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Fulltext Hide and Seek: The Interplay Between Zika Virus and the Host Immune Response

Lee LJ, Komarasamy TV, Adnan NAA, James W, Rmt Balasubramaniam V

Front Immunol, 2021;12:750365.
PMID: 34745123 DOI: 10.3389/fimmu.2021.750365

Zika virus (ZIKV) received worldwide attention over the past decade when outbreaks of the disease were found to be associated with severe neurological syndromes and congenital abnormalities. Unlike most other flaviviruses, ZIKV can spread through sexual and transplacental transmission, adding to the complexity of Zika pathogenesis and clinical outcomes. In addition, the spread of ZIKV in flavivirus-endemic regions, and the high degree of structural and sequence homology between Zika and its close cousin Dengue have raised questions on the interplay between ZIKV and the pre-existing immunity to other flaviviruses and the potential immunopathogenesis. The Zika epidemic peaked in 2016 and has affected over 80 countries worldwide. The re-emergence of large-scale outbreaks in the future is certainly a possibility. To date, there has been no approved antiviral or vaccine against the ZIKV. Therefore, continuing Zika research and developing an effective antiviral and vaccine is essential to prepare the world for a future Zika epidemic. For this purpose, an in-depth understanding of ZIKV interaction with many different pathways in the human host and how it exploits the host immune response is required. For successful infection, the virus has developed elaborate mechanisms to escape the host response, including blocking host interferon response and shutdown of certain host cell translation. This review provides a summary on the key host factors that facilitate ZIKV entry and replication and the mechanisms by which ZIKV antagonizes antiviral innate immune response and involvement of adaptive immune response leading to immunopathology. We also discuss how ZIKV modulates the host immune response during sexual transmission and pregnancy to induce infection, how the cross-reactive immunity from other flaviviruses impacts ZIKV infection, and provide an update on the current status of ZIKV vaccine development.

Matched MeSH terms: Cross Reactions
Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay

Ng LF, Barr I, Nguyen T, Noor SM, Tan RS, Agathe LV, et al.

BMC Infect Dis, 2006;6:40.
PMID: 16512903

Continuous outbreaks of the highly pathogenic H5N1 avian influenza A in Asia has resulted in an urgent effort to improve current diagnostics to aid containment of the virus and lower the threat of a influenza pandemic. We report here the development of a PCR-based assay that is highly specific for the H5N1 avian influenza A virus.

Matched MeSH terms: Cross Reactions
Fulltext Effective equine immunization protocol for production of potent poly-specific antisera against Calloselasma rhodostoma, Cryptelytrops albolabris and Daboia siamensis

Sapsutthipas S, Leong PK, Akesowan S, Pratanaphon R, Tan NH, Ratanabanangkoon K

PLoS Negl Trop Dis, 2015 Mar;9(3):e0003609.
PMID: 25774998 DOI: 10.1371/journal.pntd.0003609

Snake envenomation has been estimated to affect 1.8 million people annually with about 94,000 deaths mostly in poor tropical countries. Specific antivenoms are the only rational and effective therapy for these cases. Efforts are being made to produce effective, affordable and sufficient antivenoms for these victims. The immunization process, which has rarely been described in detail, is one step that needs to be rigorously studied and improved especially with regard to the production of polyspecific antisera. The polyspecific nature of therapeutic antivenom could obviate the need to identify the culprit snake species. The aim of this study was to produce potent polyspecific antisera against 3 medically important vipers of Thailand and its neighboring countries, namely Cryptelytrops albolabris "White lipped pit viper" (CA), Calleoselasma rhodostoma "Malayan pit viper" (CR), and Daboia siamensis "Russell's viper" (DS). Four horses were immunized with a mixture of the 3 viper venoms using the 'low dose, low volume multi-site' immunization protocol. The antisera showed rapid rise in ELISA titers against the 3 venoms and reached plateau at about the 8th week post-immunization. The in vivo neutralization potency (P) of the antisera against CA, CR and DS venoms was 10.40, 2.42 and 0.76 mg/ml, respectively and was much higher than the minimal potency limits set by Queen Soavabha Memorial Institute (QSMI). The corresponding potency values for the QSMI monospecific antisera against CA, CR and DS venoms were 7.28, 3.12 and 1.50 mg/ml, respectively. The polyspecific antisera also effectively neutralized the procoagulant, hemorrhagic, necrotic and nephrotoxic activities of the viper venoms. This effective immunization protocol should be useful in the production of potent polyspecific antisera against snake venoms, and equine antisera against tetanus, diphtheria or rabies.

