Chromatographic kinetic measurements of human serum albumin adsorption on monoclonal antibodies.

A chromatographic method was employed to study the kinetics of human serum albumin (HSA) adsorbed on immobilized monoclonal antibodies. The antibodies of various specificities were covalently bound to a high-performance liquid chromatography (HPLC) silica support. For very low desorption rates, successive amounts of the reacting protein were injected until column saturation. The analysis of the increase of the non-retained fraction calculated from peak area measurements gives the capacity of the support and the rate of the biospecific adsorption process. The model is based on a second-order Langmuir kinetic law and assumes a global mass transfer for the adsorption process. The use of a silica support of small pore size permits the reduction of the contribution for mass transfer in the stagnant fluid and the decrease in the column capacity: due to its large size, the reacting molecule is adsorbed on the external surface of the particle. The adsorption rate constants of HSA on five monoclonal anti-HSA antibodies of different specificities were determined. For all the immuno-adsorbents studied, the adsorption rate constant is significantly lower than that found on immobilized polyclonal antibodies. Measurements at different flow rates reveal that the mass transfer due to the transport to the adsorbent surface is small and can be estimated.

[Study of albumin conformational changes in human serum using an immunoenzymatic technic using monoclonal antibodies]. / Etude de modifications conformationnelles de l'albumine dans le sérum humain à l'aide d'une technique immunoenzymatique utilisant des anticorps monoclonaux.

The specificity of six peroxidase-labelled anti-albumin monoclonal antibodies was studied by allowing them to react with albumin-derived fragments of known structure comprising the whole albumin molecule. The epitopes corresponding to these antibodies were located on defined regions of the albumin molecule extending from the N to the C-terminal ends. These antibodies have been used for investigating by ELISA the conformational alterations of albumin molecule brought about by the N-B transition in twelve individual sera. The results do not show any significant differences between the sera. The N-B transition involves essentially the N-terminal portion of the albumin molecule. There also exists as modification of the C-terminal region which is however much less pronounced than the one in the N-terminal region. In addition, it is not observed when using isolated albumin.

Enzyme immunoassay using monoclonal antibodies to study conformational changes of human serum albumin.

A method for studying conformational changes induced in the human albumin molecule, either in its purified form or in serum, is described. Plates were coated with albumin or human serum at varying pHs and were reacted with peroxidase-labeled anti-albumin monoclonal antibodies of different specificities. The data showed that albumin molecules were coated in conformations induced as a result of pH changes, allowing us to demonstrate that pH modifications involved the N-terminal portion of the albumin molecule whether in its purified form or in serum. This method should be applicable to the study of conformational modifications in other proteins as well.

Fixation of penicilloyl groups to albumin and appearance of anti-penicilloyl antibodies in penicillin-treated patients.

Penicilloyl groups, which have been connected to penicillin allergy, are derived from penicillin by cleavage of the beta lactam ring and bind covalently to proteins. Fixation of penicilloyl groups was studied in seven patients given large amounts of penicillin. Penicilloyl groups were found essentially on the albumin molecule at sites not accessible to anti-penicilloyl antibodies, except after pronase digestion. The amount of penicilloyl groups was proportional to the cumulated doses of penicillin. The decline of penicilloyl groups with time after treatment interruption was exponential. The half-life of penicilloylated albumin was lower than or equal to that of normal albumin. The presence of anti-penicilloyl antibodies was demonstrated in 19 out of 34 penicillin-treated patients (including the seven mentioned above). The relative scarcity of penicillin allergy as compared with the frequent occurrence of anti-penicilloyl antibodies may be partly related to unavailable sites of penicilloyl groups within the albumin molecule.

Comparative analysis of CD4-4B4 and CD4-2H4 lymphocyte subpopulations in HIV negative homosexual, HIV seropositive and healthy subjects.

