Production and purification of polyclonal anti-chicken IgY immunoglobulins in Goats and conjugation with Horseradish Peroxidase

2021 ◽  
Vol 16 (7) ◽  
pp. 30-34
Author(s):  
P. Navaneetha ◽  
M. Anil ◽  
M. Manasa ◽  
N. Aparna Reddy ◽  
Ch. Satya Sagar Reddy ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Medical Diagnostics ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein G ◽  
Affinity Column ◽  
Igg Antibodies ◽  
Biochemical Assays ◽  
Sds Page ◽  
Polyclonal Igg

Antibodies are very important components in medical diagnostics and various biochemical assays for diagnosis of the diseases. IgY is an immunoglobulin largely present in Avians. Anti-chicken IgY immunoglobulins conjugated with gold nanoparticles are used to diagnose the chicken infectious diseases by lateral flow assay (LFA) and enzyme linked immunosorbent assay (ELISA) immunodiagnostic tests. The main aim of this study is the production of antichicken IgY antibodies in goats, purification of antiantibody and conjugation with horse radish peroxidase (HRP) enzyme. The antibody was purified using protein G sepharose affinity column and the purity of the antibody was confirmed by SDS-PAGE analysis and by spectrophotometer. The purified anti IgY antibody was also evaluated by ELISA. The optimum dilution of prepared HRP conjugated anti IgY was 1:9600. The sensitivity and specificity of the antibody were evaluated and have no cross reactivity with other IgG antibodies. This study showed that protein G affinity purification could be best useful method for purification of polyclonal IgG antibodies.

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

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.

High-affinity folate binding in human prostate

1993 ◽  
Vol 13 (2) ◽  
pp. 99-105 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen
Keyword(s):  
Human Milk ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Human Prostate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gel Chromatography ◽  
Folate Binding Protein ◽  
High Affinity ◽  
Sds Page ◽  
Triton X

Binding of 3H-folate in Triton X-100 solubilized human prostate homogenate was of a high-affinity type and displayed apparent positive cooperativity typical of specific folate binding. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. Gel chromatography reveled two major folate binding proteins (Mr≈100 and 25kDa), but only one single band (Mr ≈ 65–70 kDa) was detectable on SDS-PAGE and immunoblotting with rabbit-anti human milk folate binding protein. Concentration of folate binding protein in prostate homogenate expressed as maximum 3H-folate binding was 1.10 nmol/g protein, and the cross-reactivity with rabbit-anti human milk folate binding protein serum was 15% as determined by an enzyme-linked immunosorbent assay (median values; n = 6).

scholarly journals Immunoglobulins G, M, and A against Sporothrix schenckii Exoantigens in Patients with Sporotrichosis before and during Treatment with Itraconazole

2007 ◽  
Vol 14 (9) ◽  
pp. 1149-1157 ◽  
Author(s):  
Rodrigo Almeida-Paes ◽  
Monique Amorim Pimenta ◽  
Paulo Cezar F. Monteiro ◽  
Joshua D. Nosanchuk ◽  
Rosely Maria Zancopé-Oliveira
Keyword(s):  
Sporothrix Schenckii ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Number Of Patients ◽  
Highly Sensitive ◽  
Iga Antibodies ◽  
Itraconazole Treatment ◽  
Few Data ◽  
Humoral Responses

ABSTRACT Sporotrichosis is an important subcutaneous mycosis, with an increasing worldwide incidence. However, few data are available regarding the immunological aspects of Sporothrix schenckii infection, particularly the humoral responses to the fungus. In this study we measured immunoglobulin G (IgG), IgM, and IgA in sera from 41 patients with sporotrichosis before antifungal treatment and from another 35 patients with sporotrichosis during itraconazole treatment by using a recently described S. schenckii exoantigen enzyme-linked immunosorbent assay (ELISA). More than 95% of patients had detectable IgA antibodies, and more than 85% had IgM and IgG antibodies before treatment. The number of patients with IgG antibodies increased to 91% during treatment. Conversely, significantly fewer samples from treated patients were positive for IgM (71%) and IgA (89%). Overall, 78% of patients had detectable levels of all isotypes tested at diagnosis, and this percentage dropped to 62.9% in patients receiving itraconazole. Testing of all three isotypes improved the sensitivity; at least two isotypes were detected in 93% of patients before and 89% after treatment. The reactivity of 94 sera from patients with other diseases and healthy individuals was also tested. Cross-reactivity occurred in 33% of the heterologous sera. Most of them were positive only in one isotype, 8.5% were positive for at least two isotypes, and only one serum (1.1%) was positive for the three isotypes. Antibodies produced during S. schenckii infection are diverse, and we demonstrate that an exoantigen ELISA for the detection of combinations of IgA, IgG, and IgM antibodies is a highly sensitive and specific diagnostic assay for sporotrichosis.