Matched MeSH terms: Cross Reactions
A simple adherence test for detection of IgM antibodies in typhoid

Ong LY, Pang T, Lim SH, Tan EL, Puthucheary SD

J Med Microbiol, 1989 Jul;29(3):195-8.
PMID: 2473209

A simple adherence test to detect IgM antibodies in patients with typhoid is described. The test utilises the IgM-"capture" approach, in which the test serum is applied to microtitration plate wells previously coated with anti-human IgM, followed by application of a stained Salmonella typhi antigen suspension which shows adherence in positive cases. By this test, 58 (95%) of 61 sera from confirmed cases of typhoid possessed IgM antibodies to the H or O or both antigens of S. typhi. In patients for whom a diagnosis of typhoid was based only on a significant Widal-test titre, 31 (41%) of 76 sera had IgM antibodies to the H or O or both antigens of S. typhi. Some cross-reactivity of the IgM antibodies was detected, especially with the O antigens of S. paratyphi A and B. A total of 82 sera from non-typhoidal fevers (leptospirosis, typhus, dengue fever) showed no reactivity in this test. In normal sera there was no detectable IgM to the O antigen of S. typhi and only a small number (3.9%) had low levels of IgM to the H antigen. The significance and potential importance of this simple, sensitive, specific and economical test is discussed.

Matched MeSH terms: Cross Reactions
Serum IgA cross-reactivity between glycine-alanine repeat sequence of EBNA-1 and keratin or collagen in nasopharyngeal carcinoma

Mathew A, Cheng HM, Sam CK, Prasad U

Clin. Immunol. Immunopathol., 1994 May;71(2):164-8.
PMID: 7514112

Inhibition studies were carried out to study possible cross-reactivity between a peptide fragment of the Epstein-Barr virus nuclear antigen, EBNA-1, and keratin/collagen. The 20-amino acid peptide (pAG), derived from a glycine-alanine repeat region of EBNA-1, uniquely makes up about one-third of the viral protein and is a dominant IgA antigenic epitope in patients with nasopharyngeal carcinoma (NPC). A small percentage of normal human sera (NHS) also binds pAG and this reactivity is examined in this study. Ten percent (2/20) and 13.4% (2/15) of IgA-pAG-positive NPC sera and NHS, respectively, were significantly inhibited by keratin in a competitive ELISA system. Conversely, 31.6% (6/19) and 30.8% (4/13) of IgA-keratin-positive NPC sera and NHS, respectively, were significantly inhibited by pAG. This indicated minimum cross-reactivity between IgA serum antibodies to EBNA-1 and keratin. Using collagen as inhibitor, none of 18 and only 2/13 IgA-pAG-positive NPC sera and NHS, respectively, were inhibited. In the collagen ELISA system, only 2/19 (10.5%) and 4/25 (16%) of IgA-collagen-positive NPC sera and NHS, respectively, were inhibited with pAG. Therefore, cross-reactivity with collagen was also low. IgA-pAG-positive NHS may therefore not be a false positive phenomenon, but whether it may represent an early serological profile related to NPC carcinogenesis remains to be determined.