A comparative analysis of subsets (4B4 and 2H4) within the CD4 lymphocyte subpopulation was made by double marker analysis among 23 healthy heterosexual, 16 healthy HIV seronegative high-risk homosexuals and 82 HIV seropositive subjects. Data show that the significant increase in CD4 lymphocytes observed among seronegative homosexuals corresponds mainly to an increase in CD4-4B4 positive cells while CD4-2H4 subset levels remain comparable to healthy heterosexual controls. A decrease both in CD4-4B4 and CD4-2H4, parallel to a decrease in CD4 subpopulation, was observed in asymptomatic seropositive carriers (SPC) and patients with ARC syndrome (AIDS-related complex) or AIDS (acquired immunodeficiency syndrome). Interestingly, an absolute decrease in CD4 subpopulation in patients with asymptomatic lymph node enlargement (LAS), is chiefly accounted for by a decrease in CD4-4B4 subset. The values of CD4-2H4 subset are significantly higher than those observed for SPC patients and are close to the normal values. These observations are of interest, as no differences between SPC and LAS patients could be detected when CD3, CD4 and CD8 subpopulations were studied.

Location of penicilloyl groups on CNBr fragments of the albumin from penicillin-treated patients.

Two fixation sites for penicilloyl groups on human albumin were demonstrated. Using CNBr cleavage the first site was located between methionine 123 and methionine 297 and the second one between methionine 297 and the C-terminal residue. In both cases, penicilloyl groups were unmasked by pronase degradation or disulfide bond reduction.

[Plasmodium falciparum malaria attacks with low or negative parasitemia on returning from areas of endemic resistance to 4-aminoquinolines]. / Accès palustres à Plasmodium falciparum à parasitémie faible ou nulle au retour de régions d'endémie de résistance aux amino-4-quinoléines.

Twelve recent cases of Plasmodium falciparum malaria presented with unusual clinical and laboratory features. All patients had regularly been taking adequate doses of amino-4-quinolines as prophylaxis. In most cases the symptoms were mild as compared with those in a group of 20 control patients with typical malaria. More surprisingly, parasitaemia was either negative or very weakly positive (less than 1000 infested erythrocytes per mm3). All cases occurred in people returning from areas in which resistance of P. falciparum to these drugs is endemic. Diagnostic problems could be solved in most patients by new immunological techniques relying on the detection of parasitized erythrocytes and plasmodium antigens using a battery of monoclonal antibodies. A physiopathological model explaining why parasitaemia is negative in such cases is suggested.

Structural differences between rabbit cathepsin E and cathepsin D.

Rabbit cathepsins D and E were isolated from bone marrow. Both enzymes were purified by affinity chromatography on pepstatin-Sepharose 4B and Con A-Sepharose 4B. Purity of the enzymes was ascertained by two-dimensional gel electrophoresis after iodination. The isoelectric point of cathepsin D was found to be 6.95. Cathepsin E was shown to consist of two subunits having molecular masses each of 40 kDa and isoelectric points of 4.60 and 4.65, respectively. The amino-acid composition of cathepsin E was found to be different from that of cathepsin D.

[Subacute septicemia caused by Meningococcus of serogroup Y and acquired deficiency of complement C3 fraction]. / Septicémie subaiguë à méningocoque du sérogroupe Y et déficit acquis en facteur C3 du complément.

The high frequency of meningococcal infections in patients with congenital deficiency of a component of the complement terminal pathway emphasizes the critical role of this system in host resistance against Neisseriaceae. We report the observation of a subacute septicaemia due to N. meningitidis serogroup Y. This girl had an acquired deficit of the C3 fraction of complement due to a high titre of C3 nephritic factor (C3NeF). There was no evidence of partial lipodystrophy or biological symptoms of glomerular disease. The meningococcal infection revealed this biological abnormality.

Evidence for the presence of large amounts of cathepsin E in rat spleen.

Although cathepsin E is present in trace amounts in spleen from several species, it was found in large amounts in rat spleen. This observation can be correlated with the fact that spleen in the rat is an important organ in haemopoeisis.

Enzyme-linked immunosorbent assay for measurement of penicilloyl groups.

An enzyme-linked immunosorbent assay for measuring penicillin groups is described. Alkaline phosphatase conjugated to penicilloyl residue was reacted with purified anti-penicilloyl antibodies coated upon polystyrene plates. The inhibition of this reaction allowed a specific determination of penicilloyl residues at the picomole level. This assay was applied to the study of the penicilloyl groups linked to human albumin and to the measurement of some of these groups after enzymatic degradation or physical modifications of the albumin molecule.