Max Goyffon

A recombinant single-chain antibody fragment that neutralizes toxin II from the venom of the scorpion Androctonus australis hector

Monoclonal antibody 4C1 specifically binds to and neutralizes the most potent neurotoxin (AahII) of the scorpion Androctonus australis. The cDNAs encoding the variable regions of this antibody were isolated by PCR‐mediated cloning. A single‐chain Fv gene was engineered and expressed in Escherichia coli. The recombinant protein had neutralizing activity similar to that of the intact antibody in vitro and in vivo. We have thus neutralized the pharmacological and biological properties of a scorpion neurotoxin with a single‐chain Fv, which opens new perspectives for the treatment of envenomizations.

Production and characterization of a bivalent single chain Fv/alkaline phosphatase conjugate specific for the hemocyanin of the scorpion Androctonus australis

A102 is a monoclonal antibody raised against the hemocyanin of the Tunisian scorpion Androctonus australis. It is directed against the subunit Aa6 and does not cross-react when tested against a variety of similar scorpion hemocyanins. Here, we report the construction of a plasmid encoding a recombinant enzyme-linked antigen-binding protein with the antigen-binding specificity of antibody A102. The DNA fragments encoding the variable domains of A102 were inserted into a prokaryotic expression vector so as to produce a single chain antibody variable fragment (scFv) fused to the bacterial alkaline phosphatase. The fusion protein preserved the IgG binding and alkaline phosphatase activities. Immunoelectron microscopic analysis showed that the recombinant protein bound antigen bivalently as is the case for natural antibodies. Crude preparations containing the conjugate were used in a rapid visual immunoassay for the specific detection of A. australis hemocyanin, using a droplet of hemolymph removed from live animals by puncture. The simplicity of the test made it suitable for the direct identification of animals belonging to this species. It could be useful in areas where A. australis, the most dangerous African scorpion, is found with other species from which it is not easy to distinguish using morphological criteria.

A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin AahI.

Abstract We created a construct encoding a peptide known to mimic the binding properties of biotin fused to the carboxyterminus of a scFv fragment that binds a scorpion toxin (AahI). This fusion protein was produced in the periplasm of bacteria and purified to homogeneity by singlestep affinity chromatography on streptavidinagarose with a yield close to 1 mg/l. DNA sequencing, dot blot and mass spectrometric analyses demonstrated the integrity of the soluble immunoconjugate. Fusion to the streptavidinbinding peptide did not affect the ability of the scFv to recognize its antigen with a high affinity (Kd = 2.3x 1010M). Similarly, the streptavidinbinding property was not impaired in the fusion protein. Thus, the immunoconjugate was bifunctional and had a low molecular mass of 28 kDa. This enabled us to develop rapid and sensitive immunoassays for the specific detection of the toxin AahI accurately to 0.6 ng/ml, opening up new perspectives for the diagnosis of envenomations.

Antioxidant activity of hemocyanin; a pulse radiolysis study

In order to determine the reactivity on hemocyanin from Androctonus australis, the reaction of superoxide anion has been investigated using pulse radiolysis. The kinetics of O2- decays have been studied in aqueous buffered media at various basic pH (8, 8.5 and 9), first in the absence and then in the presence of hemocyanin (in oxygenated solutions containing formate anion 0.16 mol.l-1). We have shown that, in the presence of hemocyanin, O2- decay is a first-order process whose apparent rate constant is proportional to protein concentration (10(-7) to 10(-6) mol.l-1) and pH independent between 8 to 9. A second-order rate constant of 3.5 +/- 0.1.10(7) mol-1.l.s-1, has been deduced for the catalytic rate constant of hemocyanin with O2-. Meanwhile, this activity is smaller than that described for free copper, eukaryotic Cu-Zn-SOD or some copper chelates. We have verified that apohemocyanin--the copper deprived protein--does not exhibit such an activity vs. SOD (superoxide dismutase).

