Synthetic antibody fragment as ligand in immunoaffinity chromatography

Welling, Gjalt W.; Geurts, Ton; Gorkum, Judith Van; Damhof, R.A.; Drijfhout, Jan W.; Bloemhoff, W.; Welling‐Wester, Sytske

doi:10.1016/s0021-9673(01)89500-5

Cited by 27 publications

(3 citation statements)

References 13 publications

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“…The interaction ofthe histidine-containing peptide with the Ni2+ ions bound to the nitrilotriacetic acid resin results in an oriented immobilization of the antibodies. An interesting approach for selective purification and analysis of proteins is the use of a synthetic antibody fragment that mimics the antigenbinding site as ligand (Welling et al, 1990).…”

Section: Miscellaneous Immobilized Ligandsmentioning

confidence: 99%

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Chromatographic Techniques for the Characterization of Proteins

Holthuis¹,

Driebergen²

1995

Pharmaceutical Biotechnology

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Section: Miscellaneous Immobilized Ligandsmentioning

confidence: 99%

“…Immobilized peptides have also been used as stationary phase (Welling et al, 1990;Fleminger et al, 1992). DNA polymerase-a was immobilized on Affi-Gel 1O-activated agarose by Miles and Formosa (1992).…”

Section: Miscellaneous Immobilized Ligandsmentioning

confidence: 99%

Chromatographic Techniques for the Characterization of Proteins

Holthuis¹,

Driebergen²

1995

Pharmaceutical Biotechnology

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“…This may be because the potential MicroAb does not mimic the conformation of the CDR sufficiently well, because effective IgG binding requires more than one CDR or because the mass of the IgG is essential to its biological activity. Nonetheless, several MicroAbs that bind to their cognate antigens have been synthesized (Williams et al, 1988(Williams et al, , 1989a(Williams et al, , b, 1991aCohen et al, 1990;Welling et al, 1990Welling et al, , 1991Winter & Milstein, 1991;Jarrin et al, 1994;Smith et al, 1994;Laune et al, 1997;Monnet et al, 1999), but so far only three virus-neutralizing MicroAbs have been isolated. The first was derived from the HIV-1 gp120 envelope glycoproteinspecific mAb F58 (Broliden et al, 1990;Levi et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Analysis of a 17-amino acid residue, virus-neutralizing microantibody

Heap

Wang

Pinheiro

et al. 2005

Journal of General Virology

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The antibody-binding site, through which an antibody binds to its epitope, is a complex structure formed by the folding together of six complementarity-determining regions (CDRs). However, certain peptides derived from CDR sequences retain antibody specificity and function; these are know as microantibodies (MicroAbs). For example, the F58 MicroAb is a 17 residue, cyclized peptide (CDLIYYDYEEDYYFDYC) derived from CDR-H3 of F58, an IgG1 specific for the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). Both MicroAb and IgG recognize the same epitope in the V3 loop and, despite its small size, the MicroAb neutralizes the infectivity of HIV-1 IIIB only 32-fold less efficiently on a molar basis. The advantage of MicroAbs is that their small size facilitates structure-function analysis. Here, the F58 MicroAb was investigated using alanine scanning, mass spectroscopy and surface plasmon resonance. Neutralization of infectious IIIB was generally more sensitive to alanine substitution than binding to soluble gp120. There appeared to be a division of function within the MicroAb, with some residues involved in antigen binding (alanine substitution of 11D, 12Y or 13Y abrogated both binding and neutralization), whereas others were concerned solely with neutralization (substitution of 3L, 8Y or 14F abrogated neutralization, but not binding). The MicroAb is predominantly b-sheet and has strong conformational constraints that are probably essential for activity. The MicroAb and soluble gp120 formed a 1 : 1 complex, with an association rate that was threefold greater than that with IgG and a faster dissociation rate. Its equilibrium dissociation constant is 37?5-fold greater than that of IgG, in line with neutralization data. This study demonstrates how MicroAbs can make a useful contribution to the understanding of antigen-antibody interactions. INTRODUCTIONAn antibody binds to its cognate epitope through its paratope, a domain formed from the six complementaritydetermining regions (CDRs) situated within the variable portions of the immunoglobulin light and heavy chains. Thus, a bivalently binding IgG can attach to antigen by the combined action of 12 CDRs. A microantibody (MicroAb) is the minimum recognition unit of an antibody and comprises a peptide derived from usually just one CDR. Its activity will depend on both its sequence and conformation. Some antiviral MicroAbs retain the virus binding and infectivity neutralizing activities of the parent antibody. The small size of MicroAbs should enable residues that are key to virus binding and neutralization activities to be easily identified. MicroAbs also have a potential advantage over IgG as therapeutic antivirals as they are less immunogenic and should be useful in situations where larger antibodies cannot easily penetrate.MicroAbs have proved difficult to identify. This may be because the potential MicroAb does not mimic the conformation of the CDR sufficiently well, because effective IgG binding requires more than one CDR or because the mass of ...

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Characterization of a synthetic 37-residue fragment of a monoclonal antibody against herpes virus by capillary electrophoresis/electrospray (ionspray) mass spectrometry and252Cf plasma desorption mass spectrometry

Kostiainen

Lasonder

Bloemhoff

et al. 1994

Biol. Mass Spectrom.

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A peptide comprising 37 amino acids of the antigen binding site of a monoclonal antibody directed against glycoprotein D of herpes simplex virus was synthesized. The synthetic peptide and the impurities formed in the synthesis were characterized by capillary electrophoresis/ionspray mass spectrometry and by 252Cf plasma desorption-time of flight mass spectrometry. The measured average molecular mass of the synthetic peptide was 4627.16 Da, which was only 0.08 Da higher than the calculated value (4627.08 Da). The plasma desorption mass spectrum of the synthetic peptide showed a protonated molecule at m/z 4624.1, which was 4 Da lower than the calculated one (4628.09 Da). The amino acid sequence of the peptide was confirmed in part by electrospray (ionspray) mass spectrometry using a high nozzle skimmer voltage difference. Five impurities were separated and identified by capillary electrophoresis/mass spectrometry and two of them also appeared in the plasma desorption mass spectrum.

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Synthetic antibody fragment as ligand in immunoaffinity chromatography

Cited by 27 publications

References 13 publications

Chromatographic Techniques for the Characterization of Proteins

Chromatographic Techniques for the Characterization of Proteins

Analysis of a 17-amino acid residue, virus-neutralizing microantibody

Characterization of a synthetic 37-residue fragment of a monoclonal antibody against herpes virus by capillary electrophoresis/electrospray (ionspray) mass spectrometry and252Cf plasma desorption mass spectrometry

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