Giorgio Montagnoli

Molecular geometry of antigen binding by a monoclonal antibody against 5-methylcytidine

1. The specificity of a monoclonal IgG1 raised against a 5-methylcytidine-keyhole limpet hemocyanin conjugate was investigated by inhibition experiments with soluble competing antigens. 2. A competitive enzyme immunoassay has been set up, with the antigen immobilized on polystyrene microtitration wells. 3. The analysis of the cross-reaction profile allowed the topography of the antigen-antibody interaction to be described. 4. The binding properties of the monoclonal antibody are discussed in terms of both analytical applications and working limitations in the immunochemical study of gene methylation.

BIOLOGICAL EFFECTS OF LIGHT ON PROTEINS: ENZYME ACTIVITY MODULATION

Literature pertinent to the functional consequences of light on protein systems has been summarized from the period of June, 1976 to June, 1977, with some reference to previous studies. General review articles are those of Schopfer [62], Smith [657, Wolf [75] and Zucker [82]. Erlanger [22] has reviewed models for the photoregulation of protein systems. Although in v i m studies have so far shown that enzyme activity control is not due to a direct effect of light on the enzyme, assessment of these researches could be made by bringing together the various different aspects. This might also lead to some unifying view.

Photomodulation of azoaldolase activity.

Abstract— Rabbit muscle aldolase, modified by azo groups covalently bound to cysteinyl residues 237 and 287, shows activation of the fructose‐1,6 diphosphate cleavage reaction at 30T, after preirradiation with visible light. When the light treatment is carried out under an atmosphere of Ar to avoid oxidation, there is a reversible diminution of the Michaelis‐Menten constant, with no change in the turnover number. The direct effect of the light is the E → Z geometric isomerization of the azochromophore; thereafter the Z isomer is thermally converted to the more stable E isomer. Both regulation at the level of the enzyme‐substrate interaction and the involvement of the geometry of the chromophore have been demonstrated. The substrate, added to azoaldolase in the dark, causes E to Z isomerization of part of the extrinsic protein chromophores leading to a different isomeric composition at the thermal equilibrium. This result is consistent with higher binding energy of the enzymesubstrate complex when the modified enzyme contains azo chromophores in the Z configuration, as compared with the same chromophores in the E configuration. Azoaldolase has been discussed as a model for direct photoregulation of enzyme activity at the level of enzyme‐substrate interaction.

Time-Resolved Fluoroimmunoassay

A method is reported to set up a standard competitive TR-FIA. A simple and inexpensive way to prepare reagents and carry out operations is presented as well, with the aim to make it possible to perform a very sensitive analytical procedure in a personalized way within a nondedicated biochemistry laboratory. This protocol is general and can be easily modified with consideration to the analytical target. Once the antibody is available, both its labeling with diethylenetriaminepentaacetic acid dianhydride and Eu3+, and the setting-up of the assay with measurement of europium ion time-resolved fluorescence in a home-made enhancement solution become feasible.

Immunological and Biochemical Evidence that Blepharismin is not a Prosthetic Group

Abstract A polyclonal, multispecific antiserum was raised against a whole 3[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate-extract of nonirradiated Blepharisma japonicum cells. It was used to reveal the composition of solutions that were hypothesized to contain the photoreceptor of the ciliate. A Bio-Gel A 1.5 m fine column chromatography of the extract allowed recovery of a single elution peak isolated by recording the 580 nm light absorbance. Fused-rocket immunoelectrophoresis of this material revealed a large number of >300 kDa coeluted proteins. Blepharismin-rich material with a molecular mass of approximately 50 kDa, consisting of at least nine proteins was obtained when the same extract underwent preparative isoelectric focusing before column chromatography separation. Purification of the pigment obtained from light-exposed cells gave blepharismin-rich material with a molecular weight of approximately 200 kDa. Comparison of the materials obtained under the same conditions, either from the dark-kept or light-irradiated cells, by means of pore-gradient electrophoresis confirmed that proteins present in the two preparations were different. It revealed only a very small amount, if any, of proteins in the chromatography fractions with the highest absorbance at 600 nm. Results are discussed on the basis of the hypothesis that a specific blepharismin-binding protein does not exist in the protozoan.

