A 3D Microfluidic ELISA for the Detection of Severe Dengue: Sensitivity Improvement and Vroman Effect Amelioration by EDC–NHS Surface Modification

Serum is commonly used as a specimen in immunoassays but the presence of heterophilic antibodies can potentially interfere with the test results. Previously, we have developed a microfluidic device called: 3D Stack for enzyme-linked immunosorbent assay (ELISA). However, its evaluation was limited to detection from a single protein solution. Here, we investigated the sensitivity of the 3D Stack in detecting a severe dengue biomarker—soluble CD163 (sCD163)—within the serum matrix. To determine potential interactions with serum matrix, a spike-and-recovery assay was performed, using 3D Stacks with and without surface modification by an EDC–NHS (N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) coupling. Without surface modification, a reduced analyte recovery in proportion to serum concentration was observed because of the Vroman effect, which resulted in competitive displacement of coated capture antibodies by serum proteins with stronger binding affinities. However, EDC–NHS coupling prevented antibody desorption and improved the sensitivity. Subsequent comparison of sCD163 detection using a 3D Stack with EDC–NHS coupling and conventional ELISA in dengue patients’ sera revealed a high correlation (R = 0.9298, p < 0.0001) between the two detection platforms. Bland–Altman analysis further revealed insignificant systematic error between the mean differences of the two methods. These data suggest the potentials of the 3D Stack for further development as a detection platform.

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Bioaffinity-based Surface Immobilization of Antibodies to Capture Endothelial Colony-forming Cells

10.1101/2021.06.23.449631 ◽

2021 ◽

Author(s):

Mariève D Boulanger ◽

Mohamed A Elkhodiry ◽

Omar Bashth ◽

Gaétan Laroche ◽

Corinne A Hoesli

Keyword(s):

Surface Modification ◽

Surface Proteins ◽

Enzyme Linked Immunosorbent Assay ◽

Protein G ◽

Cell Capture ◽

Igg Antibodies ◽

Immobilized Antibodies ◽

Endothelial Colony Forming Cells ◽

Covalent Grafting

Maximizing the re-endothelialization of vascular implants such as prostheses or stents has the potential to significantly improve their long-term performance. Endothelial progenitor cell capture stents with surface-immobilized antibodies show significantly improved endothelialization in the clinic. However, most current antibody-based stent surface modification strategies rely on antibody adsorption or direct conjugation via amino or carboxyl groups which leads to poor control over antibody surface concentration and/or molecular orientation, and ultimately bioavailability for cell capture. Here, we assess the utility of a bioaffinity-based surface modification strategy consisting of a surface-conjugated cysteine-tagged protein G molecules that immobilize Immunoglobulin G (IgG) antibodies via the Fc domain to capture circulating endothelial colony-forming cells (ECFCs). The cysteine-tagged protein G was grafted onto aminated substrates at different concentrations as detected by an enzyme-linked immunosorbent assay and fluorescence imaging. Different IgG antibodies were successfully immobilized on the protein G-modified surfaces and higher antibody surface concentrations were achieved compared to passive adsorption methods. Surfaces with immobilized antibodies targeting endothelial surface proteins, such as CD144, significantly enhanced the capture of circulating ECFCs in vitro compared to surfaces with non-endothelial specific antibodies such as anti-CD14. This work presents a potential avenue for enhancing the clinical performance of vascular implants by using covalent grafting of protein G to immobilize IgG antibodies more effectively.

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Dengue Fever and Severe Dengue in Barbados, 2008–2016

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed5020068 ◽

2020 ◽

Vol 5 (2) ◽

pp. 68

Author(s):

Kirk Osmond Douglas ◽

Sudip Kumar Dutta ◽

Byron Martina ◽

Fatih Anfasa ◽

T. Alafia Samuels ◽

...

