scholarly journals RECOVERY OF ANTIBODY ACTIVITY FROM INACTIVE HYBRIDS OF H AND L CHAINS

1967 ◽  
Vol 125 (1) ◽  
pp. 191-197 ◽  
Author(s):  
O. A. Roholt ◽  
G. Radzimski ◽  
D. Pressman
Keyword(s):  
Propionic Acid ◽  
Binding Activity ◽  
Light Chains ◽  
Antibody Activity ◽  
Heavy Chains

Hybrid IgG molecules were prepared from the heavy and light chains of specifically purified antibody against two different haptens. One hybrid consisted of the H chains from the anti-p-azobenzoate antibody from one rabbit and L chains from the anti-p-azobenzenearsonate antibody from the second rabbit, and the second hybrid consisted of the opposite combination, light chains from the first rabbit and heavy chains from the second. The two hybrids were mixed at pH 8 and were found to be so stable in the mixture that even after 2 wk at 5°C there was still only low hapten-binding activity toward p-iodobenzoate and p-iodobenzenearsonate. However, exposure of the mixture of hybrids to 1 M propionic acid followed by buffer at pH 8 resulted in recovery of binding activity for p-iodobenzoate and p-iodobenzenearsonate. Thus no exchange occurred between the light and heavy chains of the hybrids in the buffer, but exchange did occur on exposure to propionic acid, and this exchange favored a preferential combination among the chains in such a manner that effective antibody sites resulted.

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 Expression of a public idiotype by human monoclonal IgM directed to myelin-associated glycoprotein and characterization of the variability subgroup of their heavy and light chains.

1989 ◽  
Vol 170 (5) ◽  
pp. 1551-1558 ◽  
Author(s):  
J C Brouet ◽  
K Dellagi ◽  
M C Gendron ◽  
A Chevalier ◽  
C Schmitt ◽  
...  
Keyword(s):  
Nonhuman Primate ◽  
Light Chains ◽  
Antibody Activity ◽  
Rheumatoid Factors ◽  
Variable Regions ◽  
Cold Agglutinins ◽  
Heavy Chains ◽  
Myelin Associated Glycoprotein ◽  
Kappa Light Chains

Most studies using rabbit or mouse antisera failed to detect CRI between human IgM directed to MAG. We show here that 9 of 10 such IgM express a public CRI as defined by a nonhuman primate antiserum. Shared idiotype is likely involved in (or close to) the combining site of those IgM since antiidiotypic serum inhibited the binding of IgM to MAG and reacted with IgM having different variable regions of light and heavy chains. Partial aminoterminal sequence of heavy and light chains showed that anti-MAG IgM use either lambda chains (one IgM) or kappa light chains (six IgM) of different variability subgroups (V kappa IV in three instances, V kappa I in two, and V kappa II in one), whereas heavy chains belong to the VHIII (six IgM) or to the VHII (1 IgM) subgroup. These features distinguish these IgM from other human monoclonal IgM with a defined antibody activity, such as rheumatoid factors or cold agglutinins.

Specificity of antigenic recognition of antibody heavy chain

1966 ◽  
Vol 166 (1003) ◽  
pp. 176-187 ◽  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Group Analysis ◽  
Binding Activity ◽  
Association Constants ◽  
Light Chains ◽  
Specific Information ◽  
Parent Molecule ◽  
Heavy Chains ◽  
Antibody Heavy Chain

The specificity of antigenic recognition of the component chains of purified dinitrophenyl and trinitrophenyl antibodies was examined. Heavy chains were rendered soluble at neutral pH, either by prior reaction of the parent antibodies with D,L-alanine N -carboxy anhydride, or by mixing heavy chain with light chain of non-specific IgG. The degree of homologous light chain contamination of these heavy chain preparations was found to be less than 2 %, either by immune precipitation, or by end-group analysis. Association constants of the heavy chains of both antibodies with several closely related haptenes were measured by fluorescence quenching. Heavy chains differentiated among these haptenes in the same manner as the parent antibodies, though considerable binding affinity was lost. When specific homologous light chains were added to the heavy chain preparations, association constants were increased, but without change in relative selectivity. Binding activity of light chains alone could not be measured. The heavy chain, then, appears to bear the specificity of its parent molecule. Whether or not homologous light chain contributes additional specific information with respect to antigenic recognition or simply plays a non-specific modulating role cannot be answered from these experiments.

Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

1988 ◽  
Vol 46 ◽  
pp. 156-157
Author(s):  
Donald A. Winkelmann
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Actin Filaments ◽  
Light Chains ◽  
Myosin Filament ◽  
Primary Role ◽  
Heavy Chains ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Globular Head

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

scholarly journals Transient IgA-lambda paraproteinemia during treatment of acute myelomonoblastic leukemia

Blood ◽  
1980 ◽  
Vol 55 (1) ◽  
pp. 21-25 ◽  
Author(s):  
B Van Camp ◽  
P Reynaerts ◽  
JP Naets ◽  
J Radl
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Clonal Expansion ◽  
Cellular Level ◽  
Light Chains ◽  
Antibody Activity ◽  
Intensive Chemotherapy ◽  
Bone Marrow Aplasia ◽  
Large Panel ◽  
Common Antigens

Abstract Monoclonal plasma cell proliferation with secretion of IgA-lambda and free lambda light chains during a phase of bone marrow aplasia following intensive chemotherapy was observed in a patient suffering from acute myelomonoblastic leukemia. The clonal expansion and regression was investigated at the cellular level by immunofluorescence using an antiserum against the idiotype of the paraportein. Although a large panel of common antigens was used for testing, no antibody activity of the paraprotein could be demonstrated.

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.

scholarly journals Affinity labelling of the binding site of rabbit antibody. Evidence for the involvement of the hypervariable regions of the heavy chain

1974 ◽  
Vol 139 (1) ◽  
pp. 135-149 ◽  
Author(s):  
Christopher E. Fisher ◽  
Elizabeth M. Press
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Binding Sites ◽  
Light Chains ◽  
Rabbit Antibody ◽  
Heavy Chains ◽  
Group 4 ◽  
Hypervariable Regions ◽  
Affinity Labelling ◽  
Antibody Complex

The binding sites of rabbit antibodies with affinity for the haptenic group 4-azido-2-nitrophenyl-lysine have been specifically labelled by photolysis of the hapten–antibody complex. The extent of covalent labelling was 0.5–0.9mol of hapten bound/mol of antibody and, by using an immunoadsorbent, antibody with 1.3mol of hapten/mol was obtained. The antibody was specifically labelled in the binding site and the ratio of labelling of heavy and light chains was in the range 3.3–5.0. The labelled heavy chains were cleaved by CNBr treatment and after reduction and alkylation of the intrachain bonds, were digested with trypsin. Evidence is presented that two regions of the heavy chain, positions 29–34 and 95–114, together contain about 80% of the label on the heavy chain; these two regions respectively include two of the hypervariable regions of rabbit heavy chain.

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 Biochemical characterization and tissue distribution of the A and B variants of the integrin alpha 6 subunit.

1993 ◽  
Vol 121 (1) ◽  
pp. 179-191 ◽  
Author(s):  
F Hogervorst ◽  
L G Admiraal ◽  
C Niessen ◽  
I Kuikman ◽  
H Janssen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Peripheral Nerves ◽  
Biochemical Characterization ◽  
Light Chains ◽  
Cytoplasmic Domains ◽  
Heavy Chains ◽  
Electrophoretic Mobilities ◽  
Reducing Conditions ◽  
Integrin Alpha 6 ◽  
Major Target

