosorbent assays (ELISAs) revealed several reactivity patterns, including individual adhesin specificity, intrasubclass specificity, intersubclass specificity, and class-wide cross-reactivity, suggesting that some conserved epitopes, including two conserved arginines, are shared by the class 5 adhesins. However, the cross-reactive MAbs had functional activities limited to strains expressing colonization factor antigen I (CFA/I), coli surface antigen 17 (CS17), or CS1, suggesting that the breadth of functional activities of the MAbs was more restricted than the repertoire of cross-reactivities measured by ELISA. The results imply that multivalent adhesin-based ETEC vaccines or prophylactics need more than one active component to reach broad protection.
Project description:Monoclonal antibody-based two-site immunoradiometric assays are described for human insulin, proinsulin, 65-66 split and 32-33 split proinsulin. The detection limits of the assays lie in the range 0.8-2.5 pM. The assays for 65-66 and 32-33 split proinsulins do not distinguish between these substances and their respective C-terminal di-desamino derivatives. The assay of 65-66 split proinsulin does not cross-react with insulin, proinsulin or 32-33 split proinsulin. This material was undetectable (less than 1.0 pM) in plasma taken after an overnight fast in eight normal male subjects and the maximum individual concentration reached in plasma taken during an oral glucose tolerance test of these subjects was 3.8 pM. The proinsulin assay cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. The 32-33 split proinsulin assay cross-reacted 84 and 60% with proinsulin and 65-66 split proinsulin respectively. The insulin assay cross-reacted 5.3, 62 and 5.0% with intact proinsulin, 65-66 split proinsulin and 32-33 split proinsulin respectively. The very low concentration of 65-66 split proinsulin meant that this derivative did not interfere significantly with the specificity of the assays of proinsulin and insulin. The concentration of 32-33 split proinsulin could be calculated by subtracting the cross-reactivity of the measured proinsulin. The mean concentrations of insulin, proinsulin and 32-33 split proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 0.3, 2.3 +/- 0.3 and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4 and 19.7 +/- 6.0 respectively.
Project description:This study investigated the use of androgen receptor (AR) reporter gene assay data in a non-animal exposure-led risk assessment in which in vitro anti-androgenic activity and exposure data were put into context using a naturally occurring comparator substance with a history of dietary consumption. First, several dietary components were screened to identify which selectively interfered with AR signaling in vitro, using the AR CALUX® test. The IC50 values from these dose-response data together with measured or predicted human exposure levels were used to calculate exposure: activity ratios (EARs) for the dietary components and a number of other well-known anti-androgenic substances. Both diindolylmethane (DIM) and resveratrol are specifically acting dietary anti-androgens. The EARs for several anti-androgens were therefore expressed relative to the EAR of DIM, and how this 'dietary comparator ratio' (DCR) approach may be used to make safety decisions was assessed using an exposure-led case study for an anti-androgenic botanical ingredient. This highlights a pragmatic approach which allows novel chemical exposures to be put into context against dietary exposures to natural anti-androgenic substances. The DCR approach may have utility for other modes of action where appropriate comparators can be identified.
Project description:The number of human avian H7N9 influenza infections has been increasing in China. Understanding their antigenic and serologic relationships is crucial for developing diagnostic tools and vaccines. Here, we evaluated the cross-reactivities and neutralizing activities among H7 subtype influenza viruses and between H7N9 and heterosubtype influenza A viruses. We found strong cross-reactivities between H7N9 and divergent H7 subtypic viruses, including H7N2, H7N3, and H7N7. Antisera against H7N2, H7N3, and H7N7 could also effectively neutralize two distinct H7N9 strains. Two-way cross-reactivities exist within group 2, including H3 and H4, whereas one-way cross-reactivities were found across other groups, including H1, H10, H9, and H13. Our data indicate that the hemaglutinins from divergent H7 subtypes may facilitate the development of vaccines for distinct H7N9 infections. Moreover, serologic diagnoses for H7N9 infections need to consider possible interference from the cross-reactivity of H7N9 with other subtype influenza viruses.
