J.-P. Dandeu

Hydrophobic interaction chromatography for isolation and purification of Equ.cl, the horse major allergen

Equ.cl, the horse (Equus caballus) major allergen, was identified in a partially purified extract obtained from a crude aqueous horse dander extract, by acetonic precipitation and a salting-out process. It was isolated and purified by size-exclusion chromatography followed by hydrophobic interaction chromatography. Equ.cl appeared as an almost pure protein in a fraction eluted at 1.2 M ammonium sulphate from a phenyl Superose column. It is a single peptide with a relative molecular mass of 20,000 and a pI of ca. 3.9.

Crystallization and preliminary crystallographic analysis of the major horse allergen Equ c 1

The secreted protein Equ c 1 is the major component responsible for the induction of specific IgE antibodies in patients sensitized to horse allergens. Equ c 1 belongs to the lipocalin superfamily of hydrophobic ligand-binding proteins, which also includes other known allergens. Equilibrium sedimentation and gel-filtration studies demonstrate that both the glycosylated form of Equ c 1 purified from horse salivary glands and the non-glycosylated recombinant form expressed in bacteria exist predominantly as dimers in solution. As observed for other dimeric lipocalins, acidic pH and low protein concentration favour dimer dissociation. The recombinant form of Equ c 1 has been crystallized using ammonium sulfate as a precipitant. The crystals belong to the tetragonal space group P41212 with cell parameters a = b = 84.0, c = 56.1 A, and contain a single molecule in the asymmetric unit. A complete data set from native crystals was collected at the synchrotron source in Hamburg to 2.9 A resolution using a frozen crystal, and structure determination is in progress.

THE RELATIONSHIPS BETWEEN THE BIOCHEMICAL PROPERTIES OF ALLERGENS AND THEIR IMMUNOGENICITY

In the past years, proofs have been accumulated to argue for a close relationship between structure and function of proteins (1). On the other hand, progress made on allergen characterization has shown that the majority of clinically important allergens are biochemically active. Among the biochemical activities associated with allergens, most are enzymes or regulatory proteins. Thus, the relatively new idea of a link between function and allergenicity of proteins (2-5) has emerged, raising again a currently debated question about what makes an antigen an allergen. It is no longer believed that there exist proteins that are allergenic and others that are not. An antigen is an allergen for a particular subject when it provokes, on a first contact, specific IgE synthesis and, later, anaphylactic or inflammatory reactions. In a healthy subject, the same molecule will behave as a classical antigen without any allergic disorders. As it is well-established, the hypersensitivity reactions are genetically controlled, but they are also dependent on numerous factors that include: the allergen itself, allergen dose and exposure frequency, environmental factors, and, finally, on the subjects immunological background (6). One of the main findings in the mechanisms of immunity is that T-helper lymphocytes (Th) can be subdivided into two subsets according to the cytokines they produce (7). Among cytokines pro-

Isolation and purification of cat albumin from cat serum by copper ion affinity chromatography: further analysis of its primary structure.

Proteins, regardless of their origin, have to be highly purified, particularly from the immunochemical point of view, if they are to be used to study their allergenicity. It is shown that cat albumin, a highly potent allergen for cat-sensitive humans, can be isolated and purified from cat serum using immobilized metal ion affinity chromatography (copper ions) instead of a salting-out process or precipitation with alcohol, techniques generally used for the preparation of serum proteins. During the process described, immunoglobulins are concomitantly isolated in a relatively pure form. Cat albumin amino acid composition and sequence were analysed after an ultimate purification by ion-exchange chromatography. The highest homology (greater than 80%) was found with the rat serum albumin.

Étude immuno-chimique de l'hémolymphe d'abeille ouvrière adulte (Apis mellifera L) saine ou infestée par Varroa jacobsoni Oud

Résumé — Varroa jacobsoni Oud, ectoparasite de l’abeille Apis mellifica L, se nourrit essentiellement par succion de l’hémolymphe de l’hôte, larve ou insecte adulte, à travers la cuticule. La composition antigénique de cette hémolymphe peut varier, soit du fait de la ponction elle-même, soit de l’action des enzymes injectées, et les modifications peuvent donc être quantitatives ou qualitatives. Nous montrons ici que des marqueurs antigéniques qui semblent ne pas provenir de l’acarien luimême, peuvent apparaître spécifiquement après l’infestation même récente de la ruche par celui-ci,

Isolation of Der pI, the Dermatophagoides pteronyssinus major mite allergen, from a crude mite culture extract, purification by ion-chromatography, and comparison between the material obtained and a cDNA-coded Der pI.

A high degree of purity is a prerequisite for an allergen preparation to be suitable for clinical diagnosis and therapy. A pure allergen can easily be obtained from a crude mite culture extract by using an immunosorbent prepared with highly specific monoclonal antibodies or from a cDNA-coded material. However, up to now none of these methods has been performed on a process scale. Here large-scale purification is defined as a process in which a crude Dermatophagoides pteronyssinus mite culture extract is essentially fractionated by acetone and ammonium sulphate precipitations followed by anion-exchange high-performance liquid chromatography. A high yield of a very pure Der pI allergen is obtained during the first isocratic run, as shown by sodium dodecylsulphate-polyacrylamide gel electrophoresis, capillary electrophoresis, chromatofocusing and a two site monoclonal antibody enzyme-linked immunosorbent assay. Microsequencing revealed that the 25-residue sequence obtained is entirely in agreement with the sequence derived from the cDNA of Der pI.

H2 genotype and IgE immune response to Fel dI, the cat major allergen, in mice

Mice of 6 strains were immunized with a highly purified Fel dI allergen adsorbed to alum. Their ability to display a significant IgE response was detected via passive cutaneous anaphylaxis (PCA) tests, performed in rats. Linkage of the responsiveness to the H2 genotype is not totally obvious. IgE response can be delayed, particularly in the SJL strain (H2-s histocompatibility allele), and lacking in the C57 B1/6 strain (H2-b). Mice with H2-k, H2-d alleles and the B6D2F1 hybrid H2-b/d are good responders, leading to the hypothesis that the IgE response to Fel dI may be related to the H2-d allele. For all good responders, individual variations are rather important. All our observations show that mice perfectly mimic human hypersensitization to the cat major allergen Fel dI, at least where the IgE response is concerned.

Simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by checkerboard immunoblotting (CBIB)

A simple technique, checkerboard immunoblotting (CBIB), is described for simultaneous quantitation of specific IgE antibodies against several allergens in human sera. Using as little as 50 μl of each of the 20 sera examined against 20 different allergens, it was possible in a single run to achieve 400 tests. To guarantee high specificity and sensitivity of the assay, this new application of CBIB employs purified allergens, cyanogen bromide‐activated nitrocellulose membrane, and Phosphorimager technology. Results are expressed both qualitatively (five classes) and quantitatively in kilo units per liter equilibrated against the World Health Organization (WHO) standard for IgE. There was excellent agreement between the results of CBIB and the results of Pharmacia Cap System, an alternative method widely used for measuring serum‐specific IgE. The CBIB method certainly could be useful in any laboratory interested in allergy clinical research for easy screening and relative quantitation of allergen‐specific human IgE. J. Clin. Lab. Anal. 11:357–362, 1997.© 1997 Wiley‐Liss, Inc.