Development of a Dengue Virus Serotype-Specific Non-Structural Protein 1 Capture Immunochromatography Method

Four serotypes of dengue virus (DENV), type 1 to 4 (DENV-1 to DENV-4), exhibit approximately 25–40% of the difference in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from specimens followed by PCR amplification is the current standard method of DENV serotype determination. However, since this method is time-consuming, rapid detection systems are desirable. We established several mouse monoclonal antibodies directed against DENV non-structural protein 1 and integrated them into rapid DENV detection systems. We successfully developed serotype-specific immunochromatography systems for all four DENV serotypes. Each system can detect 104 copies/mL in 15 min using laboratory and clinical isolates of DENV. No cross-reaction between DENV serotypes was observed in these DENV isolates. We also confirmed that there was no cross-reaction with chikungunya, Japanese encephalitis, Sindbis, and Zika viruses. Evaluation of these systems using serum from DENV-infected individuals indicated a serotype specificity of almost 100%. These assay systems could accelerate both DENV infection diagnosis and epidemiologic studies in DENV-endemic areas.

Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.

Cross-reaction between mouse and rat immunoglobulin G: does it matter in sandwich ELISA?

Background Sandwich ELISA is an ideal antigen detection and quantification assay. Recently, it was used as the basic concept for high technology diagnostics. The specificity of the assay depends on the exclusion of detection cross-reactivity which arises from using two antibodies developed in different species. Since mice and rats are the common laboratory animals used to develop antigen specific antibodies. Therefore, the questions we addressed here were (1) can one use antigen-specific antibodies raised in mice and rats in the same assay to specifically detect/quantify antigens? and (2) which antibodies of the two rodents should be placed for capturing and for detection in the antigen-detection sandwich? Results Direct ELISA assay was used to assess for the specific reaction of the HRP-conjugated antibody to the target serum. First reaction was to compare between either conjugate anti-rat IgG (homologous) or anti-mouse IgG (heterologous) for the detection of rat sera IgG. Following the dilution factor optimization, the O.D. were 0.744±0.051 and 0.604±0.05, respectively (p= .004). The difference in mean O.D. of 0.14 reflected an unaccepted non-specific reaction. The second reaction was to compare between either conjugate anti-mouse IgG (homologous) and anti-rat IgG (heterologous) for the detection of mouse sera IgG. The recorded O.D. were 0.9414±0.14 and 0.317 ±0.141, respectively (p= .0002). The improved difference in mean O.D. of 0.624 reflecting a minimized cross-reaction. Conclusions Our results suggest that it is possible to use both Swiss albino mice and albino rats in a single sandwich ELISA, given that the captured antibody species to be from the Swiss albino mice and the detection antibody to be from the albino rat. The described working details are limited to the source of the antibodies used in the study. However, the approach stresses on the importance of such optimization steps before making any interpretations based on the antigen detection. To our knowledge, this study is the first to cover the optimal order of the capturing and the detection antibodies in a sandwich ELISA assay. In addition to addressing the possible interfering cross-reactivity that result from using mouse and rat serum antibodies in a single assay. Graphical abstract

Absolute Absorption Cross-Section of the Ã←X˜ Electronic Transition of the Ethyl Peroxy Radical and Rate Constant of Its Cross Reaction with HO2

The absolute absorption cross-section of the ethyl peroxy radical C2H5O2 in the Ã←X˜ electronic transition with the peak wavelength at 7596 cm−1 has been determined by the method of dual wavelengths time resolved continuous wave cavity ring down spectroscopy. C2H5O2 radicals were generated from pulsed 351 nm photolysis of C2H6/Cl2 mixture in presence of 100 Torr O2 at T = 295 K. C2H5O2 radicals were detected on one of the CRDS paths. Two methods have been applied for the determination of the C2H5O2 absorption cross-section: (i) based on Cl-atoms being converted alternatively to either C2H5O2 by adding C2H6 or to hydro peroxy radicals, HO2, by adding CH3OH to the mixture, whereby HO2 was reliably quantified on the second CRDS path in the 2ν1 vibrational overtone at 6638.2 cm−1 (ii) based on the reaction of C2H5O2 with HO2, measured under either excess HO2 or under excess C2H5O2 concentration. Both methods lead to the same peak absorption cross-section for C2H5O2 at 7596 cm−1 of σ = (1.0 ± 0.2) × 10−20 cm2. The rate constant for the cross reaction between of C2H5O2 and HO2 has been measured to be (6.2 ± 1.5) × 10−12 cm3 molecule−1 s−1.

