Screening and characterization of specific anti-lipopolysaccharide antibodies in Belgian blood donors by enzyme-linked immunosorbent assays.

The goal of this project was to find and collect high concentrations of endotoxin-specific antibodies for therapeutic IgG- or IgM-enriched preparations. Various enzyme-linked immunosorbent assays (ELISAs) were developed to perform longitudinal studies of the serological response to a large panel of smooth and rough purified lipopolysaccharide (LPS) extracts in a population of healthy blood donors. To accomplish this, 1612 human serum samples from volunteer blood donors collected by seven different blood banks in Belgium were screened and specific IgM and IgG activities were measured. Approximately 17% of the donors had anti-LPS concentrations higher than 40 mg L-1. Of these, 10.9% had anti-smooth LPS antibodies, 3.7% had anti-rough LPS antibodies and 2.8% were found to be positive towards both types of LPS. The mean anti-LPS antibody concentration was 8 mg L-1 for rough LPS and 14 mg L-1 for smooth LPS. Age- and sex-related distributions of the activities indicated that the greatest prevalence of high anti-LPS concentration was in women aged 40-49 years and in men older than 60 years. Differential absorption experiments showed that the pooled serum of selected blood donors contained a mixture of specific and cross-reacting antibodies. We detected predominantly anti-LPS activities due to the IgG1 and IgG2 subclasses. The range of specificities to different LPS was increased by the pooling of selected sera. It was concluded that pools of naturally occurring specific anti-LPS immunoglobulin antibodies may be obtained in Belgium by screening blood donors using ELISAs that we have developed.

Sequential anti-core glycolipid immunoglobulin antibody activities in patients with and without septic shock and their relation to outcome.

OBJECTIVE: This study follows the sequential changes in anti-lipopolysaccharide antibodies in infected patients with and without septic shock. SUMMARY BACKGROUND DATA: A relation between high endogenous levels of anti-LPS antibodies and protection against bacteremia and septic shock in at-risk patient groups has been observed. However, information on the daily follow-up and kinetics of apparition or disappearance of anti-LPS antibody activities and their relations with the protective properties of the different immunoglobulin classes has not been clearly investigated. METHODS: Two hundred and five septic surgical patients were studied during their stay in the intensive care unit during a period of 3 years. Among these patients, septic shock developed in 54 and 47 died. A sensitive ELISA was used to study circulating IgM and IgG antibodies to the core glycolipid (CGL) region of Salmonella minnesota R595. The activities were measured each day when sepsis occurred and every hour during septic shock. RESULTS: Anti-CGL IgM activity was found in 32% of the septic patients. This response, however, most often appeared to be transient. A strong correlation was observed between the occurrence of septic shock and the absence of anti-CGL IgM activity on admission to the ICU (p < 0.02). Anti-CGL IgG activity was detected in 82% of the patients and better correlated with outcome for patients with high or rising activities during their hospitalization (p < 0.0005). In patients with septic shock or irreversible organ failure, a fall in the anti-CGL IgG activity was observed before death, suggesting that the IgG antibodies were consumed during this acute event. Therefore, the anti-CGL IgG activity measured by ELISA could be used as a marker of the evolution of the illness. CONCLUSIONS: Our observations demonstrate the interest to follow-up the evolution of the anti-CGL antibodies during sepsis. The fall of these antibodies during septic shock and in patients who died was an additional argument to perform, as an additive form, passive antibody therapy to decrease lethality in this group of patients.

Protective effects of polyclonal sera and of monoclonal antibodies active to Salmonella minnesota Re595 lipopolysaccharide during experimental endotoxemia.

Mice were passively immunized with sera from blood donors active for rough lipopolysaccharides (LPS), the J5 (Rc chemotype) mutant of Escherichia coli O111:B4, and the Re595 (Re chemotype) mutant of Salmonella minnesota. All protected the mice against lethal challenge with smooth E. coli WF96 LPS, E. coli and Salmonella rough mutant LPS, or free lipid A. Epitopes recognized by monoclonal antibodies (MAbs) reacting with the LPS of S. minnesota Re595 or lipid A were localized in the 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) region and on lipid A. Core-reactive MAbs reacted with their homologous Re LPS and with free lipid A. One, GL11, cross-reacted with the KDO alone. MAbs GL6, GL11, L.4, L.6, and L.8 protected the actinomycin D-sensitized mice against the lethal effects of LPS from E. coli WF96, Salmonella enteritidis, E. coli J5, S. minnesota Re595, and free lipid A. The GL11 antibody was also protective when injected after LPS challenge. These results indicate that antibodies directed against the core glycolipid of S. minnesota Re595 LPS may be useful as an additive form of therapy that may enable decreased mortality during gram-negative bacterial sepsis.

