scholarly journals Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains.

1986 ◽  
Vol 164 (5) ◽  
pp. 1809-1814 ◽  
Author(s):  
V Agnello ◽  
J L Barnes
Keyword(s):  
Rheumatoid Factor ◽  
Primary Structure ◽  
Binding Activity ◽  
Light Chains ◽  
Heat Denaturation ◽  
Antigen Binding ◽  
Rheumatoid Factors ◽  
Simple Method ◽  
Heat Labile

Evidence was obtained that both the WA and BLA crossidiotype (XId) groups are conformational antigens requiring both L and H chains and that with heat denaturation the antigens that define the XIds and antigen-binding activity are lost in parallel. In contrast, the primary structure-dependent crossreactive idiotype (CRI), PSL2, which is only weakly detected on native Wa and Bla monoclonal rheumatoid factors (mRFs), became prominently detected on the heated Wa and Bla mRFs. Heat denaturation may provide a simple method for distinguishing Ids determined by conformational antigen from primary structure-dependent Ids. In addition to heat denaturation, some acid conditions commonly used for preparation of RFs were also found to cause marked loss of Id antigen. The finding of PSL2-CRI on Bla mRF indicates that this Id is not unique to the WA XId.

Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris

Bioimpacts ◽  
2021 ◽  
Author(s):  
Shirin Movaghar Asareh ◽  
Tahereh Savei ◽  
Sareh Arjmand ◽  
Seyed Omid Ranaei Siadat ◽  
Fataneh Fatemi ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Fluorescent Protein ◽  
Antibody Fragment ◽  
Binding Activity ◽  
Age Related Macular Degeneration ◽  
Light Chains ◽  
Antigen Binding ◽  
Active Structure ◽  
Age Related ◽  
Fragment Antigen Binding

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD).Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

scholarly journals Human rheumatoid factor crossidiotypes. II. Primary structure-dependent crossreactive idiotype, PSL2-CRI, present on Wa monoclonal rheumatoid factors is present on Bla and other IgM kappa monoclonal autoantibodies.

1987 ◽  
Vol 165 (1) ◽  
pp. 263-267 ◽  
Author(s):  
V Agnello ◽  
F Goñi ◽  
J L Barnes ◽  
M T de la Vega ◽  
B Frangione
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rheumatoid Factor ◽  
Primary Structure ◽  
Rheumatoid Factors ◽  
Allotypic Marker ◽  
Complementarity Determining Region

The amino acid sequence of the L-CDR2 (complementarity-determining region) of Bla mRF (monoclonal rheumatoid factor) is identical to that of the Wa mRFs. The PSL2-CRI (crossreactive idiotype), as determined by anti-PSL2, which has been shown to be present on all Wa mRFs, is also present on the Bla mRF and other monoclonal autoantibodies. PSL2-CRI is, therefore, not unique to Wa mRFs and may be present on most IgM kappa monoclonal autoantibodies. Whether PSL2-CRI is a crossidiotype (XId) that is selectively present on autoantibodies or represents an allotypic marker for a V kappa III gene is undetermined.

Framework peptides fromxIIIb rheumatoid factor light chains with binding activity for aggregated IgG

1991 ◽  
Vol 21 (8) ◽  
pp. 1837-1841 ◽  
Author(s):  
Frank Charles Hay ◽  
Andzriej Jan Soltys ◽  
Gordon Tribbick ◽  
H. Mario Geysen
Keyword(s):  
Rheumatoid Factor ◽  
Binding Activity ◽  
Light Chains

scholarly journals Broadly Distributed Chemical Reactivity of Natural Antibodies Expressed in Coordination with Specific Antigen Binding Activity

2003 ◽  
Vol 278 (22) ◽  
pp. 20436-20443 ◽  
Author(s):  
Stephanie Planque ◽  
Hiroaki Taguchi ◽  
Gary Burr ◽  
Gita Bhatia ◽  
Sangeeta Karle ◽  
...  
Keyword(s):  
Chemical Reactivity ◽  
Specific Antigen ◽  
Binding Activity ◽  
Natural Antibodies ◽  
Antigen Binding

Antibodies to Rotaviruses in Chickens' Eggs: A Potential Source of Antiviral Immunoglobulins Suitable for Human Consumption

PEDIATRICS ◽  
1988 ◽  
Vol 81 (2) ◽  
pp. 291-295
Author(s):  
Robert H. Yolken ◽  
Flora Leister ◽  
Siok-Bi Wee ◽  
Robin Miskuff ◽  
Steven Vonderfecht
Keyword(s):  
Binding Activity ◽  
Human Consumption ◽  
Antigen Binding ◽  
Rotavirus Gastroenteritis ◽  
Potential Source ◽  
Egg Products ◽  
Egg Yolks ◽  
The Individual ◽  
Antibody Levels ◽  
Infants And Young Children

