BINDING OF AGGREGATED IgG Fab-FRAGMENTS AND LIGHT CHAINS TO SOME GROUP A, C and G STREPTOCOCCI

2009 ◽  
Vol 91B (1-6) ◽  
pp. 27-33
Author(s):  
CLAËS SCHALÉN ◽  
ULF ZÄTTERSTRÖM ◽  
MAJ-LIS SVENSSON ◽  
POUL CHRISTENSEN
Keyword(s):  
Light Chains ◽  
Fab Fragments ◽  
Group A

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 AN IDIOTYPIC CROSS-REACTION BETWEEN ALLOTYPE a3 AND ALLOTYPE a NEGATIVE RABBIT ANTIBODIES TO STREPTOCOCCAL CARBOHYDRATE

1973 ◽  
Vol 137 (3) ◽  
pp. 636-648 ◽  
Author(s):  
Thomas J. Kindt ◽  
David G. Klapper ◽  
Michael D. Waterfield
Keyword(s):  
Chemical Analysis ◽  
Cross Reaction ◽  
The Other ◽  
Light Chains ◽  
Binding Affinities ◽  
Variable Regions ◽  
Heavy Chains ◽  
Relative Binding ◽  
Group A

Two antibodies to Group C streptococcal carbohydrate isolated from an individual rabbit had similar relative binding affinities for a Group C immuno-adsorbent column. Their light chains were similar, if not identical, as were the constant regions of their heavy chains. Differences in the variable regions of the H chains of the two antibodies were detected by chemical analysis. The two antibodies had serologically identical idiotypic determinants although one antibody possessed the a3 allotype and the other had no detectable group a marker. The occurrence of such antibodies indicates the absence of obligatory associations between group a allotypes and idiotypic specificities, despite the fact that both determinants have antigenic components in the VH region of the H chain.

scholarly journals STUDIES ON HUMAN ANTIBODIES

1969 ◽  
Vol 129 (5) ◽  
pp. 871-896 ◽  
Author(s):  
Carlos Moreno ◽  
Elvin A. Kabat
Keyword(s):  
Density Gradient ◽  
Blood Group ◽  
Density Gradient Centrifugation ◽  
Equilibrium Dialysis ◽  
Gradient Centrifugation ◽  
Light Chains ◽  
Human Antibodies ◽  
Blood Group A ◽  
Group A

Human antibodies to blood group A substance were purified by absorption on columns of insoluble polyleucyl hog blood group A + H substance and eluted first with N-acetylgalactosamine and then with an A active reduced pentasaccharide ARL0.52. The γM and γG antibodies in these eluates were separated by density gradient centrifugation. The antibodies were studied for their relative capacities to be inhibited by various blood group A active oligosaccharides. Antibodies eluted by the N-acetylgalactosamine could be inhibited by N-acetylgalactosamine, as well as by lower concentrations of A active tri- and pentasaccharides, while those eluted by the pentasaccharide ARL0.52 could only be inhibited by the two oligosaccharides, but not by N-acetylgalactosamine, indicating that the N-acetylgalactosamine eluate had more antibodies with smaller size combining sites than the ARL0.52 eluate. Measurements by equilibrium dialysis gave values ranging from 2 x 103 to 1 x 105 M–1 and the values obtained with the ARL0.52 eluate were somewhat higher than those with the GalNAc eluate. Only one of three anti-A sera had γM anti-A in the ARL0.52 eluate, while all three had γM in the N-acetylgalactosamine eluate. Data on the precipitating, hemagglutinating, complement fixing, hemolytic properties of the eluted antibodies, and of their content of κ and λ light chains are given.

scholarly journals Protein crystallization with microseed matrix screening: application to human germline antibody Fabs

2014 ◽  
Vol 70 (8) ◽  
pp. 1107-1115 ◽  
Author(s):  
Galina Obmolova ◽  
Thomas J. Malia ◽  
Alexey Teplyakov ◽  
Raymond W. Sweet ◽  
Gary L. Gilliland
Keyword(s):  
Structure Determination ◽  
Protein Crystallization ◽  
Light Chains ◽  
Human Antibody ◽  
Initial Screening ◽  
Heavy Chains ◽  
Fab Fragments ◽  
Macromolecular Crystallization

The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.

scholarly journals THE INDIVIDUAL ANTIGENIC SPECIFICITY OF ANTIBODIES TO STREPTOCOCCAL CARBOHYDRATES

1968 ◽  
Vol 128 (5) ◽  
pp. 969-989 ◽  
Author(s):  
Dietmar G. Braun ◽  
Richard M. Krause
Keyword(s):  
Disc Electrophoresis ◽  
Antigenic Specificity ◽  
Light Chains ◽  
Fab Fragment ◽  
Additional Absorption ◽  
Group A ◽  
The Individual ◽  
Restricted Pattern

Although a single electrophoretically uniform antibody component with specificity for the group carbohydrate may comprise the bulk of the γ-globulin in rabbits immunized with streptococcal vaccines, this is not always the case. Not infrequently, electrophoresis may reveal multiple antibody components. Nevertheless, it has been feasible by various preparative procedures to isolate from a single antiserum at least two antibody components with similar reactivity for the carbohydrate both of which are electrophoretically monodisperse. Light chains from such antibodies reveal a restricted pattern when examined by disc electrophoresis. Antibodies to streptococcal carbohydrates have been examined for their individual antigenic specificity. Goats were immunized with isolated Group C and Group A-variant antibodies raised in rabbits. Individual antigenic specificity of these antibodies was brought out by absorption of the goat anti-antiserum with Fr II of pooled normal rabbit sera. Additional absorption of the goat anti-antisera with Fr II diminished but did not eliminate the reactivity for the homologous antibody. Immunoelectrophoretic studies with papain fragments of purified streptococcal antibodies localized the specificity to the Fab fragment. Specificity was not confined to the isolated light chains of the antibody.