Keyword(s):

Dengue Virus ◽

Dengue Fever ◽

Small Sample Size ◽

Health Planning ◽

Case Fatality ◽

Small Sample ◽

Severe Dengue ◽

Medical Attention ◽

Enzyme Linked Immunosorbent Assay ◽

Virus Serotype

Analysis of the temporal, seasonal and demographic distribution of dengue virus (DENV) infections in Barbados was conducted using national surveillance data from a total of 3994 confirmed dengue cases. Diagnosis was confirmed either by DENV–specific real time reverse transcriptase polymerase chain reaction (rRT–PCR), or non–structural protein 1 (NS1) antigen or enzyme linked immunosorbent assay (ELISA) tests; a case fatality rate of 0.4% (10/3994) was observed. The dengue fever (DF) prevalence varied from 27.5 to 453.9 cases per 100,000 population among febrile patients who sought medical attention annually. DF cases occurred throughout the year with low level of transmission observed during the dry season (December to June), then increased transmission during rainy season (July to November) peaking in October. Three major dengue epidemics occurred in Barbados during 2010, 2013 and possibly 2016 with an emerging three–year interval. DF prevalence among febrile patients who sought medical attention overall was highest among the 10–19 years old age group. The highest DF hospitalisation prevalence was observed in 2013. Multiple serotypes circulated during the study period and Dengue virus serotype 2 (DENV–2) was the most prevalent serotype during 2010, whilst DENV–1 was the most prevalent serotype in 2013. Two DENV–1 strains from the 2013 DENV epidemic were genetically more closely related to South East Asian strains, than Caribbean or South American strains, and represent the first ever sequencing of DENV strains in Barbados. However, the small sample size (n = 2) limits any meaningful conclusions. DF prevalence was not significantly different between females and males. Public health planning should consider DENV inter–epidemic periodicity, the current COVID–19 pandemic and similar clinical symptomology between DF and COVID–19. The implementation of routine sequencing of DENV strains to obtain critical data can aid in battling DENV epidemics in Barbados.

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An Antigen Capture Enzyme-Linked Immunosorbent Assay Reveals High Levels of the Dengue Virus Protein NS1 in the Sera of Infected Patients

Journal of Clinical Microbiology ◽

10.1128/jcm.38.3.1053-1057.2000 ◽

2000 ◽

Vol 38 (3) ◽

pp. 1053-1057 ◽

Cited By ~ 289

Author(s):

Paul R. Young ◽

Paige A. Hilditch ◽

Cheryl Bletchly ◽

Wendy Halloran

Keyword(s):

Monoclonal Antibodies ◽

Dengue Virus ◽

Acute Phase ◽

Immunosorbent Assay ◽

Nonstructural Protein ◽

Surrogate Marker ◽

Detection Sensitivity ◽

Severe Dengue ◽

Clinical Samples ◽

Enzyme Linked Immunosorbent Assay

We describe the development of a capture enzyme-linked immunosorbent assay for the detection of the dengue virus nonstructural protein NS1. The assay employs rabbit polyclonal and monoclonal antibodies as the capture and detection antibodies, respectively. Immunoaffinity-purified NS1 derived from dengue 2 virus-infected cells was used as a standard to establish a detection sensitivity of approximately 4 ng/ml for an assay employing monoclonal antibodies recognizing a dengue 2 serotype-specific epitope. A number of serotype cross-reactive monoclonal antibodies were also shown to be suitable probes for the detection of NS1 expressed by the remaining three dengue virus serotypes. Examination of clinical samples demonstrated that the assay was able to detect NS1 with minimal interference from serum components at the test dilutions routinely used, suggesting that it could form the basis of a useful additional diagnostic test for dengue virus infection. Furthermore, quantitation of NS1 levels in patient sera may prove to be a valuable surrogate marker for viremia. Surprisingly high levels of NS1, as much as 15 μg/ml, were found in acute-phase sera taken from some of the patients experiencing serologically confirmed dengue 2 virus secondary infections but was not detected in the convalescent sera of these patients. In contrast, NS1 could not be detected in either acute-phase or convalescent serum samples taken from patients with serologically confirmed primary infection. The presence of high levels of secreted NS1 in the sera of patients experiencing secondary dengue virus infections, and in the context of an anamnestic antibody response, suggests that NS1 may contribute significantly to the formation of the circulating immune complexes that are suspected to play an important role in the pathogenesis of severe dengue disease.

