Selection and characterisation of FVIII-specific single chain variable fragments

2013 ◽  
Vol 33 (S 01) ◽  
pp. S39-S45 ◽  
Author(s):  
A. Naumann ◽  
A. K. Scherger ◽  
J. Neuwirth ◽  
A. Orlowski ◽  
J. Kahle ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Tolerance Induction ◽  
Igg Subclasses ◽  
Igg Subclass ◽  
Single Chain ◽  
Relative Contribution ◽  
Phage Display Libraries ◽  
Specific Igg ◽  
Single Chain Variable Fragments ◽  
A2 Domain

SummaryThe development of inhibitory anti-FVIII antibodies is currently the most severe complication in the treatment of haemophilia A patients. Inhibitor eradication can be achieved by immune tolerance induction (ITI). Recent findings suggest a correlation between the FVIII-specific IgG subclass distribution and the duration or outcome of ITI. To quantify FVIII-specific IgG subclasses in patients’ plasma FVIII-specific IgG standards are required. Here, the isolation of FVIII-specific single chain variable fragments (scFvs) from synthetic phage display libraries and the characterisation of their FVIII domain specificity are described. The isolated scFv 1G10, which binds to the FVIII A2 domain, was cloned into the context of the four human IgG (hIgG) subclasses and expressed in mammalian cells. Purified 1G10-hIgG1, -hIgG2, -hIgG3 and -hIgG4 are used as standards to determine the absolute amounts and relative contribution of the different FVIII-specific IgG subclasses in future studies. The results from these studies will eventually add to understanding the role of the FVIII-specific IgG subclass distribution as prognostic factor for the outcome of ITI.

Immunoglobulin G (IgG) and IgG subclass reference intervals in children, using Optilite® reagents

2018 ◽  
Vol 56 (8) ◽  
pp. 1319-1327 ◽  
Author(s):  
Olivier Grunewald ◽  
Benjamin Lopez ◽  
Séverine Brabant ◽  
Stéphanie Rogeau ◽  
Antoine Deschildre ◽  
...  
Keyword(s):  
Immunoglobulin G ◽  
Age Groups ◽  
Reference Intervals ◽  
Igg Subclasses ◽  
Specific Reference ◽  
Igg Subclass ◽  
Serum Samples ◽  
Gender Specific Differences ◽  
Specific Igg ◽  
Changes Over Time

Abstract Background: Immunoglobulin G (IgG) and IgG subclass assays are indicated in patients with suspected primary immunodeficiency (PID). Commercially available assays for IgG subclass determination are calibrated against various preparations, and so specific reference values are required for each of them. Using Optilite® reagents from The Binding Site Group Ltd., we sought to determine the pediatric IgG and IgG subclass reference intervals with respect to the ERM-DA470k certified reference material. Methods: Levels of IgG and IgG subclasses were analyzed in serum samples collected from a large cohort of PID-free children and adolescents. Reference intervals were calculated for previously published age groups (6–12 months, 12–18 months, 18 months–2 years, 2–3 years, 3–4 years, 4–6 years, 6–9 years, 9–12 years and 12–18 years), according to the Clinical and Laboratory Standards Institute’s C28-A3c protocol. Results: A total of 456 serum samples were analyzed. The correlation between the total IgG and the sum of the IgG subclasses was good (r2=0.96). No statistically significant gender-specific differences were observed. Our results for the changes over time in IgG and IgG subclass levels are consistent with previous reports. The differences between our lower/upper reference limits and those in the literature are probably due to variations in calibration. Conclusions: Our present results provide a reliable basis for the diagnosis of PIDs in childhood and for the accreditation of laboratories using Optilite® immunoturbidimetric reagents for IgG subclass measurement. Laboratory scientists and clinicians should be aware of the need for manufacturer-specific IgG subclass reference intervals.

scholarly journals Genetic Variation in the Magnitude and Longevity of the IgG Subclass Response to a Diphtheria-Tetanus-Acellular Pertussis (DTaP) Vaccine in Mice

Vaccines ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 124 ◽  
Author(s):  
Mosley ◽  
Radder ◽  
HogenEsch
Keyword(s):  
Antibody Production ◽  
Genetic Background ◽  
Genetic Factors ◽  
Biological Function ◽  
Important Determinant ◽  
Igg Subclasses ◽  
Mouse Strains ◽  
Igg Subclass ◽  
Antibody Titers ◽  
Specific Igg

The type of IgG subclasses induced by vaccination is an important determinant of vaccine efficacy because the IgG subclasses vary in their biological function. The goal of this study was to determine the influence of the genetic background on the production and duration of vaccine-induced IgG subclasses. IgG1, IgG2b, and IgG3 titers against diphtheria toxoid (DT), pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (Prn) were measured in mice from 28 different inbred and wild-derived strains vaccinated with an aluminum hydroxide-adjuvanted DTaP vaccine. The titers and duration of vaccine-specific IgG subclass responses were different among mouse strains, indicating that genetic factors contribute to this variation. Statistical associations were used to identify potential mechanisms that contribute to antibody production and longevity. This analysis showed that the mechanisms guiding the magnitude of antibody production were antigen-dependent for IgG1 but antigen-independent for IgG2b and IgG3. However, the mechanisms driving the longevity of antibody titers were antigen-independent for IgG1, IgG2b, and IgG3. The ratio of IgG1 and IgG3 titers identified Th1 and Th2-prone mouse strains. TLR4-deficient C3H/HeJ mice had an enhanced IgG1 response compared with C3H/HeOuJ mice with intact TLR4. This work demonstrates that the genetic background contributes significantly to the magnitude and longevity of vaccine-induced IgG1, IgG2b, and IgG3 titers in mice.