An Enzyme-Linked Immunosorbent Assay for the Detection of Aspergillus parasiticus and Aspergillus flavus

1997 ◽  
Vol 60 (8) ◽  
pp. 978-984 ◽  
Author(s):  
GUO-JANE TSAI ◽  
SHOU-CHIN YU
Keyword(s):  
Aspergillus Flavus ◽  
Immunosorbent Assay ◽  
False Positive ◽  
Aspergillus Parasiticus ◽  
False Negative ◽  
Zealand White Rabbit ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Weights ◽  
Sds Page

An enzyme-linked immunosorbent assay (ELISA) was established for the specific detection of Aspergillus parasiticus and Aspergillus flavus. A New Zealand white rabbit was immunized intravenously with 100 μg of A. parasiticus CCRC 30117 mycelial protein extracts. The antibodies were separated and purified. The optimal concentration of the antibody and antibody-peroxidase conjugate used in the established ELISA was 10 μg/ml with a detection limit of 1 μg/ml. Among the 126 strains tested (including 21 strains of A. parasiticus, 11 strains of A. flavus, 34 isolates of A. parasiticus/A. flavus from cereals, and 60 strains of non-A. parasiticus/A. flavus fungi), the false-negative and false-positive rates were 1.5 and 3.3%, respectively. Strains of Aspergillus flavofrucatis and Aspergillus sojae produced false-positive reactions. However, their antigens had much lower cross-reactivity with the antibodies raised against A. parasiticus, as shown from I50 values. The molecular weights of the main antigens of A. parasiticus were 94, 82, and 40 kDa. The two heavier antigens had higher sugar contents, as demonstrated by SDS-PAGE and immunoblotting. A good correlation (r = 0.97) was found between mycelium measurement by weighing and by ELISA for A. parasiticus grown in yeast extract sucrose broth (YESB) at 25°C.

scholarly journals Sensitivity and Kinetics of an NS1-Based Zika Virus Enzyme-Linked Immunosorbent Assay in Zika Virus-Infected Travelers from Israel, the Czech Republic, Italy, Belgium, Germany, and Chile

2017 ◽  
Vol 55 (6) ◽  
pp. 1894-1901 ◽  
Author(s):  
Yaniv Lustig ◽  
Hana Zelena ◽  
Giulietta Venturi ◽  
Marjan Van Esbroeck ◽  
Camilla Rothe ◽  
...  
Keyword(s):  
Czech Republic ◽  
Immunosorbent Assay ◽  
Zika Virus ◽  
False Negative ◽  
Cross Reactivity ◽  
Diagnostic Methods ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Kinetics Of

ABSTRACT Serological diagnosis of Zika virus is challenging due to high cross-reactivity of Zika virus with other flavivirus antibodies. Recently, a Zika NS1-based enzyme-linked immunosorbent assay (ELISA) was developed and shown to be highly specific for Zika antibody detection; however, sensitivity was evaluated for only a small number of confirmed Zika-infected patients. In this study, we measured the sensitivity and kinetics of Zika IgM and IgG antibodies using the Zika NS1-based ELISA in 105 samples from 63 returning travelers infected with Zika virus (proven by PCR or neutralization assay) from Israel, Czech Republic, Italy, Belgium, Germany, and Chile. Zika virus IgM was detected from 2 to 42 days post-symptom onset (PSO) with an overall sensitivity of 79% in the first month and 68% until 2 months PSO, while IgG antibodies were detected from 5 days to 3 years PSO with 79% sensitivity. Interestingly, significant differences in IgM sensitivity and IgM detection period were observed between Israeli and European/Chilean Zika-infected travelers, adding to the complexity of Zika infection diagnosis and suggesting that other diagnostic methods should be complemented to reduce false-negative results.

scholarly journals Identification and Characterization of Immunogenic Proteins of Mycoplasma genitalium

2006 ◽  
Vol 13 (8) ◽  
pp. 913-922 ◽  
Author(s):  
Helle Friis Svenstrup ◽  
Jørgen Skov Jensen ◽  
Kris Gevaert ◽  
Svend Birkelund ◽  
Gunna Christiansen
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mycoplasma Genitalium ◽  
Cross Reactivity ◽  
Rabbit Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Immunogenic Proteins ◽  
Recombinant Fragments