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Effect of Carrier Surface Modification on the Resultant Antibody to Folic Acid

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.581-582.77 ◽

2012 ◽

Vol 581-582 ◽

pp. 77-80

Author(s):

Qiu Shi Tang ◽

Hong Tao Lei ◽

Long Bin Huang ◽

Yuan Ming Sun ◽

Jin Yi Yang

Keyword(s):

Surface Modification ◽

Folic Acid ◽

Ultraviolet Spectrum ◽

Enzyme Linked Immunosorbent Assay ◽

Carrier Protein ◽

Coupling Ratio ◽

Hexamethylene Diamine ◽

Artificial Antigen ◽

Sds Page ◽

Surface Modified

For the generation of antibodies against small hapten molecules, the hapten is cross-linked with some carrier protein to make it immunogenic. However, it was rarely systematically studied about the effect of modified carrier protein on the obtained antibody nature. In this study, folic acid as the model hapten was coupled to natural and surface-modified bovine serum albumin (BSA) using modifying agent amidocaproic acid (ACA) and hexamethylene diamine (HDA). The three immunogens of FA-BSA, FA-ACA-BSA, FA-HDA-BSA were confirmed by SDS-PAGE and ultraviolet spectrum (UV). The Balb/C mice were immunized with the artificial antigen to obtain three specific antibody with indirect competitive enzyme-linked immunosorbent assay (icELISA) method to compare the performance of antibodies obtained based on different imunogen. The results indicate that the conjugates were successfully synthesized and the coupling ratio from the high to the low was HDA-BSA > ACA-BSA > BSA, while the titer of antibody was ACA-BSA > BSA >HDA-BSA and the IC50was HDA-BSA < ACA-BSA < BSA. This suggests that sensitivity of antibody was improved by cationizing the carrier protein.

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Factors predicting the severity of dengue in patients with warning signs in Rio de Janeiro, Brazil (1986–2012)

Transactions of the Royal Society of Tropical Medicine and Hygiene ◽

10.1093/trstmh/trz066 ◽

2019 ◽

Vol 113 (11) ◽

pp. 670-677 ◽

Cited By ~ 1

Author(s):

Bianca De Santis Gonçalves ◽

Rita Maria Ribeiro Nogueira ◽

Ana Maria Bispo de Filippis ◽

Marco Aurélio Pereira Horta

Keyword(s):

Rio De Janeiro ◽

Dengue Infection ◽

Warning Signs ◽

Severe Dengue ◽

Enzyme Linked Immunosorbent Assay ◽

Infection Status ◽

Viral Loads ◽

Dengue Virus Type 3 ◽

Dengue With Warning Signs ◽

Type 3

AbstractBackgroundSince 1981, >12 million cases of dengue have been reported in Brazil. Early prediction of severe dengue with no warning signs is crucial to avoid progression to severe dengue. Here we aimed to identify early markers of dengue severity and characterize dengue infection in patients in Rio de Janeiro.MethodsWe evaluated early severity markers, serotypes, infection status, number of days of illness and viral loads associated with dengue fever in patients from Rio de Janeiro, Brazil through an observational retrospective study (1986–2012). We compared dengue without warning signs and dengue with warning signs/severe dengue (DWWS/SD). Infection status was classified by enzyme-linked immunosorbent assay and viraemia was quantified by quantitative real-time reverse transcription polymerase chain reaction.ResultsThe presence of DWWS/ SD was significantly associated with younger age; patients 13–19 y of age had a significantly greater chance of presenting warning signs. Dengue virus type 3 (DENV3) was more likely to induce DWWS/SD, which was more frequent on days 4–5 of illness.ConclusionsDENV3, 4–5 d of illness and 13–19 y of age were early biomarkers of dengue severity. To our knowledge, this was the first study to analyse the characteristics of dengue severity in the state of Rio de Janeiro over 27 y of epidemics since the introduction of DENV.