scholarly journals Differential Decline in Leishmania Membrane Antigen-Specific Immunoglobulin G (IgG), IgM, IgE, and IgG Subclass Antibodies in Indian Kala-Azar Patients after Chemotherapy

1999 ◽  
Vol 67 (12) ◽  
pp. 6663-6669 ◽  
Author(s):  
Khairul Anam ◽  
Farhat Afrin ◽  
Dwijadas Banerjee ◽  
Netai Pramanik ◽  
Subhasis K. Guha ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Immunoglobulin G ◽  
Fold Increase ◽  
Igg Subclasses ◽  
Igg Subclass ◽  
Kala Azar ◽  
Specific Immunoglobulin ◽  
Strong Stimulation ◽  
Specific Igg ◽  
Stimulation Of

ABSTRACT Pathogenesis in kala-azar is associated with depressed cellular immunity and significant elevation of antileishmanial antibodies. Since these antibodies are present even after cure, analysis of the parasite-specific isotypes and immunoglobulin G (IgG) subclasses in kala-azar patients may shed new light on the immune responses during progression and resolution of infection. Using leishmanial membrane antigenic extracts, we investigated the relative levels of specific IgG, IgM, IgA, IgE, and IgG subclasses in Indian kala-azar patient sera during disease, drug resistance, and cure. Acute-phase sera showed strong stimulation of IgG, followed by IgE and IgM and lastly by IgA antibodies. IgG subclass analysis revealed expression of all of the subclasses, with a predominance of IgG1 during disease. Following sodium stibogluconate (SAG) resistance, the levels of IgG, IgM, IgE, and IgG4 remained constant, while there was a decrease in the titers of IgG2 and IgG3. In contrast, a significant (2.2-fold) increase in IgG1 was observed in these individuals. Cure, in both SAG-responsive and unresponsive patients, correlated with a decline in the levels of IgG, IgM, IgE, and all of the IgG subclasses. The stimulation of IgG1 and the persistence, most importantly, of IgE and IgG4 following drug resistance, along with a decline in IgE, IgG4, and IgG1 with cure, demonstrate the potential of these isotypes as possible markers for monitoring effective treatment in kala-azar.

scholarly journals Toxoplasma-SPECIFIC IgG SUBCLASS ANTIBODY RESPONSE IN CEREBROSPINAL FLUID SAMPLES FROM PATIENTS WITH CEREBRAL TOXOPLASMOSIS

2015 ◽  
Vol 57 (5) ◽  
pp. 439-442
Author(s):  
Fernanda S. NASCIMENTO ◽  
Lisandra A. SUZUKI ◽  
Nilson BRANCO ◽  
Regina M.B. FRANCO ◽  
Paula D. ANDRADE ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Immune System ◽  
Toxoplasma Gondii ◽  
Antibody Response ◽  
Igg Subclasses ◽  
Igg Subclass ◽  
Cerebral Toxoplasmosis ◽  
Arithmetic Means ◽  
Antigen Preparation ◽  
Specific Igg

SUMMARY Cerebral toxoplasmosis can be highly debilitating and occasionally fatal in persons with immune system deficiencies. In this study, we evaluated the Toxoplasma gondii-specific IgG subclass antibody response in 19 cerebrospinal fluid (CSF) samples from patients with cerebral toxoplasmosis who had a positive IgG anti-T. gondii ELISA standardized with a cyst antigen preparation. There were no significant differences between the rates of positivity and the antibody concentrations (arithmetic means of the ELISA absorbances, MEA) for IgG1 and IgG2, but the rates of positivity and MEA values for these two IgG subclasses were significantly higher than those for IgG3 and IgG4. The marked IgG2 response in CSF from patients with cerebral toxoplasmosis merits further investigation.

scholarly journals SELECTION OF SPECIFIC SINGLE CHAIN VARIABLE FRAGMENTS (SCFV) AGAINST POLYMYXA BETAE FROM PHAGE DISPLAY LIBRARIES

2013 ◽  
Vol 53 (4) ◽  
pp. 357-363 ◽  
Author(s):  
Mohammad Reza Safarnejad ◽  
Hossein Safarpour ◽  
Fatemeh Shahryari ◽  
Marzieh Basirat ◽  
Meisam Tabatabaei ◽  
...  
Keyword(s):  
Sugar Beet ◽  
Phage Display ◽  
Recombinant Antibodies ◽  
Diagnostic Methods ◽  
Polymyxa Betae ◽  
Single Chain ◽  
Glutathione S Transferase ◽  
Phage Display Libraries ◽  
Single Chain Variable Fragments ◽  
Selection Of

Abstract Sugar beet is one of the most important industrial crops in Iran. For the last two decades it has been mainly affected by a destructive virus, beet necrotic yellow vein virus (BNYVV). The Polymyxa betae is the only natural transmitting agent of the disease among the plants. Developing accurate diagnostic methods may have a major impact on the rising of resistant germplasms. In the present study, specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFv) were obtained from naïve phage display libraries. The fungus specific glutathione-S-transferase (GST) protein was chosen as an antigen for developing antibodies and diagnostic purposes. To generate specific scFv, screening of Tomlinson phage display libraries was performed by applying both recombinant and native fungal GST. Using the recombinant GST in the panning process resulted in the isolation of an antibody only bound to recombinant GST but it failed to detect native GST in the infected plants. Alternatively, the process of panning was carried out by applying native fungal GST trapped to immunotubes through specific polyclonal antibody intermediate. The recent approach resulted in the selection of a specific scFv binding to native GST which was able to detect the presence of the fungi within infected plants. To the best of our knowledge, this is the first report on the generation of recombinant antibodies against Polymyxa betae, fungal vector of sugar beet rhizomania disease.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

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