ABSTRACT Mycoplasma genitalium causes nonchlamydial nongonococcal urethritis. M. genitalium was detected by PCR in 17 urethral swabs obtained from 99 men with and without urethritis (J. S. Jensen, R. Orsum, B. Dohn, S. Uldum, A. M. Worm, and K. Lind, Genitourin. Med. 69:265-269, 1993), and later, four M. genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed a specific ELISA for detection of M. genitalium antibodies. This antigen did not bind M. pneumoniae antibodies. Using serum samples from the 99 men with and without urethritis, we found that 26 had immunoglobulin G (IgG) antibodies to M. genitalium. There was a strong statistically significant correlation between PCR and IgG antibodies to M. genitalium (odds ratio [OR], 5.9; 95% confidence interval [CI], 2.3 to 21.5; P = 0.002). Furthermore, men with recurrent urethritis were more likely to have antibodies to M. genitalium than were those without recurrent urethritis (OR, 4.0; 95% CI, 1.1 to 14.5; P = 0.0383) and they had significantly higher antibody titers. By use of the rMgPa ELISA, this study further substantiates the importance of M. genitalium as a cause of male urethritis.

scholarly journals First detection of Zika virus infection in a Croatian traveler returning from Brazil, 2016

2017 ◽  
Vol 11 (08) ◽  
pp. 662-667
Author(s):  
Tatjana Vilibic-Cavlek ◽  
Ljiljana Betica-Radic ◽  
Giulietta Venturi ◽  
Claudia Fortuna ◽  
Stjepan Djuricic ◽  
...  
Keyword(s):  
Zika Virus ◽  
High Titer ◽  
Disease Onset ◽  
Negative Control ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Grade ◽  
Igg Antibodies ◽  
Zikv Infection

In the last few years, several imported cases of Zika virus (ZIKV) infection were reported in European countries. We report the first imported ZIKV infection case in a Croatian traveler returning from Brazil. The patient presented with a low-grade fever, pruritic rash, general weakness, myalgia, arthralgia and edema of the legs and recovered completely within a week. ZIKV infection was confirmed by detection of IgM/IgG antibodies using enzyme-linked immunosorbent assay (ELISA) and confirmed by plaque-reduction neutralization test (PRNT). ZIKV IgM antibodies cross-reacted with dengue virus (DENV), West Nile virus (WNV) and tick-borne encephalitis virus (TBEV) in ELISA. In indirect immunofluorescence assay (IFA), IgM cross-reactivity was found only with DENV-3.  ZIKV IgG antibodies cross-reacted with DENV in both ELISA and IFA. PRNT for DENV was negative. Control serology performed on days 64 and 98 after disease onset showed a decline in cross-reactive heterologous DENV IgG antibodies compared to persistently high titer of homologous ZIKV IgG antibodies.

scholarly journals Biochemical and biological characterization of Bothriechis schlegelii snake venoms from Colombia and Costa Rica

2016 ◽  
Vol 241 (18) ◽  
pp. 2075-2085 ◽  
Author(s):  
José P Prezotto-Neto ◽  
Louise F Kimura ◽  
André F Alves ◽  
José María Gutiérrez ◽  
Rafael Otero ◽  
...  
Keyword(s):  
Costa Rica ◽  
Cross Reactivity ◽  
Phospholipase A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Biological Parameters ◽  
Biological Characterization ◽  
Snake Venoms ◽  
Sds Page ◽  
Local Necrosis

Snakebites inflicted by the arboreal viperid snake Bothriechis schlegelii in humans are characterized by pain, edema, and ecchymosis at the site of the bite, rarely with blisters, local necrosis, or defibrination. Herein, a comparative study of Bothriechis schlegelii snake venoms from Colombia (BsCo) and Costa Rica (BsCR) was carried out in order to compare their main activities and to verify the efficacy of Bothrops antivenom produced in Brazil to neutralize them. Biochemical (SDS-PAGE and zymography) and biological parameters (edematogenic, lethal, hemorrhagic, nociceptive, and phospholipase A2 activities) induced by BsCo and BsCR snake venoms were evaluated. The presence of antibodies in Bothrops antivenom that recognize BsCo and BsCR snake venoms by enzyme-linked immunosorbent assay and Western blotting, as well as the ability of this antivenom to neutralize the toxic activities were also verified. SDS-PAGE showed differences between venoms. Distinctive caseinolytic and hyaluronidase patterns were detected by zymography. BsCo and BsCR showed similar phospholipase A2 activity. Strong cross-reactivity between BsCo and BsCR was detected using Bothrops antivenom with many components located between 150 and 35 kDa. BsCR was more edematogenic and almost fourfold more hemorrhagic than BsCo, and both venoms induced nociception. BsCR (LD50 5.60 mg/kg) was more lethal to mice than BsCo (LD50 9.24 mg/kg). Bothrops antivenom was effective in the neutralization of lethal and hemorrhagic activities of BsCo and BsCR and was partially effective in the neutralization of edematogenic and nociceptive activities. In conclusion, geographic distribution influences the composition and activities of Bothriechis schlegelii venoms. Bothrops antivenom cross-reacted with these venoms and was partially effective in neutralizing some toxic activities of BsCo and BsCR.