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Evaluation of a Novel Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Salmonella, Employing a Stable Coating of Lipopolysaccharide-Derived Antigens Covalently Attached to Polystyrene Microwells

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870001200205 ◽

2000 ◽

Vol 12 (2) ◽

pp. 130-135 ◽

Cited By ~ 13

Author(s):

Camilla Wiuff ◽

Eva Stenbæk Jauho ◽

Henrik Stryhn ◽

Lars Ole Andresen ◽

Karina Thaulov ◽

...

Keyword(s):

Microtiter Plate ◽

Enzyme Linked Immunosorbent Assay ◽

O Antigen ◽

Salmonella Infections ◽

Specificity And Sensitivity ◽

Conventional Elisa ◽

Polysaccharide Antigens ◽

Serological Surveillance ◽

Coated Surface ◽

High Degree

Polysaccharides derived from Salmonella typhimurium lipopolysaccharide (LPS) representing the O-antigen factors 1, 4, 5, and 12 and the O-antigen factors 6 and 7 from Salmonella choleraesuis LPS were derivatized with the photoreactive compound anthraquinone and subsequently covalently coupled to microtiter polystyrene plates by ultraviolet irradiation. Both polysaccharide antigens could be coupled simultaneously to the same microtiter plate. The coated surface was used in indirect ELISA for the determination of serum antibodies from pigs infected with bacteria of the two Salmonella groups and from uninfected pigs. This ELISA proved itself by having a good long-term durability and a high degree of reproducibility, including low day-to-day variations and low interplate variations. Furthermore, the ELISA showed good specificity and sensitivity when data were compared with the optical density levels of a panel of pig sera as determined by a conventional ELISA on the basis of passive coating of the two Salmonella LPS antigens (the mix-ELISA). The covalent anthraquinone mix-ELISA shows promise as a stable and durable alternative to the existing conventional ELISA for serological surveillance of Salmonella infections in pigs.

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Serodiagnosis of Anthroponotic Cutaneous Leishmaniasis (ACL) Caused by Leishmania tropica in Sanliurfa Province, Turkey, Where ACL Is Highly Endemic

Clinical and Vaccine Immunology ◽

10.1128/cvi.00133-07 ◽

2007 ◽

Vol 14 (11) ◽

pp. 1409-1415 ◽

Cited By ~ 14

Author(s):

Fadile Yildiz Zeyrek ◽

Metin Korkmaz ◽

Yusuf Özbel

Keyword(s):