Correlations between the catalase-like activity, and the H2O2-ATP production of haemocyanin and its subunits. Implications with the radioresistance of the scorpion Androctonus australis

Abstract 1. 1. Desert scorption are exceedingly resistant to ionizing irradation. It has previously been assumed that scorpion haemocyanin(blue blood pigment) plays an important role in this radioresistance because of its copper content, oxyphoric properties and catalase-like activity. 2. 2. In the present paper we describe eight monomers and a 55,000 mol. wt proteolytic fragment that have high catalase-like activity. 3. 3. Purified apohaemocyanin (copper-free protein) does not display oxyphoric and/or catalase-like properties. The concerted synthesis of nucleotide triphosphate in the presence of H 2 O 2 and catalase-like activity is reported for haemocyanin and its subnits. 4. 4. Measurements were carried out using luciferin-luciferase assays and HPLC in order to analyze the mechanism of 3 H-ADP transformation into 3 H-ATP. 5. 5. The catalase-like activity and H 2 O 2 disproportionation of haemocyanin and its subunits, which are thus strictly copper dependent, seem to be the two main functions of haemocyanin and its subunits (monomers and the proteolytic fragments) in the resistance of the scorpion to ionizing radiation. 6. 6. This is probably because ionizing radiation induces production of superoxide ions, hydroxyl-free radicals and hdyrogen peroxide, which oxidize susceptible target molecules, and hence induces biological changes. 7. 7. Haemocyanin, representing 80–90% of the haemolymph proteins in the scorpion, may play an important role in cellular detoxication of H 2 O 2 due to its catalase-like activity. 8. 8. Thus it is possible that this pigment and its subunits play a role in the high resistance of the scorpion to ionizing radiation.

Catalatic properties of hemocyanin in helping to account for the Scorpion's radioresistance

1. 1. Scorpions are exceedingly resistant to ionizing irradiations. 2. 2. Hemocyanin, the blue pigment of hemolymph, is undoubtedly an important factor in this resistance because of its copper content and oxyphoric properties, and of its catalatic activity demonstrated here. 3. 3. These characteristics, found in crude or purified hemocyanin and in its heavy dissociated products (i.e. dodecamers and hexamers) made it possible to show that hemocyanin neutralizes the effects of irradiation by disproportionation of the toxic H2O2 product. 4. 4. The finding that catalase is a component of this complex oxyphoric metalloprotein is an exciting discovery that warrants further study, since catalase is considered essential to cell protection against all types of irradiation.

Affinity capture using chimeric membrane proteins bound to magnetic beads for rapid ligand screening by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The rapid and specific detection of therapeutically important ligands in complex mixtures, that may bind to membrane proteins, remains challenging for many research laboratories and pharmaceutical industries. Through its use in the development of screening assays, mass spectrometry (MS) is currently experiencing a period of tremendous expansion. In the study presented here, we took advantage of the remarkable stability properties of a bacterial membrane protein, the KcsA K+ channel, produced in E. coli and purified as a tetrameric protein in the presence of a detergent. This membrane protein can subserve as a molecular template to display the pore-forming region of human K+ channels, which are considered as targets in the search for inhibitory ligands. The engineered chimeric proteins were linked to metal-bound magnetic beads, for the screening of complex peptide mixtures, such as that of scorpion venoms. The affinity-captured scorpion toxins were eluted prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and to nano-electrospray ionization tandem mass QqTOF mass spectrometry (MS/MS) analysis. The de novo sequence of the toxins was deduced by combining the MS/MS fragmentation of the reduced form (up to the 33 first residues) and the trypsin digest peptides of the native toxins. This affinity-capture screening assay led to the isolation and characterization of potent and specific ligands of the human K+ channel, Kv1.3. The affinity-capture procedure is fast and reproducible. When linked to magnetic beads, the chimeric membrane protein can be re-used several times without losing any of its selectivity or specificity. This assay also benefits from the fact that it requires minimal amounts of animal venoms or complex mixtures, which can be expensive or difficult to procure.

Immunolabeling of CD3-Positive Lymphocytes with a Recombinant Single-Chain Antibody/Alkaline Phosphatase Conjugate

Abstract G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigenbinding specificity of antibody G3(3). A gene encoding a singlechain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.

Cross-reactivity of a monoclonal antibody to the haemocyanin of various scorpion species

Abstract 1. 1. A monoclonal antibody has been produced against the external subunit Aa 6 of the haemocyanin of the scorpion Androctonus australis (Buthidae). 2. 2. This monoclonal antibody cross-reacts with some, but not all, the haemocyanins of the family Buthidae using ELISA and PAGE immunoblotting. 3. 3. The intramolecular localization of the epitope is well-preserved within all the haemocyanins on which it is present. 4. 4. These results are correlated with morphological characteristics of the animals and may be used for a revision of the scorpion systematics.