scholarly journals Purification of anti-glycoconjugate monoclonal antibodies using newly developed porous zirconia particles

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tetsuya Okuda ◽  
Katsuya Kato ◽  
Masahiro Kitamura ◽  
Shinjiro Kasahara
Keyword(s):  
Monoclonal Antibodies ◽  
Functional Group ◽  
Mammalian Species ◽  
Light Chains ◽  
Purification Method ◽  
Elution Buffer ◽  
Porous Zirconia ◽  
Group A ◽  
Culture Supernatants ◽  
Antibody Function

AbstractHere, we describe porous zirconia particles (PZPs) optimized for the purification of immunoglobulins. PZPs, with a pore size of approximately 10 nm, were designed to specifically interact with antibodies via surface modification with a phosphate functional group. A simple PZP purification method based on precipitation enabled efficient purification of mouse anti-glycosphingolipid globoside/Gb4Cer monoclonal IgM (κ-light chains) from hybridoma culture supernatants. Over 99% of contaminating proteins were removed by the PZP purification process, and approximately 50% of the IgM was recovered in the purified fraction after eluting the PZP-adsorbed antibodies with 100 mM phosphate buffer. Other IgG3 and IgM monoclonal antibodies that react with Gb4Cer or α2,6-sialyl LacNAc-modified glycoproteins could also be purified using PZPs and elution buffer at concentrations of 100–500 mM. All of the purified antibodies retained their antigen reactivity and specificity, indicating that PZP purification does not affect antibody function. As PZP purification is also suitable for purification of IgM consisting of λ-light chains and IgG derived from other mammalian species, it is expected to be applied to the purification of a variety of antibodies, including anti-glycoconjugate IgMs.

scholarly journals An Expression System for the E. coli Fermentation of Recombinant Antibody Fab Fragments from Mice and Rabbits

2010 ◽  
Vol 93 (1) ◽  
pp. 80-88 ◽  
Author(s):  
Julia C Wiebe ◽  
Carolin Schüller ◽  
Jana A Reiche ◽  
Karl Kramer ◽  
Arne Skerra ◽  
...  
Keyword(s):  
Recombinant Antibody ◽  
Expression System ◽  
Coli Strain ◽  
Vector System ◽  
Light Chains ◽  
Selection Marker ◽  
Specific Class ◽  
Fab Fragment ◽  
E Coli ◽  
Fab Fragments

Abstract A mutually compatible vector system was developed for the bench-top fermenter production of mouse and rabbit Fab fragments comprising pASK85-pro-K411B and pASK85Rab-pro-WR13. These vectors provide a mouse- and rabbit-specific cloning site, respectively, the tetA promoter/operator and the proBA operon that complements the Pro biosynthetic deficiency of the Escherichia coli strain JM83 and can serve as an additional selection marker. Fermentation at elevated cell density (OD600 = 2040) of the atrazine-specific mouse Fab fragment K411B using JM83 harboring pASK85-pro-K411B in a 2 L bench-top vessel resulted in a yield of 240 g/L OD600 affinity-purified protein (13.8 mg). In contrast, expression of leporid Fab fragments using pASK85Rab-pro-WR13 was unsuccessful due to the aggregation of rabbit light chains, which probably relates to a general problem of this specific class of immunoglobulins with their additional Cys residues. Coexpression of rabbit Fab fragments together with four periplasmic folding-helper proteins encoded on pTUM4 led to a significantly improved folding efficiency, resulting in a yield of 50 µg/L OD600 affinity-purified rabbit Fab fragment (3.3 mg) from the 2 L bench-top fermenter.

scholarly journals Epitopes of streptococcal M proteins shared with cardiac myosin.

1985 ◽  
Vol 162 (2) ◽  
pp. 583-591 ◽  
Author(s):  
J B Dale ◽  
E H Beachey
Keyword(s):  
Acute Rheumatic Fever ◽  
M Protein ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Myosin Heavy Chains ◽  
Cardiac Myosin ◽  
Poststreptococcal Glomerulonephritis ◽  
Heavy Chains ◽  
M Proteins ◽  
Group A

We present evidence that M proteins from three different serotypes of group A streptococci share epitopes with cardiac myosin. Rabbit antisera evoked by a purified fragment of type 5 M protein crossreacted with myosin, but not alpha-tropomyosin, actin, or myosin light chains. In enzyme-linked immunosorbent assays, the myosin-crossreactive antibodies were totally inhibited by type 5 M protein and partially inhibited by types 6 and 19 M proteins. The affinity-purified myosin antibodies opsonized type 5 streptococci, indicating that they were directed against protective M protein epitopes on the surface of the organisms. Immunoblot analyses demonstrated the binding of the crossreactive antibodies to myosin heavy chains. Sera from patients with acute rheumatic fever showed significantly stronger reactions with myosin than did sera from their siblings, hospitalized controls, or patients with poststreptococcal glomerulonephritis.

scholarly journals Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies: Mouse on Mouse Ab Staining

2021 ◽  
pp. 002215542110332
Author(s):  
Francesco Mascadri ◽  
Roberta Ciccimarra ◽  
Maddalena M. Bolognesi ◽  
Fabio Stellari ◽  
Francesca Ravanetti ◽  
...  
Keyword(s):  
Detection System ◽  
Cross Reactivity ◽  
Mouse Tissue ◽  
Light Chains ◽  
Specific Detection ◽  
Specific Antibodies ◽  
Fab Fragments ◽  
Secondary Antibodies

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.

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