Detection by enzyme-linked immunosorbent assay of specific immunoglobulin G isotypes in primary and established cytomegalovirus infections.

An enzyme-linked immunosorbent assay using monoclonal antibodies was developed to study the subclass distribution of immunoglobulin G (IgG) to cytomegalovirus (CMV) in individuals from a number of clinical groups. Most CMV-seropositive individuals had IgG1 and IgG3. IgG2 and IgG4 were detected less frequently at very low levels of activity, mostly among mothers at delivery and renal patients. Most seroconversions were accompanied by an important increase of the IgG1 activity, whereas IgG3 appeared at lower levels; neither IgG2 nor IgG4 occurred. This suggests that these isotypes play a secondary role in the response to the CMV infection and that they may be considered markers of past infections. Anti-CMV IgG1 is the most efficiently transmitted through the placenta. Whether infected or not, newborns had the same subclass distribution and activity levels as their mothers. Isotype determination did not offer a decisive explanation of a number of discrepancies observed between CMV IgG enzyme-linked immunosorbent assay and complement fixation test results.

Thermophilic semisolid anaerobic digestion of municipal refuses.

Comparison of both mesophilic (35 degrees C) and thermophilic (55 degrees C) anaerobic digestions of the organic fraction of municipal refuses in pilot digesters designed to process in a semisolid phase at total solids concentrations of ca. 25% shows that the average gas production is 20-25% higher in thermophilic conditions than in mesophilic conditions even for a retention time of 10 days. These results and the data recorded during long periods of experimentation indicate that the process allows to increase the net energy production and to improve the economical balance of an industrial plant.

Enzyme-linked immunosorbent assay for immunoglobulin G subclass antibodies specific for enterobacterial Re core glycolipid in healthy individuals and in patients infected by gram-negative bacteria.

An enzyme-linked immunosorbent assay was developed to study the subclass distribution of immunoglobulin G (IgG) specific to the core glycolipid (CGL) of the Re mutant of Salmonella minnesota R595 in serum samples from individuals with an IgG response toward these antigens. In a group of healthy blood donors, we detected predominantly the IgG2 and IgG1 subclasses. In a group of patients in an intensive care unit who developed infectious complications due to gram-negative bacteria, the anti-CGL IgG activity was due mainly to the IgG2 and IgG3 subclasses. In all serum samples found to be IgG positive, the assay for anti-CGL IgG2 was positive. This subclass was revealed to play a predominant role in patients displaying a seroconversion or a significant rise in their antibody response toward CGL. IgG4 was found or appeared only in patients with confirmed bacteremia. In addition, we observed a drop in anti-CGL IgG2 before the death of patients undergoing a septic shock or an irreversible organ failure, suggesting that the anti-CGL IgG2 activity could be used as a marker of the evolution of the illness in this group of patients.

A direct enzyme-linked immunosorbent assay (ELISA) for antibodies to enterobacterial Re core glycolipid and lipid A. Results in healthy subjects and in patients infected by gram-negative bacteria.

We have developed an ELISA for IgM and IgG antibodies to the core glycolipid (CGL) of the Re mutant Salmonella minnesota R 595, and to lipid A. Anti-CGL antibodies have been detected in sera from 37% of healthy blood donors, whereas anti-lipid A activities were found in 13% of individuals only. The anti-CGL and anti-lipid A activities were examined in patients in a surgical intensive care unit, selected on the basis of a definite risk of infectious complications due to Gram-negative bacteria. Of the patients who developed such infections, the rate of favourable outcome was significantly higher in patients with either stable positive or increasing anti-CGL activities than in patients found to be negative. Our results provide clear evidence that anti-CGL antibodies contribute to host defence against various Gram-negative bacteria.

Monoclonal antibodies detect M-protein epitopes on the surface of influenza virions.

Various data obtained with activable hydrophobic probes, proteolytic treatments and anti M-protein polyclonal antibodies strongly suggest that M-protein of influenza A is an integral part of the lipid bilayer of native virions and somehow spans at the surface of the virions. Therefore we have looked for the presence of M-protein epitopes on the surface of influenza A virion by using four type A M-protein monoclonal antibodies. We developed a specific and sensitive competition ELISA where intact virions, dodecyl-sulfate disrupted virions and spikeless particles obtained after proteolytic treatment with caseinase C were used to test their ability to inhibit the reaction between these monoclonal antibodies and pure M-protein. Intact virions or SDS disrupted virions prevented three monoclonal antibodies from reacting with the M-protein. Spikeless particles also inhibited the specific binding of two of these antibodies, whereas the other fourth antibody was inhibited by contact with SDS disrupted particles only. Data presented show that at least three distinct M-protein epitopes were detected, of which at least two are exposed on the surface of intact virions. Of these two epitopes, one is inactivated by the proteolytic treatment. The third epitope could only react with its monoclonal antibody when the virus particles were solubilized with SDS. This work provides a clear demonstration that a substantial part of the M-protein spans the lipid bilayer and that the rest, protected by lipids, resists proteolytic enzymes and is prevented from binding with anti M-protein monoclonal antibodies.