Matched MeSH terms: Cross Reactions
An investigation on the antigenic cross-reactivity of Calloselasma rhodostoma (Malayan pit viper) venom hemorrhagin, thrombin-like enzyme and L-amino acid oxidase using enzyme-linked immunosorbent assay

Tan NH, Ponnudurai G

Toxicon, 1994 Oct;32(10):1265-9.
PMID: 7846697

Indirect ELISA shows that the antibodies to Calloselasma rhodostoma venom hemorrhagin (CR-HMG), thrombin-like enzyme (CR-TLE) and L-amino acid oxidase (CR-LAAO) exhibited strong to moderate cross-reactions with most crotalid and viperid venoms, but only anti-CR-LAAO cross-reacted with the elapid venoms. However, the indirect ELISA failed to detect some antigenic similarities demonstrable by cross-neutralization study. The double-sandwich ELISA for the three anti-C. rhodostoma venom components exhibited a much lower level of cross-reactions than the indirect ELISA.

Matched MeSH terms: Cross Reactions
Cross-reactivity of monovalent and polyvalent Trimeresurus antivenoms with venoms from various species of Trimeresurus (lance-headed pit viper) snake

Tan NH, Choy SK, Chin KM, Ponnudurai G

Toxicon, 1994 Jul;32(7):849-53.
PMID: 7940592

Trimeresurus bite is a serious medical problem in Asia. However, at present only a few monospecific Trimeresurus antivenoms are available. Investigation of the cross-neutralization capacity of three Trimeresurus antivenoms indicates that the antivenoms exhibit broad cross-reactivity. A polyvalent Trimeresurus antivenom was also found to be effective in neutralization of the haemorrhagic, necrotizing and thrombin-like activities of heterologous Trimeresurus venoms.

Matched MeSH terms: Cross Reactions
Detection of antibodies against Salmonella typhi outer membrane protein (OMP) preparation in typhoid fever patients

Verdugo-Rodriguez A, Gam LH, Devi S, Koh CL, Puthucheary SD, Calva E, et al.

Asian Pac J Allergy Immunol, 1993 Jun;11(1):45-52.
PMID: 8216558

An indirect ELISA was used to detect antibodies against outer membrane protein preparations (OMPs) from Salmonella typhi. Sera from patients with a definitive diagnosis of typhoid fever (TF) gave a mean absorbance reading, at 414 nm, of 1.52 +/- 0.23 as compared to 0.30 +/- 0.11 for sera from healthy individuals. This gave a positive to negative ratio of absorbance readings of approximately 5.1. Suspected TF patients (no isolation of S. typhi), with positive and negative Widal titers had mean absorbance readings of 1.282 +/00.46 and 0.25 +/- 0.19, respectively. Sera from patients with leptospirosis, rickettsial typhus, dengue fever, and other infections gave mean absorbances of 0.20 +/- 0.08, 0.24 +/- 0.08, 0.27 +/- 0.08, and 0.31 +/- 0.16, respectively. The sensitivity, specificity, positive and negative predictive values were 100%, 94%, 80% and 100%, respectively. The antibody response detected in the definitive TF cases was predominantly IgG in nature and no cross-reactivity was seen with OMP preparations extracted from E. coli. Variable reactivity was noted with OMP preparations obtained from other Salmonella spp. Three major OMPs are presented in the antigen preparation and strong binding of positive sera was detected to all three bands.

Matched MeSH terms: Cross Reactions
Fulltext Identification of tropomyosin and arginine kinase as major allergens of Portunus pelagicus (blue swimming crab)