Two cytoplasmic variants of the alpha 6 integrin, alpha 6A and alpha 6B, have been identified previously (Hogervorst, F., I. Kuikman, A. G. van Kessel, and A. Sonnenberg. 1991. Eur. J. Biochem. 199:425-433; Cooper, H. M., R. N. Tamura, and V. Quaranta. 1991. J. Cell Biol. 115:843-850). Using synthetic peptides, containing sequences of their cytoplasmic domains, we have produced mAbs specific for either of the variants. These antibodies reacted with a variety of different epithelial tissues. In some tissues (e.g., salivary gland) both variants could be detected while in others only one of the variants was found (e.g., alpha 6A in epidermis and alpha 6B in kidney). Among nonepithelial cells and tissues, perineural fibroblasts and Schwann cells in peripheral nerves and platelets reacted with anti-alpha 6A, while microvascular endothelia reacted with both anti-alpha 6A and anti-alpha 6B. From our immunohistochemical results there is not evidence that combination with beta 1 or beta 4 is restricted to one of the two variants of alpha 6. This was confirmed by immunoprecipitation studies which showed that both beta 1 and beta 4 were coprecipitated by both anti-alpha 6A or anti-alpha 6B antibodies from cells. Also, the distribution of alpha 6A and alpha 6B subunits associated with beta 1 on cells attached to laminin was similar: both were found in focal contacts colocalizing with vinculin. In contrast, the alpha 6A subunit, associated with beta 4 in cultures of a squamous cell carcinoma cell line, was found to codistribute with bullous pemphigoid antigen 230 in hemidesmosomal-like structures. The alpha 6A and alpha 6B variants, immunoprecipitated from various cell lines, exhibited slightly different electrophoretic mobilities. Analysis of the antigens under reducing conditions showed that the mobility of the light chains, but not of the heavy chains, is different. In addition, in some cells the light chains of alpha 6A and alpha 6B, each are of two different sizes. Treatment with N-glycanase showed that these two light chain variants of alpha 6A and alpha 6B are not due to differences in N-linked glycosylation, and may therefore represent alternative proteolytic products of the alpha 6 precursor. We further demonstrate that alpha 6A, but not alpha 6B, is a major target for PMA-induced phosphorylation. Phosphorylated alpha 6A contained phosphoserine and a small amount of phosphotyrosine. There are also two variants of the integrin alpha 3 subunit with different cytoplasmic domains, but in the cell lines examined only alpha 3A could be demonstrated by RT-PCR.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Fate of surrogate light chains in B lineage cells.

1996 ◽  
Vol 183 (2) ◽  
pp. 421-429 ◽  
Author(s):  
K Lassoued ◽  
H Illges ◽  
K Benlagha ◽  
M D Cooper
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
Light Chain ◽  
Light Chains ◽  
Receptor Complex ◽  
Surrogate Light Chain ◽  
Heavy Chains ◽  
Receptor Component ◽  
B Lineage ◽  
Receptor Assembly

Biosynthesis of the immunoglobulin (Ig) receptor components and their assembly were examined in cell lines representative of early stages in human B lineage development. In pro-B cells, the nascent surrogate light chain proteins form a complex that transiently associates in the endoplasmic reticulum with a spectrum of unidentified proteins (40, 60, and 98 kD) and Bip, a heat shock protein family member. Lacking companion heavy chains, the surrogate light chains in pro-B cells do not associate with either the Ig(alpha) or Ig(beta) signal transduction units, undergo rapid degradation, and fail to reach the pro-B cell surface. In pre-B cells, by contrast, a significant portion of the surrogate light chain proteins associate with mu heavy chains, Ig(alpha), and Ig(beta) to form a stable receptor complex with a relatively long half-life. Early in this assembly process, Bip/GRP78, calnexin, GRP94, and a protein of approximately 17 kD differentially bind to the nascent mu heavy chains. The 17-kD intermediate is gradually replaced by the surrogate light chain protein complex, and the Ig(alpha) and Ig(beta) chains bind progressively to the mu heavy chains during the complex and relatively inefficient process of pre-B receptor assembly. The results suggest that, in humans, heavy chain association is essential for surrogate light chain survival and transport to the cell surface as an integral receptor component.

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