Elimination of nonspecific cytomegalovirus immunoglobulin M activities in the enzyme-linked immunosorbent assay by using anti-human immunoglobulin G.

Direct enzyme-linked immunosorbent assay methods offer several advantages in assessing past or recent exposure to cytomegalovirus (CMV) infection, but there persist many pitfalls in the use of these methods for determining specific immunoglobulin M (IgM). The efficiency of absorption of sera by IgG-coated latex beads, aggregated human IgG, or Staphylococcus aureus, i.e., for removing nonspecific CMV IgM activities, was evaluated in comparison with the effect of an anti-human IgG hyperimmune serum. Large routine series comprising serum samples from patients of various clinical groups and healthy individuals were examined. The CMV IgM-positive samples were at first treated with latex or aggregated IgG, but these absorptions left too many CMV IgM-positive individuals. S. aureus increased the nonspecific activity of some sera and, in other cases, removed or impaired specific IgM activities. The anti-IgG treatment caused the disappearance of nonspecific CMV IgM activities that had resisted the other treatments, whereas specific activities remained intact. Utilizing this method, only 1.03% of the routine series patients remained CMV IgM positive by the enzyme-linked immunosorbent assay, a figure in good agreement with a mean probability of CMV antibody acquisition of 0.33% for the population living in Belgium. On the other hand, in a series of patients who were investigated for serological response to several viruses, eight individuals displayed multiple IgM activities after anti-IgG treatment. In these cases, most IgM activities were found in patients who had IgG toward the related antigen for a long time before transient IgM was detected. This result implies that to assess a diagnosis of primary infection, it is necessary to examine serial specimens for IgG acquisition accompanying specific IgM.

Anti M-protein antibody response to type A or B natural influenza detected by solid phase enzyme linked immunosorbent assay and by complement fixation.

Anti M-protein antibody response has been looked for in sera from individuals with serological evidence of A or B influenza infection using pure M-protein (M) in complement fixation tests (CF), in IgG and in IgA specific enzyme linked immunosorbent assays (ELISA). Mp ELISA (IgG specific) antibodies are not restricted to people with history of recent respiratory infection. Individuals under 15 years are less prone than those older to display M ELISA activity. Most M ELISA positive individuals are also nucleoprotein (NP) positive. There are more M than NP ELISA positives in the influenza A series whereas the reverse is observed in the influenza B series. Most of the M ELISA positives are also S CF (standard soluble antigen CF) positive indicating that M ELISA IgGs are related to recent infections. Some sera exhibit M ELISA activity with no other evidence of influenza experience than V CF (viral antigen CF) or HI (haemagglutination inhibition), suggesting that some recent influenza infections are better traced with M ELISA than with S CF. Amongst chronic bronchitis patients with V CF or HI antibodies to A2 influenza virus but no type A S CF activity, the proportion of M ELISA positives averages 40 per cent. This fact as well as two other features of that group i.e. cases with long lasting S CF activity and occasional virus isolation several months after the initial acute infection, suggest that influenza virus might cause prolonged infection in some patients with chronic bronchitis.

Ligands for antibody to M-protein are exposed at the surface of influenza virions: effect of proteolytic treatment on their activity.

Antiserum to pure M-protein extracted from PR8 virions neutralized the infectivity and inhibited the haemagglutinating activity of various influenza A virions. It agglutinated concentrated suspensions of these virions and fixed complement in their presence. Antibodies to M-protein were readily absorbed by intact virions or by spikeless particles obtained after proteolytic treatment, giving clear evidence that M-protein is exposed at the surface of the virus envelope. The data suggest that when antibodies to M-protein occupy specific ligands exposed at the surface of the virion they interfere with sites critical for infectivity and haemagglutinating activity.

The amino acid sequence of the major parvalbumin of the whiting (Gadus merlangus).

1. The amino acid sequence of the major parvalbumin of the Whiting has been determined; the polypeptide chain is made of 108 residues, the terminal amino acid group is acetylated, there is no disulfide bridges, the structure of the two calcium binding sites is preserved and the distribution along the polypeptide chain of the hydrophobic residues implicated in the compact hydrophobic core of the protein is also maintained. 2. The comparison of this amino acid sequence with other parvalbumins indicates that it belongs to the beta type and that within the Gadidae family two types of parvalbumins also occur.