Rosmilah M, Shahnaz M, Zailatul HM, Noormalin A, Normilah I

Trop Biomed, 2012 Sep;29(3):467-78.
PMID: 23018510

Crab is an important source of food allergen. Tropomyosin represents the main crab allergen and is responsible for IgE cross-reactivity between various species of crustaceans. Recently, other new crab allergens including arginine kinase have been identified. However, information on allergens of the local Portunidcrab is not available. Thus, the aim of this study was to identify the major allergens of Portunus pelagicus (blue swimming crab) using the allergenomics approach. Raw and cooked extracts of the crab were prepared from the crab meat. Protein profile and IgE binding pattern were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from 30 patients with crab allergy. The major allergens of the crab were then identified by two-dimensional electrophoresis (2-DE), followed by mass spectrometry analysis of the peptide digests. The SDS-PAGE of raw extract revealed approximately 20 protein fractions over a wide molecular weight range, while cooked extract demonstrated fewer protein bands. The raw extract also demonstrated a higher number of IgE reactive bands than the cooked extract. A heat-resistant protein of 36 kDa has been identified as the major allergen in both raw and cooked extracts. In addition, a heat-sensitive protein of 41 kDa was also recognized as a major allergen in raw crab. The 2-DE gel profile of the raw extract demonstrated about >100 distinct proteins spots and immunoblotting of the 2-DE profile demonstrated at least 12 different major IgE reactive spots with molecular masses between 13 to 250 kDa and isoelectric point (pI) values ranging from 4.0 to 7.0. The 36 and 41 kDa proteins were identified as the crab tropomyosin and arginine kinase, respectively by mass spectrometry. Therefore, this study confirmed that tropomyosin and arginine kinase are the major allergens of the local Portunid crab, P. pelagicus.

Matched MeSH terms: Cross Reactions/immunology
Limited diagnostic value of two commercial rapid tests for acute leptospirosis detection in Malaysia

Chang CH, Riazi M, Yunus MH, Osman S, Noordin R

Diagn Microbiol Infect Dis, 2014 Dec;80(4):278-81.
PMID: 25241641 DOI: 10.1016/j.diagmicrobio.2014.08.012

This study evaluated 2 rapid leptospirosis serological tests, Leptorapide® (Linnodee, Northern Ireland) and VISITECT®-LEPTO (Omega Diagnostics, Scotland, UK), which are commonly used in Malaysia. A total of 183 samples comprised 113 sera from leptospirosis patients, and 70 sera from other infections and healthy controls were used. The leptospirosis sera were grouped into 2 serum panels, i.e., Group I (MAT+, PCR+) and Group II (MAT+). When inconclusive results were interpreted as positives, both tests showed lower diagnostic sensitivities (≤ 34%) with Group I sera, as compared to Group II sera (Leptorapide®, 93%; VISITECT®-LEPTO, 40%). When inconclusive results were interpreted as negatives, the 2 tests showed ~20% sensitivity with both serum panels. The diagnostic specificity of VISITECT®-LEPTO (94%) was superior to Leptorapide® (69%). Since both tests had misdiagnosed a large proportion of Group I patients and showed many inconclusive results among Group II patients, they have limited diagnostic value in detecting acute leptospirosis.

Matched MeSH terms: Cross Reactions
Fulltext Laboratory detection of strongyloidiasis: IgG-, IgG4 - and IgE-ELISAs and cross-reactivity with lymphatic filariasis

Norsyahida A, Riazi M, Sadjjadi SM, Muhammad Hafiznur Y, Low HC, Zeehaida M, et al.

Parasite Immunol., 2013 May-Jun;35(5-6):174-9.
PMID: 23448095 DOI: 10.1111/pim.12029

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG, IgG4 and IgE antibodies against Strongyloides stercoralis. A commercial ELISA (IVD Research, USA) was also used, and the sensitivities and specificities of the four assays were determined. Serum samples from 26 patients with S. stercoralis infection and 55 patients with other infections or no infection were analysed. Sensitivities of the IgG4 , IgG, IgE and IgG (IVD) assays were 76.9%, 84.6%, 7.7% and 84.6%, respectively, while the specificities were 92.7%, 81.8%, 100% and 83.6%, respectively. If filariasis samples were excluded, the specificities of the IgG4 -ELISA and both IgG-ELISAs increased to 100% and 98%, respectively. A significant positive correlation was observed between IgG- and IgG4 -ELISAs (r = 0.4828; P = 0.0125). IgG- and IgG- (IVD) ELISAs (r = 0.309) were positively correlated, but was not significant (P = 0.124). Meanwhile there was no correlation between IgG4 - and IgG- (IVD) ELISAs (r = 0.0042; P = 0.8294). Sera from brugian filariasis patients showed weak, positive correlation between the titres of antifilarial IgG4 and the optical densities of anti-Strongyloides IgG4 -ELISA (r = 0.4544, P = 0.0294). In conclusion, the detection of both anti-Strongyloides IgG4 and IgG antibodies could improve the serodiagnosis of human strongyloidiasis. Furthermore, patients from lymphatic filariasis endemic areas who are serologically diagnosed with strongyloidiasis should also be tested for filariasis.