The prevalence of antibodies to human rotaviruses in commercially available eggs and egg products that are suitable for human consumption was investigated. The yolks of virtually all of the individual eggs and pasteurized pooled egg preparations contain antirotavirus antibodies detectable by means of enzyme immunoassay systems. Also, the eggs and egg preparations are capable of inhibiting the growth of two strains of rotaviruses in tissue culture. Chromatographic studies indicated that the antigen-binding activity is limited largely to the immunoglobulin fractions of the egg yolks. The antibody levels in eggs can be increased by the immunization of hens with purified rotavirus preparations, and the immunoglobulins isolated from the eggs of immunized hens can prevent the development of rotavirus gastroenteritis in experimentally infected animals. Egg preparations might serve as a practical source of antiviral antibodies suitable for consumption by infants and young children.

scholarly journals SPECIFICITY IN THE COMBINATION OF FD FRAGMENTS WITH L CHAINS TO FORM HAPTEN-BINDING SITES

1966 ◽  
Vol 123 (5) ◽  
pp. 921-934 ◽  
Author(s):  
O. A. Roholt ◽  
G. Radzimski ◽  
D. Pressman
Keyword(s):  
Amino Acid ◽  
Propionic Acid ◽  
Binding Sites ◽  
Amino Acid Analysis ◽  
Binding Activity ◽  
Light Chains ◽  
Antibody Activity ◽  
Fc Fragment ◽  
Fab Fragments ◽  
Correct Alignment

In the work reported here we have shown that light chains and Fd fragments can be separated completely in propionic acid and then recombined to form Fab fragments with antibody activity. This experiment indicates that in the recombination a correct alignment of the Fd fragments and the L chains occurs to give a competent antibody site, just as occurs with the recombination of separated heavy and light chains of the antibody; thus the Fc fragment is not required for correct alignment. Fd fragments of antibody alone show very low binding activity toward the specific hapten. As is the case for the combination of heavy and light chains, the combination of Fd fragments and light chains also requires that both components come from antibody from the same rabbit in order to give binding sites. When they are derived from different rabbits producing antibody against the same antigen, they still give Fab fragments as shown by immunoelectrophoresis but do not have competent binding sites. An important observation is that the subunits of the papain digest fractions, FabI and FabII, have the capacity to cross-combine to form active Fab fragments with competent binding sites. FdI from FabI combines with LII chains from FabII to give the composite (FdI-LII) with good binding activity. Likewise, the composite (FdII-LI) has good binding activity. The composites from the two types of antibody molecules yielding different Fab fragments have antibody activity although heretofore these molecules have appeared to be different on the bases of chromatography and amino acid analysis. There is also a preferential combination of the Fd fragments to combine with the correct L fragments to give binding sites since this combination takes preference over the combination of Fd fragments of antibody with light chains of normal globulin (or of light chains of antibody with Fd fragments of normal globulin).

scholarly journals A simple method to measure CLOCK-BMAL1 DNA binding activity in tissue and cell extracts

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1316 ◽  
Author(s):  
Maud Gillessen ◽  
Pieter Bas Kwak ◽  
Alfred Tamayo
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Casein Kinase I ◽  
Simple Method ◽  
Dna Binding Activity ◽  
Tissue Extracts ◽  
Cell Extracts ◽  
Specialized Equipment ◽  
Dna Binding Assay

The proteins CLOCK and BMAL1 form a heterodimeric transcription factor essential to circadian rhythms in mammals.  Daily rhythms of CLOCK-BMAL1 DNA binding activity are known to oscillate with target gene expression in vivo. Here we present a highly sensitive assay that recapitulates native CLOCK-BMAL1 DNA binding rhythms from crude tissue extracts, which we call the Clock Protein-DNA Binding Assay (CPDBA). This method can detect less than 2-fold differences in DNA binding activity, and can deliver results in two hours or less using 10 microliters or less of crude extract, while requiring neither specialized equipment nor expensive probes. To demonstrate the sensitivity and versatility of this assay, we show that enzymatic removal of phosphate groups from proteins in tissue extracts or pharmacological inhibition of casein kinase I in cell culture increased CLOCK-BMAL1 DNA binding activity by ~1.5 to ~2 fold, as measured by the CPDBA. In addition, we show that the CPDBA can measure CLOCK-BMAL1 binding to reconstituted chromatin. The CPDBA is a sensitive, fast, efficient and versatile probe of clock function.

scholarly journals The generation of the human mab RabD4 specific to the rabies virus glycoprotein and characterization thereof

2019 ◽  
Vol 485 (3) ◽  
pp. 370-373
Author(s):  
Е. N. Ilina ◽  
E. V. Solopova ◽  
Т. К. Aliev ◽  
М. V. Larina ◽  
D. S. Balabashin ◽  
...  
Keyword(s):  
B Cells ◽  
Rabies Virus ◽  
Binding Activity ◽  
Antigen Binding ◽  
Rabies Virus Glycoprotein ◽  
Neutralizing Activity ◽  
Virus Glycoprotein ◽  
Peripheral B Cells

We generated a novel human neutralizing human mAb RabD4 against rabies virus glycoprotein using in vitro stimulation human peripheral B cells produced from immunized donor. It was revealed that the human mAb RabD4 demonstrated high antigen-binding activity and virus-neutralizing activity in the FAVN test with the CVS-11 rabies virus.

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