Cutaneous Leishmaniasis ◽

Western Blotting ◽

Blood Donors ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests ◽

Western Blot Test ◽

Conventional Elisa ◽

Negative Controls ◽

Positive Controls

ABSTRACT In this study, we aimed to evaluate the validity of the conventional enzyme-linked immunosorbent assay (ELISA) and the Western blotting test for the diagnosis of anthroponotic cutaneous leishmaniasis (ACL) using serum samples obtained from 51 patients with parasitologically proven nontreated CL (NonT-CL patients) and 62 patients under treatment for CL (UT-CL patients). Additionally, 29 serum samples obtained from patients with parasitologically and serologically proven visceral leishmaniasis (VL) were also used as positive controls, and serum samples from 43 blood donors were used as negative controls. All sera were diluted to the same dilution (1/100). Leishmania infantum MON-1 was used as the antigen in the conventional ELISA. The sera of 27 (93.1%) of 29 VL patients were seropositive by ELISA, while the sera of 40 (78.4%) of 51 NonT-CL patients and 43 (69.3%) of 62 UT-CL patients were seropositive by the conventional ELISA. The absorbance values of the CL patients' sera were significantly lower than the absorbance values of the VL patients' sera. Bands between 15 and 118 kDa were detected in two groups of CL patients. Among all bands, the 63-kDa band was found to be more sensitive (88.5%). When we evaluated the Western blotting results for the presence of at least one of the diagnostic antigenic bands, the sensitivity was calculated to be 99.1%. By using serological tests, a measurable antibody response was detected in most of the CL patients in Sanliurfa, Turkey. It is also noted that this response can be changed according to the sizes, types, and numbers of lesions that the patient has. The Western blot test was found to be more sensitive and valid than the conventional ELISA for the serodiagnosis of ACL. In some instances, when it is very difficult to demonstrate the presence of parasites in the smears, immunodiagnosis can be a valuable alternative for the diagnosis of ACL.

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Serum Proteins Opsonization and Phagocytic Uptake of PEG-Modified PLGA Nanoparticles: Effect of Particle Size

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.393-395.939 ◽

2011 ◽

Vol 393-395 ◽

pp. 939-942 ◽

Cited By ~ 1

Author(s):

An Shu Yang ◽

Wei Liu ◽

Xiang Liang Yang

Keyword(s):

Particle Size ◽

Serum Protein ◽

Complement Activation ◽

Serum Proteins ◽

Enzyme Linked Immunosorbent Assay ◽

Spontaneous Emulsification ◽

Protein Assay ◽

Effect Of Particle Size ◽

Serum Dependent

The purpose of this study is to evaluate the effect of particle size on serum protein opsonization and in vitro macrophage uptake of polyethyleneglycol modified poly (D, L-lactide-co-glycolide) nanoparticles (PEG-PLGA-NPs). PEG-PLGA-NPs were prepared by modified-spontaneous emulsification solvent diffusion (modified-SESD) method. Serum protein adsorptions to PEG-PLGA-NPs were evaluated by bicinchoninic acid (BCA) protein assay and enzyme-linked immunosorbent assay (ELISA). Complement activation was also investigated by ELISA for complement fragments iC3b. Uptake of PEG-PLGA-NPs by macrophages was measured by fluorescence spectrometer. The results showed that serum protein adsorption and complement activation were augmented for nanoparticles with a larger size below 400 nm. Phagocytosis of PEG-PLGA-NPs by murine peritoneal macrophages involved serum-independent and serum-dependent phagocytosis. Serum-independent phagocytosis decreased, while serum-dependent phagocytosis increased with the increase of particle size in the nanometer and submicrometer range. Consequently, nanoparticles with size of about 400 nm were phagocytosed more readily than either smaller or larger particles

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Monoclonal antibody-based capture ELISA in the diagnosis of previous dengue infection

Virology Journal ◽

10.1186/s12985-019-1222-9 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 2

Author(s):

Chukiat Sirivichayakul ◽

Kriengsak Limkittikul ◽

Pornthep Chanthavanich ◽

Sutee Yoksan ◽

Anuttarasakdi Ratchatatat ◽

...

Keyword(s):

