Increase In Antibody Concentration Research Articles

Natural Development of Antibodies to Pneumococcal Capsular Polysaccharides Depends on the Serotype: Association with Pneumococcal Carriage and Acute Otitis Media in Young Children

To study the natural development of antibodies to pneumococcal capsular polysaccharides of types 1, 6B, 11A, 14, 19F, and 23F and its association with pneumococcal carriage and acute otitis media (AOM), 329 children were followed-up prospectively during their first 2 years of life. Nasopharyngeal carriage was determined by cultures of nasopharyngeal swab samples, and etiology of AOM was determined by cultures of middle ear fluid. Antibodies were measured in serum samples collected at 6, 12, 18, and 24 months by EIA. Antibodies increased modestly but significantly with age. Contact with serotypes 11A and 14 was associated with increased antibody concentration as early as age 6 months. Children with contact with serotypes 6B, 19F, and 23F had antibody levels similar to those in children without contact. Antibodies increased modestly, even in children without known contact with Streptococcus pneumoniae and in children with contact with heterologous serotypes. Antibody concentrations were equal after carriage or AOM.

Impaired immunoglobulin M production by incubation of hybridoma cells with ethanol

Several reports have presented results that demonstrate suppression of the immune system by ethanol. Using a hybridoma cell model, we studied the effects of ethanol on cell proliferation and on the production of immunoglobulin M (IgM) antibodies. The number of cells decreased while incubated with as little as 25 mM ethanol but not in a clonal subline incapable of IgM production, indicative of an increased vulnerability associated with the antibody-producing machinery. Levels of antibodies in cell culture supernatants were monitored by μ-heavy-chain-specific and κ-light-chain-specific enzyme-linked immunosorbent assays. We found a significant decrease in antibody concentration at 200 mM ethanol compared with findings for nonexposed cells. In addition, lower κ-chain compared with μ-chain values were monitored at ethanol concentrations of 50 mM and higher. This difference suggests irregular composition of the antibodies in the supernatant. Determination of IgM levels within the hybridoma cells revealed a linear increase in antibody concentrations by as much as three times the control levels with increasing ethanol concentrations when correlated with cell numbers. Analysis of the mRNA levels of two ethanol-inducible stress proteins, the 78-kilodalton glucose-regulated protein (GRP78) and the 70-kilodalton heat-shock protein (HSC70), by quantitative Northern hybridization yielded increased mRNA in a nonlinear fashion. The results demonstrate that ethanol impairs IgM composition, whereas antibody production within hybridoma cells is increased and the assembling machinery is activated, indicating compensating processes.

Serum and salivary anti-capsular antibodies in infants and children immunized with the heptavalent pneumococcal conjugate vaccine.

To study the ability of seven-valent experimental pneumococcal polysaccharide CRM197 protein conjugate vaccine (PncCRM) to induce antibodies in serum and saliva of infants. Sixty Finnish infants received Pnc-CRM vaccine at 2, 4 and 6 months of age and were boosted with PncCRM (n = 30) or pneumococcal polysaccharide (PncPS) (n = 29) vaccine at the age of 15 months. Serum IgG antibody concentrations to vaccine serotypes 4, 6B, 9V, 14, 18C, 19F and 23F were measured by enzyme immunoassay at 2, 4, 6, 7, 15, 16 and 24 months of age. Salivary IgA, IgG and secretory Ig antibody titers at 7 and 16 months of ages were analyzed by enzyme immunoassay against the same serotypes, except 23F. PncCRM induced systemic immune responses and immunologic memory. At 7 months of age 69 to 100% of children, depending on the serotype, had serum IgG antibody concentrations exceeding the value of 1.0 microg/ml. At 15 months the titers were still higher than before the vaccinations. Booster doses of either PncPS or PncCRM induced an increase in antibody concentrations. The titers were still elevated at 24 months of age. Salivary IgA and IgG antibodies were found rarely at 7 months of age, but in up to 80% of samples taken at 16 months of age, depending on the serotype and nature of the booster vaccine. Salivary IgG correlated with IgG in serum, supporting the theory that salivary IgG is derived from serum. Salivary IgA and secretory Ig correlated positively, which indicates that IgA was locally produced. PncCRM induces both systemic and mucosal immune responses in infants.

A rheo-optical method for the study of aggregated particles: application to the characterization of ABO blood group monoclonal antibodies

The adhesive energy of monoclonal antibody induced agglutination can be assessed from the shear dissociation of agglutinates. Anti-A, anti-B, and anti-AB monoclonal antibodies agglutinate erythrocytes with variable strength, depending on the affinity of antibodies and on the antigenic density on the cell surface. The time required for an applied shear stress to dissociate cell agglutinates completely is depending on the strength of the agglutination reaction. The dissociation process under constant shear stress can be followed by measuring intensity of light backscattered by suspended particles. The obtained dissociation curves could be adjusted by an exponential expression governed by a parameter called characteristic time of dissociation. This parameter decreases with increasing antibody concentration. Integration of dissociation curves provided an approach of adhesive energy induced by monoclonal antibody.