Matched MeSH terms: Cross Reactions
Lateral Flow Dipstick Test for Serodiagnosis of Strongyloidiasis

Yunus MH, Arifin N, Balachandra D, Anuar NS, Noordin R

Am J Trop Med Hyg, 2019 08;101(2):432-435.
PMID: 31218996 DOI: 10.4269/ajtmh.19-0053

The conventional method of detecting Strongyloides stercoralis in fecal samples has poor diagnostic sensitivity. Detection of Strongyloides-specific antibodies increases the sensitivity; however, most tests are ELISAs that use parasite extract which may cross-react with the sera of other helminth infections. To improve the serological diagnosis of strongyloidiasis, this study aimed at developing a sensitive and specific lateral flow rapid dipstick test. Two recombinant proteins, recombinant NIE (rNIE) and recombinant Ss1a (rSs1a), were used in preparing the dipstick, with gold-conjugated antihuman IgG4 as detector reagent. In parallel, the corresponding ELISA was performed. Both assays demonstrated diagnostic sensitivity of 91.3% (21/23) when tested with serum samples of patients with Strongyloides infection, and 100% specificity with 82 sera of asymptomatic (healthy) and those with other parasitic infections. The ELISA and dipstick test results were positively correlated to each other (r = 0.6114, P = 0.0019). The developed lateral flow dipstick test may improve the serodiagnosis of strongyloidiasis and merit further validation studies.

Matched MeSH terms: Cross Reactions
IgA antiphospholipid antibodies in normal human saliva cross-react with bacterial lipopolysaccharide

Cheng HM, Ngeow YF

Int Arch Allergy Immunol, 1993;101(3):297-8.
PMID: 8324391
Matched MeSH terms: Cross Reactions
Fulltext Cross-reactivity in AED-Induced Severe Cutaneous Adverse Reaction: A Case Report

Khor AH, Lim KS, Tan CT, Kwan Z, Ng CC

J Investig Allergol Clin Immunol, 2016;26(5):329-331.
PMID: 27763865 DOI: 10.18176/jiaci.0085
Matched MeSH terms: Cross Reactions/immunology*
Evaluation of the cross-neutralization capacity of Thai green pit viper antivenom against venom of Myanmar green pit viper

Yee KT, Maw LZ, Kyaw AM, Khow O, Oo AW, Oo TKK, et al.

Toxicon, 2020 Apr 15;177:41-45.
PMID: 32056833 DOI: 10.1016/j.toxicon.2020.02.003

Green pit viper (Trimeresurus sp.) bite occurred throughout Myanmar, but there is no specific antivenom produced in the country for related envenomation. Instead, Myanmar Russell's viper antivenom (Anti-MRV) was often misused because of prolonged clotting time was observed from both species. Thai green pit viper antivenom (Anti-TGPV) raised against Trimeresurus albolabris was found to be effective against venoms of more than ten Trimeresurus sp. from Thailand, Malaysia and Indonesia. The present study compared the neutralization capacities of Anti-TGPV and Anti-MRV towards the venom from T. erythrurus from Myanmar. Anti-TGPV was more efficacious than Anti-MRV in cross-neutralizing the lethal and haemorrhagic activities of the venom by a potency of a least 1.4 times higher. Although Anti-TGPV effectively cross-neutralized the coagulation activity of the venom, Anti-MRV failed to do so. Immunodiffusion and immunoblot experiments showed that Anti-TGPV cross-reacted with more protein components of the venom than Anti-MRV. In conclusion, Anti-TGPV is a better choice for patients bitten by Myanmar green pit viper, but further clinical investigation is required. The current findings highlight the development of a specific antivenom against Myanmar green pit viper venom.