Highly specific polyclonal antisera against estriol: Cross-reactivity restriction following affinity chromatography

An immunosorbent technique was developed to attenuate cross-reactivity of a polyclonal antiserum against a 4(2) (rho-carboxyphenylazo)-1,3,5[10]-estratrien-3,16 alpha,17 beta-triol-bovine serum albumin conjugate. The chromatographic separation of antiserum through stationary phases having either rho(carboxymethyl)phenylazo-phenol or rho(carboxymethyl)-phenylazo-2-naphthol side residues reduced the antiserum avidity, while increasing the apparent antiserum affinity and decreasing the residual cross-reactivities against heterologous ligands. The highly specific antiserum obtained allowed the development of a competitive binding assay over an extended analytical range, which opens up the possibility of direct measurement of estriol from the early pregnancy to delivery. The significance of the attenuation of antiserum cross-reactions after affinity chromatography is discussed with reference to epitope-paratope interaction in the case of small endogenous molecules like estrogens.

A SOURCE OF FALSE-NEGATIVE RESULTS IN CLENBUTEROL ANALYSIS IN TISSUES OF VEAL CALVES

The possibility of false-negative results in clenbuterol analysis was investigated in bovine tissues. An extraction procedure currently in use was adapted to process 100 specimens of different tissues each time. Its efficiency and accuracy were investigated radiometrically by means of a series of different molar concentration of the tritiated drug. In samples not submitted to extensive delipidation, unreliability of the analysis was evident. The measurement of tissue clenbuterol content, by a competitive ELISA, gave results numerically similar to those existing in literature, but with an accuracy high enough to minimize the frequency of false-negative results.

Shared reaction in solid-phase immunoassay for estriol determination

In the search of factors responsible for the experimental difficulties in developing accurate and sensitive solid-phase immunoassay of steroids, an experimental model has been set up for the study of nonspecific interaction of the steroid analyte with the coating protein. Along with the development of a highly sensitive enzyme-linked, solid-phase immunoassay for estriol measurement, we observed evidence of shared reactions. This property, to our knowledge not previously described for monomeric, low-molecular-weight antigens like estrogens, has been attributed to the presence of bovine serum albumin, which is capable of binding estrogens through hydrophobic interactions. The addition of estriol in solution in large excess did not reach a complete inhibition of the binding, so the possibility was excluded that the antibody simply binds to the adsorbed estrogen. The simplest explanation for the occurrence of the reaction is the hypothesis that a family of antigen determinants arises when the estriol is conjugated to a protein carrier. The corresponding antibodies are revealed only when the estrogen participates to the actual analytical system in the form of a steroid-protein conjugate. In the experiment, the estriol has been recognized as being coupled with one or more amino acid side chains present around its site of covalent linkage to the immunogen protein. The discussed results may be of help in developing a solid-phase immunoassay of small antigens as steroids, but also in applying the hybridoma and phage display technologies, the screening methods of which are based on sensitized solid phases.

Specificity in protein-ligand interactions: a model from estrone-antiserum binding

The specificity of protein-ligand interactions has been investigated using the binding of structurally related steroids to a polyclonal anti-estrone antiserum as a model. The cross-reaction profile of the native antiserum was compared with profiles obtained for the same antiserum preparation following affinity separation on stationary phases carrying structures which mimic parts of the antigen molecule: the coupling bridge, the A ring of the estrogen and the carrier protein. This fractionation produced antibody mixtures with different specificities from that observed for the pre-affinity antiserum. The changes in specificity observed and, more importantly, the direction of each variation detected, suggested the basis for a description of the nature of molecular recognition of small ligands by a protein as discrete rather than continuous. This methodology has revealed some recognition mechanisms.

Relevance of oestrone presentation to the specificity of the elicited antisera, as revealed by affinity separation

Polyclonal antisera raised against two different azobenzoyl-oestrone derivatives were analysed to investigate both the latency/intensity relationship of the immune response and the influence of antigen presentation on the specificity of the antisera elicited. Elongation of the azo-bridge of the hapten ([p(carboxyphenyl)-azo]-1,3,5[10]- oestratrien-3 ol-17 one) with a short aliphatic chain (4-amino-n-butyric acid) resulted in a marginal increase in the antibody yield, without affecting the time required to attain the maximum titre. The increased flexibility and mobility of the extended azo-bridge was shown to result in the appearance of antisera which cross-reacted with oestrogens with D ring structures different to that of oestrone. Antiserum fractionation by affinity chromatography through a stationary phase exposing the carrier protein determinants, as modified by the addition of the coupling bridge and the phenol ring, resulted in a reduction in its specificity. These findings are discussed with regard to the phenomena underlying the specificity of a polyclonal antiserum.