Cesar Cambiaso

Assessment of damage to the central nervous system by determination of S-100 protein in the cerebrospinal fluid.

S-100 protein was determined by Particle Counting ImmunoAssay in the CSF of patients with various neurological disorders. With a limit of sensitivity of 2.5 micrograms/l this brain-specific protein was detected only in samples from patients with acute damage of the central nervous system, particularly in compression of the spinal cord by tumour, ischaemic disorders, subarachnoïd bleeding and haematoma, and viral or suspected viral infections. Our results support the assumption that S-100 is a reliable index of central nervous system damage and that changes in its concentration could have a prognostic value.

Particle counting immunoassay (Pacia). I. A general method for the determination of antibodies, antigens, and haptens

By using a device designed for counting blood cells, it is possible to measure the agglutination of polystyrene beads (0.8 mu) with accuracy and great sensitivity, the agglutination resulting in a reduction in the number of particles. The latter coated with antigen can be used for determining IgM or IgG antibodies e.g. human rheumatoid factor or rabbit anti-bovine serum albumin antibodies. Macromolecules with multiple antigenic determinants agglutinate particles carrying specific antibodies. This system has been applied for determining HPL and alpha1-fetoprotein with a threshold of sensitivity of about 10 microgram/1. However the agglutination was decreased by serum factors which led to a 10-fold loss of sensitivity. The interference of rheumatoid factor which agglutinated the particles coated with rabbit or goat immunoglobulins could be avoided by reduction of the serum to be analyzed with 5mM dithiothreitol for 5 min. Haptens, i.e. DNP-lysine and T4, were determined by their inhibitory activities toward their specific antibodies, the agglutinator being a hapten-macromolecule conjugate or the antibodies themselves.

The concentration of IgM in the cerebrospinal fluid of neurological patients.

The level of IgM was determined by Particle Counting Immunoassay in the cerebrospinal fluid. In non-neurological patients (N = 20) the mean was 97.5 micrograms/l with the upper reference limit at 380 micrograms/l. The mean IgM index was 0.021 with the upper reference limit at 0.071. Of 21 patients with stroke, 5 had an IgM index exceeding the reference limit. High levels and indices of IgM were observed in most patients (N = 27) with infectious meningo-encephalitis. In this group, the IgM index was abnormal in about 30% of cases with a normal total protein content, and was more often increased than the IgG index. In multiple sclerosis patients (N = 80), the IgM index was increased in 32%. In this disease very high values of IgM index (greater than 0.13) were never associated with very high values of IgG index (greater than 1.8). A significantly higher proportion of males was found in the group of patients with very high values of IgM index (N = 11). No significant influence of the age of onset, the interval between onset and sampling and clinical state was observed. However, of 10 patients with a multiple sclerosis history exceeding 15 years none had an IgM index exceeding the upper reference limit. Four patients with multiple sclerosis had a high IgM index without either an increase of the IgG index or the presence of oligoclonal bands.

The Recognition By Monoclonal-antibodies of Various Portions of a Major Antigenic Site of Human Growth-hormone

The reactivities of five mouse monoclonal antibodies against human growth hormone (hGH) were defined by either a competitive radioimmunoassay with insolubilized antibodies or by an agglutination-inhibition method with hGH-coated polystyrene particles. The five antibodies reacted significantly but to various degrees with human placental lactogen and at least three antibodies reacted with human prolactin and three synthetic peptides extending from residues 19 to 128, 73 to 128 and 98 and 128 of hGH. Four tested monoclonal antibodies failed to react with bovine growth hormone and with hGH oxidized by performic acid. The antibodies were further distinguished by their different reactions with hGH modified by reduction and alkylation or by adsorption on a polystyrene surface. The unique specificity of each antibody was confirmed for most of them by an agglutination method in which the agglutinating activity of hGH was tested on latex particles coated with various paired combinations of the monoclonal antibodies. The lack of agglutination with certain combinations suggested that the specificities of such a pair of antibodies overlapped each other. These results suggest that the sequences corresponding to the synthetic peptides participate in the structure of a major antigenic site of which various portions are recognized by the monoclonal antibodies.

The clinical relevance of ferritin concentration in the cerebrospinal fluid.