Increased t-PA yields using ultrafiltration of an inhibitory product from CHO fed-batch culture.

Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The feed, an isotonic concentrate, was improved to obtain 2- to 2.5-fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5-fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed-batch results. The concentrate addition rate was based on a constant 4 pmol/cell.day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t-PA concentrations compared to batch. The fed-batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4-fold increase in viable cell index yielded a 4-fold increase in antibody concentration. The half-life of t-PA decreased from 43 to 15 days with decreasing cell viability (from 92% to 71%), but this was not sufficient to explain the apparent t-PA threshold. Instead, the CHO results were explained by a reduction in t-PA production at higher extracellular t-PA concentrations that limited the fed-batch maximum at 35 mg/L for the cell line investigated. Analysis of both the total and t-PA mRNA levels revealed no response to increasing extracellular t-PA concentrations upon exogenous additions. Instead, intracellular t-PA levels were increased, revealing a possible secretory pathway limitation. A new reactor configuration was developed using an acoustic filter to retain the cells in the reactor while an ultrafiltration module stripped t-PA from the clarified medium before the permeate was returned to the reactor. By adding this harvesting step, the t-PA fed-batch production was increased over 2-fold, up to a yield of 80 mg/L.

Health complaints and immunological markers of exposure to bioaerosols among biowaste collectors and compost workers

OBJECTIVESIn a cross sectional study, work related health complaints and diseases of 58 compost workers and 53 biowaste collectors were investigated and compared with 40 control subjects. Levels of specific...

Serum antibodies to Helicobacter hepaticus and Helicobacter pylori in patients with chronic liver disease

BACKGROUNDBile tolerant helicobacter species such as H hepaticus andH bilis have frequently been reported to cause hepatitis in mice and other rodents.AIMSTo investigate the possible pathogenic role of these and...

Polymer-coated optical fibres for application in a direct evanescent wave immunoassay

Three different polymers were tested for application in a direct evanescent wave immunoassay. For this purpose, 50 mm of a 200 μm plastic clad silica fibre was stripped off the cladding and coated with either 7% (w/w) polystyrene ( n = 1.59) in tetrahydrofuran (THF), 7% (w/w) carboxylated poly(vinyl chloride) (PVC-COOH) ( n = 1.55) in THF or chloropropyl-modified sol–gel ( n = 1.56). Human IgG was immobilised on the polymer-coated fibres. The binding of immobilised IgG to TRITC-labelled anti-IgG was measured, and was highest on the sol–gel coated fibres. However, 80% of the measured signal was due to non-specific adsorption. Non-specific binding was negligible on polystyrene, but increased with increasing antibody concentration on PVC-COOH (from 9% to 45% for 0.5–5 μg ml −1 TRITC-labelled anti-IgG). 8 M urea, 0.1 M glycine/HCl and 0.1 M triethylamine were used for regeneration of the antigen–antibody complex 4 M NaCl. The solutions of NaCl and urea did not affect the stability of the complex. The use of 0.1 M glycine/HCl, however, resulted in a 65% regeneration of the complex on PVC-COOH and in a 35% regeneration on polystyrene. Sol–gel coated fibres could not be reused, because the porous structure of the gel was destroyed due to intrapore condensation of the regeneration solutions. The results were correlated to the characteristics of the polymers.

Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma.

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures.

Immunogenicity of pneumococcal vaccine in persons with spinal cord injury

Objective: To determine immunogenicity and optimum timing for administering the 23-valent pneumococcal vaccine after spinal cord injury (SCI). Design: Double-blind, randomized, placebo control study. Setting: SCI unit in a tertiary care medical center and community. Participants: Eighty-seven persons with recent SCI. Intervention: Participants were randomized to receive either placebo or pneumococcal vaccine at 16 to 18 days versus 4 to 6 months postinjury. Main Outcome Measures: Antibody concentrations were measured prior to intervention and 1, 2, and 12 months afterward to evaluate the immune response to five serotypes of Streptococcus pueumoniae. Effects of demographic and injury-related variables on immune response were also evaluated. Results: Timing of vaccination did not influence mean antibody concentrations for any serotype ( p > .05). Ninety-five percent of vaccinated persons had twofold or greater increases in antibody concentration for at least one serotype when measured 1 month after vaccination versus 35% of placebo groups ( p < .01). After 12 months, 93% of vaccinated persons in both groups maintained antibody concentrations twofold or greater than baseline values. Conclusions: Most participants developed an immune response to at least one serotype that was maintained for at least 12 months. Immune response varied according to serotype. Given the favorable immune response and no effect of timing, persons with SCI should receive pneumococcal vaccine during initial hospitalization.