Matched MeSH terms: Cross Reactions
Fulltext Development of TaqMan-based real-time RT-PCR assay based on N gene for the quantitative detection of feline morbillivirus

Makhtar ST, Tan SW, Nasruddin NA, Abdul Aziz NA, Omar AR, Mustaffa-Kamal F

BMC Vet Res, 2021 Mar 23;17(1):128.
PMID: 33757494 DOI: 10.1186/s12917-021-02837-6

BACKGROUND: Morbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.
RESULTS: A specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34-0.53% and 1.38-2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.
CONCLUSIONS: The TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.

Matched MeSH terms: Cross Reactions
Component-resolved diagnosis (CRD): Is it worth it? frequency and differentiation in rhinitis patients with mite reactivity

Mohamad Yadzir ZH, Misnan R, Abdullah N, Bakhtiar F, Leecyous B, Murad S

Iran J Allergy Asthma Immunol, 2014 Aug;13(4):240-6.
PMID: 24659159

Component-resolved diagnosis (CRD) using microarray technology has recently been introduced with the aim to improve diagnosis of allergy. The aim of this study was to compare performance of this allergen microarray to those of an established extract-based skin prick testing (SPT).45 patients with allergic rhinitis were studied (16 children and 29 adults). SPT to Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis extracts and allergen microarray ImmunoCAP ISAC were carried out for all patients. Forty out of 45 patients demonstrated positive SPT to all mite extracts tested. These 40 patients were considered to be mite-allergic based on the positive SPT results. The remaining 5 patients with negative SPT to any mite extracts were classified as non-mite allergic. Comparatively, based on the microarray results, only 34 mite-allergic patients had detectable serum IgE to at least one of the mite allergen components tested whereas 6 patients with positive SPT to mite extracts showed no detectable IgE reactivity to any of the components tested. One non-mite allergic patient had a positive test- Blo t 5. Der p 10-positive patients also reacted to other cross-reactive tropomyosin from anisakis (Ani s 3) (25%), cockroach (Bla g 7) (50%) and shrimp (Pen m 1) (75%). CRD is a reliable tool for the diagnosis of allergy to mites. Der p 10 might be a useful indicator to identify a subset of mite-allergic patient that have additional sensitization due to cross-reactivity and thus allows selection of patients for immunotherapy.

Matched MeSH terms: Cross Reactions
Fulltext Cross-reactivity of Malaysian rat cytomegalovirus strains with its human counterpart

Camalxaman SN, Zeenathul NA, Quah YW, Loh HS, Zuridah H, Sheikh-Omar AR, et al.

Trop Biomed, 2011 Dec;28(3):661-7.
PMID: 22433897 MyJurnal

This study probes into the prospect of cross-reactivity of HCMV with RCMV which has not been acknowledged to date. We describe the uncovering of a protein with an estimated size of between 61-68 kDa from local RCMV strains which reacted with HCMV positive sera. Our findings are a first disclosure of a plausible immunological cross-reactivity between RCMV with its human counterpart which grounds substantial interest implying existence of conserved determinants between rat and human CMV polypeptides. The cross-reactive protein most likely represents an enveloped glycoprotein, though the precise identification and its degree of similarity needs to be evidently defined and further elucidated in forthcoming experiments.

Matched MeSH terms: Cross Reactions*
Fulltext An evaluation of a recombinant multiepitope based antigen for detection of Toxoplasma gondii specific antibodies

Hajissa K, Zakaria R, Suppian R, Mohamed Z

BMC Infect Dis, 2017 12 29;17(1):807.
PMID: 29284420 DOI: 10.1186/s12879-017-2920-9