Monoclonal Antibody ◽

Neutralization Test ◽

Dengue Infection ◽

Severe Dengue ◽

Enzyme Linked Immunosorbent Assay ◽

Dengue Vaccine ◽

Serum Samples ◽

Specific Immunoglobulin ◽

Study Population ◽

Dengue Prevention

Abstract Background Dengue is an important mosquito-borne disease. There is currently only one licensed vaccine for dengue prevention. The vaccine provides higher efficacy in pre-vaccination dengue-seropositive persons but a higher risk of subsequent more severe dengue in dengue-seronegative persons. It is recommended that the dengue vaccine may be given in dengue-seropositive individuals or as mass vaccination without individual pre-vaccination screening in areas where the dengue seroprevalence is > 80% in children aged 9 years. We evaluated a dengue specific immunoglobulin G monoclonal antibody-based capture enzyme-linked immunosorbent assay (MAb-ELISA) in the diagnosis of previous dengue infection using serum samples from the cohort study in Ratchaburi Province, Thailand. Methods The MAb-ELISA was compared to 70% plaque reduction neutralization test (PRNT70) in 453 serum samples from children aged 3–11 years in Ratchaburi Province, Thailand. Results The sensitivity and specificity of MAb-ELISA at the positive to negative (P/N) ratio cut-off level of > 3 were both 0.91 in the diagnosis of previous dengue infection, compared to PRNT70. The false positivity was mainly in Japanese encephalitis (JE) seropositive subjects. Conclusions This research provides evidence that MAb-ELISA is useful for dengue seroprevalence study and dengue pre-vaccination screening. JE seropositivity was the major cause of false positive result in the study population.

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Serum proteins differentially expressed in early- and late-onset preeclampsia assessed using iTRAQ proteomics and bioinformatics analyses

PeerJ ◽

10.7717/peerj.9753 ◽

2020 ◽

Vol 8 ◽

pp. e9753

Author(s):

Chengcheng Tu ◽

Feng Tao ◽

Ying Qin ◽

Mingzhu Wu ◽

Ji Cheng ◽

...

Keyword(s):

Mass Spectrometry ◽

Lipid Metabolism ◽

Pregnant Women ◽

Serum Proteins ◽

Late Onset ◽

Differentially Expressed ◽

Coagulation Cascade ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Bioinformatics Analyses

Background Preeclampsia remains a serious disorder that puts at risk the lives of perinatal mothers and infants worldwide. This study assessed potential pathogenic mechanisms underlying preeclampsia by investigating differentially expressed proteins (DEPs) in the serum of patients with early-onset preeclampsia (EOPE) and late-onset preeclampsia (LOPE) compared with healthy pregnant women. Methods Blood samples were collected from four women with EOPE, four women with LOPE, and eight women with normal pregnancies, with four women providing control samples for each preeclampsia group. Serum proteins were identified by isobaric tags for relative and absolute quantitation combined with liquid chromatography–tandem mass spectrometry. Serum proteins with differences in their levels compared with control groups of at least 1.2 fold-changes and that were also statistically significantly different between the groups at P < 0.05 were further analyzed. Bioinformatics analyses, including gene ontology and Kyoto Encyclopedia of Genes and Genomes signaling pathway analyses, were used to determine the key proteins and signaling pathways associated with the development of PE and to determine those DEPs that differed between women with EOPE and those with LOPE. Key protein identified by mass spectrometry was verified by enzyme linked immunosorbent assay (ELISA). Results Compared with serum samples from healthy pregnant women, those from women with EOPE displayed 70 proteins that were differentially expressed with significance. Among them, 51 proteins were significantly upregulated and 19 proteins were significantly downregulated. In serum samples from women with LOPE, 24 DEPs were identified , with 10 proteins significantly upregulated and 14 proteins significantly downregulated compared with healthy pregnant women. Bioinformatics analyses indicated that DEPs in both the EOPE and LOPE groups were associated with abnormalities in the activation of the coagulation cascade and complement system as well as with lipid metabolism. In addition, 19 DEPs in the EOPE group were closely related to placental development or invasion of tumor cells. Downregulationof pregnancy-specific beta-1-glycoprotein 9 (PSG9) in the LOPE group was confirmed by ELISA. Conclusion The pathogenesis of EOPE and LOPE appeared to be associated with coagulation cascade activation, lipid metabolism, and complement activation. However, the pathogenesis of EOPE also involved processes associated with greater placental injury. This study provided several new proteins in the serum which may be valuable for clinical diagnosis of EOPE and LOPE, and offered potential mechanisms underpinning the development of these disorders.

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