Project description:There are currently 7 known serotypes of botulinum neurotoxin (BoNT) classified upon non-cross reactivity of neutralizing immunoglobulins. Non-neutralizing immunoglobulins, however, can exhibit cross-reactivities between 2 or more serotypes, particularly mosaic forms, which can hamper the development of highly specific immunoassays, especially if based on polyclonal antisera. Here we employ facile recombinant antibody technology to subtractively select ligands to each of the 7 BoNT serotypes, resulting in populations with very high specificity for their intended serotype.A single llama was immunized with a cocktail of 7 BoNT toxoids to generate a phage display library of single domain antibodies (sdAb, VHH or nanobodies) which were selected on live toxins. Resulting sdAb were capable of detecting both toxin and toxin complex with the best combinations able to detect 100s-10s of pg per 50 microL sample in a liquid bead array. The most sensitive sdAb were combined in a heptaplex assay to identify each of the BoNT serotypes in buffer and milk and to a lesser extent in carrot juice, orange juice and cola. Several anti-A(1) sdAb recognized A2 complex, showing that subtype cross-reactivity within a serotype was evident. Many of our sdAb could act as both captor and tracer for several toxin and toxin complexes suggesting sdAb can be used as architectural probes to indicate BoNT oligomerisation. Six of 14 anti-A clones exhibited inhibition of SNAP-25 cleavage in the neuro-2A assay indicating some sdAb had toxin neutralizing capabilities. Many sdAb were also shown to be refoldable after exposure to high temperatures in contrast to polyclonal antisera, as monitored by circular dichroism.Our panel of molecularly flexible antibodies should not only serve as a good starting point for ruggedizing assays and inhibitors, but enable the intricate architectures of BoNT toxins and complexes to be probed more extensively.
Project description:Urine drug testing by immunoassay is widely used to detect nonmedical drug use and to monitor patients prescribed controlled substances. A key attribute of urine drug testing immunoassays is cross-reactivity, namely the response of various compounds compared to the target of the assay. In this report, we analyzed the variability in how manufacturer cross-reactivity data are summarized in package inserts for commercially available amphetamines, benzodiazepines, and opiates immunoassays, 3 broad drug classes commonly included in routine drug testing panels. Specifically, we determined the number of compounds tested for cross-reactivity, manner in which cross-reactivity is measured, concentration units used, how often compounds known to be cross-reactive with marketed urine drug testing immunoassays prior to 2010 were tested, availability of the package insert online, and how often cross-reactivity on "designer drugs" was found in the package inserts. There was wide variability in the number of compounds tested (both positive and negative), with the highest number of tested compounds generally found in point-of-care urine drug testing applications. Most package inserts used ng/mL as the concentration units and expressed cross-reactivity in terms of equivalent concentrations to the assay calibrator. Approximately 50% of package inserts were directly available online. Cross-reactivity data were sparse with respect to "off-target" drugs known to be cross-reactive prior to 2010 (an example being quinolone antibiotics and opiates immunoassays) and designer drugs. The present study indicates lack of consistency in cross-reactivity information in package inserts, complicating the interpretation of urine drug testing results. We use 3 example clinical cases to illustrate practical challenges accessing and interpreting cross-reactivity data.
Project description:BACKGROUND: Immunoassays are widely used in clinical laboratories for measurement of plasma/serum concentrations of steroid hormones such as cortisol and testosterone. Immunoassays can be performed on a variety of standard clinical chemistry analyzers, thus allowing even small clinical laboratories to do analysis on-site. One limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Interfering molecules include structurally related endogenous compounds and their metabolites as well as drugs such as anabolic steroids and synthetic glucocorticoids. METHODS: Cross-reactivity of a structurally diverse set of compounds were determined for the Roche Diagnostics Elecsys assays for cortisol, dehydroepiandrosterone (DHEA) sulfate, estradiol, progesterone, and testosterone. These data were compared and contrasted to package insert data and published cross-reactivity studies for other marketed steroid hormone immunoassays. Cross-reactivity was computationally predicted using the technique of two-dimensional molecular similarity. RESULTS: The Roche Elecsys Cortisol and Testosterone II assays showed a wider range of cross-reactivity than the DHEA sulfate, Estradiol II, and Progesterone II assays. 6-Methylprednisolone and prednisolone showed high cross-reactivity for the cortisol assay, with high likelihood of clinically significant effect for patients administered these drugs. In addition, 21-deoxycortisol likely produces clinically relevant cross-reactivity for cortisol in patients with 21-hydroxylase deficiency, while 11-deoxycortisol may produce clinically relevant cross-reactivity in 11?-hydroxylase deficiency or following metyrapone challenge. Several anabolic steroids may produce clinically significant false positives on the testosterone assay, although interpretation is limited by sparse pharmacokinetic data for some of these drugs. Norethindrone therapy may impact immunoassay measurement of testosterone in women. Using two-dimensional similarity calculations, all compounds with high cross-reactivity also showed a high degree of similarity to the target molecule of the immunoassay. CONCLUSIONS: Compounds producing cross-reactivity in steroid hormone immunoassays generally have a high degree of structural similarity to the target hormone. Clinically significant interactions can occur with structurally similar drugs (e.g., prednisolone and cortisol immunoassays; methyltestosterone and testosterone immunoassays) or with endogenous compounds such as 21-deoxycortisol that can accumulate to very high concentrations in certain disease conditions. Simple similarity calculations can help triage compounds for future testing of assay cross-reactivity.