scholarly journals Case Report: Allergic Reactivity to Mahaleb (Prunus mahaleb) Spice in a Subject With Almond and Other Tree Nut Allergies

2020 ◽  
Vol 11 ◽  
pp. 215265672095908
Author(s):  
Lora Benoit ◽  
Jongkit Masiri ◽  
Harish Janagama ◽  
Steven M. Gendel ◽  
Mansour Samadpour
Keyword(s):  
Skin Prick Testing ◽  
Middle Eastern ◽  
Specific Ige ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tree Nut ◽  
Polyclonal Igg ◽  
History Of ◽  
Specific Igg ◽  
Food Recalls

Background Mahaleb is an aromatic spice prepared from the fruit stone of the St. Lucie Cherry that is used as a flavoring agent in traditional Turkish and Middle Eastern baking. Immunodiagnostic kits for almond, which are based on polyclonal almond-specific IgG antibodies, have been shown to demonstrate considerable cross-reactivity with mahaleb as was incidentally discovered during a cluster of allergen-related food recalls in 2015. Objective Though acute allergy to almond is somewhat common, allergies to mahaleb have not been previously documented. However, based on antigenic similarity observed with almond-specific IgG, it is predicted that mahaleb nut proteins would exhibit some level of cross-reactivity with almond-specific IgE and may therefore potentiate acute allergic symptoms in individuals with food allergy to almond. Case Presentation: Herein, we report on a 40-year old Caucasian female with longitudinal history of multiple tree nut allergies including allergy to almond, presenting with moderate pruritus and oropharyngeal swelling shortly following ingestion of mahaleb seed kernels. Methods and Results Skin-prick testing using extracts compounded from pistachio, almond, and mahaleb revealed positive wheals measuring 8, 3, and 7 mm respectfully. Indirect enzyme-linked immunosorbent assay (ELISA) using plate-bound antigens prepared from pistachio, almond, and mahaleb revealed IgG positive responses to all three targets. ELISA and Western blot analysis performed using goat anti-almond polyclonal IgG demonstrated significant cross-reactivity between almond and mahaleb, but not to pistachio. Conclusion This is the first documented case of acute allergy to mahaleb, co-occurring in the context of plural tree nut allergies, providing novel evidence that mahaleb may pose a risk to nut-allergic individuals and indicating a need for awareness of spice contamination with nut and mahaleb residues.

scholarly journals Enzyme-Linked Immunosorbent Assay for Detection of Filovirus Species-Specific Antibodies

2010 ◽  
Vol 17 (11) ◽  
pp. 1723-1728 ◽  
Author(s):  
Eri Nakayama ◽  
Ayaka Yokoyama ◽  
Hiroko Miyamoto ◽  
Manabu Igarashi ◽  
Noriko Kishida ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Cross Reactivity ◽  
Marburg Virus ◽  
Diagnostic Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Species ◽  
Igg Antibodies ◽  
Specific Antibodies ◽  
Species Specific ◽  
Respective Species

ABSTRACT Several enzyme-linked immunosorbent assays (ELISAs) for the detection of filovirus-specific antibodies have been developed. However, diagnostic methods to distinguish antibodies specific to the respective species of filoviruses, which provide the basis for serological classification, are not readily available. We established an ELISA using His-tagged secreted forms of the transmembrane glycoproteins (GPs) of five different Ebola virus (EBOV) species and one Marburg virus (MARV) strain as antigens for the detection of filovirus species-specific antibodies. The GP-based ELISA was evaluated by testing antisera collected from mice immunized with virus-like particles as well as from humans and nonhuman primates infected with EBOV or MARV. In our ELISA, little cross-reactivity of IgG antibodies was observed in most of the mouse antisera. Although sera and plasma from some patients and monkeys showed notable cross-reactivity with the GPs from multiple filovirus species, the highest reactions of IgG were uniformly detected against the GP antigen homologous to the virus species that infected individuals. We further confirmed that MARV-specific IgM antibodies were specifically detected in specimens collected from patients during the acute phase of infection. These results demonstrate the usefulness of our ELISA for diagnostics as well as ecological and serosurvey studies.

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