Non-Specific Binding and Cross-Reaction of ELISA: A Case Study of Porcine Hemoglobin Detection

Different types of enzyme-linked immunosorbent assays (ELISA) have been widely used to control food safety and quality. To develop an accurate and reproducible ELISA, false immunodetection results caused by non-specific binding (NSB) and cross-reaction must be prevented. During the case study of sandwich ELISA development for the detection of porcine hemoglobin (PHb), several critical factors leading to NSB and cross-reaction were found. First, to reduce the NSB of the target analyte, the selection of microplate and blocker was discussed. Second, cross-reactions between enzyme-labeled secondary antibodies and sample proteins were demonstrated. In addition, the function of (3-aminopropyl)triethoxysilane (APTES) was evaluated. Overall, this study highlights the essence of both antibody and assay validation to minimize any false-positive/negative immunodetection results.

Cross-Reaction or Co-Infection? Serological Discrimination of Antibodies Directed against Dugbe and Crimean-Congo Hemorrhagic Fever Orthonairovirus in Nigerian Cattle

Dugbe orthonairovirus (DUGV) and Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) are tick-borne arboviruses within the order Bunyavirales. Both viruses are endemic in several African countries and can induce mild (DUGV, BSL 3) or fatal (CCHFV, BSL 4) disease in humans. Ruminants play a major role in their natural transmission cycle. Therefore, they are considered as suitable indicator animals for serological monitoring studies to assess the risk for human infections. Although both viruses do not actually belong to the same serogroup, cross-reactivities have already been reported earlier—hence, the correct serological discrimination of DUGV and CCHFV antibodies is crucial. In this study, 300 Nigerian cattle sera (150 CCHFV seropositive and seronegative samples, respectively) were screened for DUGV antibodies via N protein-based ELISA, indirect immunofluorescence (iIFA) and neutralization assays. Whereas no correlation between the CCHFV antibody status and DUGV seroprevalence data could be demonstrated with a newly established DUGV ELISA, significant cross-reactivities were observed in an immunofluorescence assay. Moreover, DUGV seropositive samples did also cross-react in a species-adapted commercial CCHFV iIFA. Therefore, ELISAs seem to be able to reliably differentiate between DUGV and CCHFV antibodies and should preferentially be used for monitoring studies. Positive iIFA results should always be confirmed by ELISAs.

Absolute Absorption Cross Section of the Ã←X ̃ Electronic Transition of the Ethyl Peroxy Radical and Rate Constant of its Cross Reaction with HO2

The absolute absorption cross section of the ethyl peroxy radical, C2H5O2, in the Ã←X ̃ electronic transition with the peak wavelength at 7596 cm-1, has been determined by the method of dual wavelengths time resolved continuous wave cavity ring down spectroscopy. C2H5O2 radicals were generated from pulsed 351 nm photolysis of C2H6/Cl2 mixture in presence of O2 and detected on one of the CRDS paths. Two methods have been applied for the determination of the C2H5O2 absorption cross section: (i) based on Cl-atoms being converted alternatively to either C2H5O2 by adding C2H6 or to hydro peroxy radicals, HO2, by adding CH3OH to the mixture, whereby HO2 was reliably quantified on the second CRDS path in the 21 vibrational overtone at 6638.2 cm-1 (ii) based on the reaction of C2H5O2 with HO2, measured under either excess HO2 or under excess C2H5O2 concentration. Both methods lead to the same peak absorption cross section of C2H5O2,7596 cm-1 = (1.0±0.2) × 10-20 cm2. The rate constant for the cross reaction between of C2H5O2 and HO2 has been measured to be (6.5±1.6) × 10-12 cm3 molecule-1 s-1.