BACKGROUND: The inefficiency of the current tachyzoite antigen-based serological assays for the serodiagnosis of Toxoplasma gondii infection mandates the need for acquirement of reliable and standard diagnostic reagents. Recently, epitope-based antigens have emerged as an alternative diagnostic marker for the achievement of highly sensitive and specific capture antigens. In this study, the diagnostic utility of a recombinant multiepitope antigen (USM.TOXO1) for the serodiagnosis of human toxoplasmosis was evaluated.
METHODS: An indirect enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the usefulness of USM.TOXO1 antigen for the detection of IgG antibodies against Toxoplasma gondii in human sera. Whereas the reactivity of the developed antigen against IgM antibody was evaluated by western blot and Dot enzyme immunoassay (dot-EIA) analysis.
RESULTS: The diagnostic performance of the new antigens in IgG ELISA was achieved at the maximum values of 85.43% and 81.25% for diagnostic sensitivity and specificity respectively. The USM.TOXO1 was also proven to be reactive with anti- T. gondii IgM antibody.
CONCLUSIONS: This finding makes the USM.TOXO1 antigen an attractive candidate for improving the toxoplasmosis serodiagnosis and demonstrates that multiepitope antigens could be a potential and promising diagnostic marker for the development of high sensitive and accurate assays.

Matched MeSH terms: Cross Reactions
Validation of an enzyme-linked immunosorbent assay screening method and a liquid chromatography-tandem mass spectrometry confirmation method for the identification and quantification of ketamine and norketamine in urine samples from Malaysia

Harun N, Anderson RA, Miller EI

J Anal Toxicol, 2009 8 6;33(6):310-21.
PMID: 19653934 DOI: 10.1093/jat/33.6.310

An ELISA and a liquid chromatography-tandem mass spectrometry (LC-MS-MS) confirmation method were developed and validated for the identification and quantitation of ketamine and its major metabolite norketamine in urine samples. The Neogen ketamine microplate ELISA was optimized with respect to sample and enzyme conjugate volumes and the sample preincubation time before addition of the enzyme conjugate. The ELISA kit was validated to include an assessment of the dose-response curve, intra- and interday precision, limit of detection (LOD), and cross-reactivity. The sensitivity and specificity were calculated by comparison to the results from the validated LC-MS-MS confirmation method. An LC-MS-MS method was developed and validated with respect to LOD, lower limit of quantitation (LLOQ), linearity, recovery, intra- and interday precision, and matrix effects. The ELISA dose-response curve was a typical S-shaped binding curve, with a linear portion of the graph observed between 25 and 500 ng/mL for ketamine. The cross-reactivity of 200 ng/mL norketamine to ketamine was 2.1%, and no cross-reactivity was detected with 13 common drugs tested at 10,000 ng/mL. The ELISA LOD was calculated to be 5 ng/mL. Both intra- (n = 10) and interday (n = 50) precisions were below 5.0% at 25 ng/mL. The LOD for ketamine and norketamine was calculated statistically to be 0.6 ng/mL. The LLOQ values were also calculated statistically and were 1.9 ng/mL and 2.1 ng/mL for ketamine and norketamine, respectively. The test linearity was 0-1200 ng/mL with correlation coefficient (R(2)) > 0.99 for both analytes. Recoveries at 50, 500, and 1000 ng/mL range from 97.9% to 113.3%. Intra- (n = 5) and interday (n = 25) precisions between extracts for ketamine and norketamine were excellent (< 10%). Matrix effects analysis showed an average ion suppression of 5.7% for ketamine and an average ion enhancement of 13.0% for norketamine for urine samples collected from six individuals. A comparison of ELISA and LC-MS-MS results demonstrated a sensitivity, specificity, and efficiency of 100%. These results indicated that a cutoff value of 25 ng/mL ketamine in the ELISA screen is particularly suitable and reliable for urine testing in a forensic toxicology setting. Furthermore, both ketamine and norketamine were detected in all 34 urine samples collected from individuals socializing in pubs by the Royal Malaysian Police. Ketamine concentrations detected by LC-MS-MS ranged from 22 to 31,670 ng/mL, and norketamine concentrations ranged from 25 to 10,990 ng/mL. The concentrations of ketamine and norketamine detected in the samples are most ikely indicative of ketamine abuse.

Matched MeSH terms: Cross Reactions

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