By means of a new technique (Particle Counting Immunoassay), we have determined the level of ferritin in 470 samples of cerebrospinal fluid of patients with various neurological disorders. The median value obtained in a control group was 2.3 ng/ml with an upper limit at 5.5 ng/ml. the concentrations in the serum and cerebrospinal fluid were independent, but that in cerebrospinal fluid correlated with its total protein content. High values of ferritin were found in infectious meningo-encephalitis, in vascular diseases of the central nervous system, and, unexpectedly, in several cases of dementia without obvious vascular pathology.

Evaluation of monoclonal antibodies with specificity for human IgA, IgA subclasses and allotypes and secretory component. Results of an IUIS/WHO collaborative study.

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN (n = 24), IgA1 (n = 7), IgA2 (n = 3), IgA2m(2) (n = 2), non-IgA2m(2) (n = 4) and SC or secretory IgA (n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, kappa and lambda by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.

A MAGE-1 peptide recognized on HLA-DR15 by CD4(+) T cells.

Antigens encoded by MAGE genes and recognized by T cells are of interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Several MAGE‐1 peptide that are recognized by CD8+ cytolytic T lymphocytes have been used in therapeutic vaccination trials. To obtain anti‐tumor immune response, vaccines combining peptides recognized by CD8+ and peptides recognized by CD4+ T cells might be optimal. We focused therefore on the identification of MAGE peptides recognized by CD4+ T cells. We report here the identification of MAGE‐1 epitope EYVIKVSARVRF, which is presented to CD4+ T lymphocytes by HLA‐DR15. This HLA allele is present in 29 % of Asians and 17 % of Caucasians.

Natural killer cell activation after infection with lactate dehydrogenase-elevating virus.

Early after infection, lactate dehydrogenase-elevating virus (LDV) alters the immune system by polyclonally activating B lymphocytes, which leads to IgG2a-restricted hypergammaglobulinaemia, and by suppressing the secretion of Th2 cytokines. Considering that these alterations may involve cells of the innate immune system and cytokines such as interferon-gamma (IFN-gamma), we analysed the effect of LDV on natural killer (NK) cells. Within a few days of infection, a strong and transient NK cell activation, characterized by enhanced IFN-gamma message expression and cytolysis, was observed. LDV triggered a large increase in serum IFN-gamma levels. Because NK cells and IFN-gamma may participate in the defence against virus infection, we analysed their possible role in the control of LDV titres with a new agglutination assay. Our results indicate that neither the activation of NK cells nor the IFN-gamma secretion affect the early and rapid virus replication that follows LDV inoculation.

Automated nephelometric immunoassay II. Its application to the determination of hapten

Abstract The principle of a novel method for the immunological assay of haptens, based on nephelometry, has been tested, using ϵ-dinitrophenyl-lysine and progesterone as model substances. It is proposed to call this method the nephelometric inhibition immunoassay (NINIA). In an automated continous flow system where aliquots of antigen are discontinuosly injected into a constant stream of diluted antiserum, the nephelometric effect of the antigen-antibody complexes formed markedly increases with the concentration of antibody relative to antigen, as well as with the affinity of the antibodies. Haptens inhibiting this reaction can be titrated by measuring the resulting decrease of the nephelometric peaks. In practice, the precipitin reaction is carried out with a ‘developer antigen’, BGG, HGG or ferritin carrying numerous haptenic groups, and antiserum preabsorbed with graded, known, amounts of hapten, or with the unknown sample. Means of increasing the sensitivity include the use of (i) high affinity antibodies, (ii) large, heavily substituted, developer antigens, (iii) 4% polyethylene glycol as the diluent of the antiserum, and (iv) adjusting the proportions of the reactants to near-equivalence. The threshold of the method as applied to progesterone is about 10 ng.

Automated nephelometric immunoassay (ANIA) I. Importance of antibody affinity

Abstract The reason why certain antisera displaying good precipitating properties in gels may be quite inefficient in automated nephelometric immunoassays has been sought. Antibodies against dinitrophenyl groups, gamma chains, or haptoglobin were eluted by increasing concentrations of ammonium thiocyanate from their respective antigens coupled to Sepharose. It was found that those antibodies that required the highest concentrations of thiocyanate to dissociate them from their antigen, were also those that formed aggregates with the most elevated nephelometric effect. Antibodies against dinitrophenyl groups were also eluted increasingconcentrations of the specific hapten. After dissociation from the latter, these antibodies displayed a nephelometric effect clearly dependent on their affinity. By increasing the concentration of antibodies (isolated on immunoadsorbents), and lowering the reaction temperature, it was possible to compensate for the lack of affinity of certain antisera and to make them suitable for automated nephelometric immunoassay.