Modification of monoclonal and polyclonal IgG with palladium (II) coproporphyrin I: stimulatory and inhibitory functional effects induced by two different methods

doi:10.1016/0022-1759(95)00154-3

Journal of Immunological Methods

Volume 186, Issue 2, 26 October 1995, Pages 293-304

https://doi.org/10.1016/0022-1759(95)00154-3 Get rights and content

Abstract

Antibodies conjugated with porphyrins and metalloporphyrins have a great potential for applications in fluorescence or phosphorescence immunoassays as well as in photodynamic therapy, radioimaging and internal radiation therapy of cancer. Here we describe how the new preactivated metalloporphyrin, palladium (II) coproporphyrin I-tetra-N-hydroxysuccinimide ester, can be covalently attached to mouse monoclonal and rabbit anti-human ferritin antibodies. The advantages of the proposed reagent over the previously reported carboxylic porphyrins coupled through carbodiimide activation are indicated. Conformational changes in antibodies caused by each of the two methods were assessed from their binding to the antigen (a probe for the antibody Fv domain) and anti-IgG antibodies probing the global conformation of the C_H2 domain in the Fc fragment. Porphyrin coupling through carbodiimide activation resulted in a decrease in both functional activities of modified antibodies even at low levels of modification. In contrast, when the N-hydroxysuccinimide (NHS) derivative of porphyrin was used, enhancement of the antigen-binding affinity of porphyrin-antibody conjugates occurred due to an increase in the conformational mobility (flexibility) of the modified antibodies. The stimulatory effect of conjugation was maximal when one porphyrin molecule was coupled to an antibody molecule. Coupling of NHS-activated porphyrin at pH 7.4, 7.8 and pH 8.5 suggested that the high efficiency of the reaction at pH 8.5 could be attributed predominantly to the formation of antibody aggregates, only 50% of which were covalently cross-linked. The lowest percentage of aggregates in porphyrin-antibody conjugates was found at pH 7.4 and a molar reagent-to-protein ratio in the 10:1–40:1 range. Thus, the use of the NHS-activated carboxylic porphyrin provides a mild, simple and convenient procedure for preparing antibody conjugates with enhanced antigen-binding affinity.

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¹: Present address: Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

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Research reportModification of monoclonal and polyclonal IgG with palladium (II) coproporphyrin I: stimulatory and inhibitory functional effects induced by two different methods

Abstract

J. Immunol. Methods

Mol. Immunol.

Immunol. Today

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Immunol. Methods

Anal. Biochem.

Mol. Immunol.

J. Immunol. Methods

Biochem. Biophys. Res. Commun.

Immunochemical evidence for conformational flexibility and its modulation by specific ligands in the β2 subunit of E. coli tryptophan synthase

Biochemistry

Adsorbents for affinity chromatography. Use of N-hydroxysuccinimide esters of agarose

Biochemistry

Photosensitizers. Therapy and detection of malignant tumors

Photochem. Photobiol.

Structure-function relationships in immunoglobulins

The development of high sensitivity pulsed light, time-resolved fluoroimmunoassay

Pure Appl. Chem.

Synthese und Eigenschaften tragerfixierter Enzyme. I. Kovalente Binding von Trypsin on Dialehydzelluloze

Acta Biol. Med. Germ.

Light, oxygen and toxity

The effects of complete modification of amino groups on the antibody activity of antihapten antibodies. Reversible inactivation with maleic anhydride

Biochemistry

Research report
Modification of monoclonal and polyclonal IgG with palladium (II) coproporphyrin I: stimulatory and inhibitory functional effects induced by two different methods

Immunochemical evidence for conformational flexibility and its modulation by specific ligands in the β₂ subunit of E. coli tryptophan synthase