Integrin activation protects pulmonary endothelial cells from the genotoxic effects of bleomycin.

Integrin activation promotes the survival of endothelial cells undergoing diverse forms of stress. Here we determined the ability of integrins to inhibit DNA strand breakage by bleomycin (BLM), a DNA-cleaving antitumor antibiotic that causes acute endothelial injury and subsequent pulmonary fibrosis. We found that BLM produced DNA breakage in cultured murine lung endothelial cells (MLEC) within 45 min of treatment as measured by DNA sedimentation and in situ labeling of 3'-OH by nick translation (ISNT). Two hours after the removal of BLM, we found a marked but incomplete reduction in DNA strand breakage as measured by ISNT, indicating that the damage was reversible. DNA sedimentation and ISNT demonstrated that strand breakage due to BLM was inhibited in MLEC cultured on fibronectin, and no evidence of breakage was found 2 h after removal of the drug in ISNT experiments. Gelatin, type IV collagen, laminin, and the integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro, but not the inactive Gly-Arg-Ala-Asp-Ser-Pro peptide, also inhibited DNA strand breakage. Activation of integrins, either by coating surfaces with antibodies to alpha 5-, beta 1-, or beta 3-integrin subunits or by receptor clustering with the soluble antibodies, inhibited BLM-induced DNA breakage. Inhibition of BLM-induced DNA strand breakage by soluble beta 1-integrin antibody increased with increasing antibody concentration and duration of receptor clustering before BLM treatment. Thus integrin activation protects pulmonary endothelial cells from the genotoxic effects of BLM.

Randomised trial of effect of vitamin A supplementation on antibody response to measles vaccine in Guinea-Bissau, west Africa

WHO has recommended vitamin A supplementation for children aged 6 months or older in developing countries at the same time as immunisation. One study has reported significantly lower seroconversion ratios among children who have received vitamin A supplements with measles vaccine at age 6 months. The aim of our study was to assess the effect of vitamin A supplementation on antibody response to measles vaccination at age 9 months, which is the more common age for immunisation in developing countries. In an urban community in Guinea-Bissau, we did a randomised, double-blind, placebo-controlled study of the effect of simultaneous vaccination and vitamin A supplementation in 462 children who received either a two-dose schedule of measles vaccine at the ages of 6 months and 9 months (150 infants) or one dose of measles vaccine at age 9 months (312 infants). Children were followed up to the age of 18 months and a blood sample was then collected to assess the antibody response. 397 (86%) of the children took part in the follow-up (52 [11%] had moved and 13 [3%] had died). Among children who received a two-dose vaccine schedule, seroconversion was 98%. There was no difference in seroconversion or geometric mean titre (GMT) for children receiving vitamin A compared with children receiving no supplement. Among children receiving only one dose of measles vaccine at age 9 months, seroconversion was 95%. The GMT was significantly higher in children receiving vitamin A than in those receiving no supplement (3704 vs 2439 mIU; GMT ratio 1.52 [1.22-1.88]). The effect on plasma antibody concentration in the blood was stronger for boys (3902 vs 1916 mIU; GMT ratio 2.04 [1.53-2.72]) than for girls (3502 vs 3017 mIU; GMT ratio 1.16 [0.85-1.58]) who had received vitamin A with measles vaccine. In a multivariate analysis of variance adjusted for sex, vitamin A supplementation was associated with higher antibody titres (p < 0.001). There was a significant interaction between vitamin A supplementation and sex (p = 0.02). There is no indication that simultaneous administration of measles vaccine and vitamin A supplements has a negative effect on measles immunity. Among the children who had received two doses of measles vaccine at the ages of 6 months and 9 months, supplements of vitamin A had no significant effect. Among children only receiving one dose of measles vaccine at age 9 months, 100,000 IU vitamin A increased antibody concentrations, especially for boys.

Use of a modified binding model for the investigation of affinity dependence on antibody concentration in immunoassay systems

Affinity parameters obtained from standard calibration curves of radioimmunoassays (RIA) have been shown to decrease with increasing antibody concentration. A modified binding model was introduced in which not only binding capacity but also affinity was influenced by the antibody concentration. This allows a consistent overall description of RIA standards and titration curves for the characterization of binding assays.

Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores ofPhytophthora cinnamomi

The role of tubular mastigonemes in the reversal of thrust of the anterior flagellum ofPhytophthora cinnamomi was analysed using mastigoneme-specific monoclonal antibodies and immunoflu-orescence and video microscopy. Exposure of live zoospores ofP. cinnamomi to the mastigoneme-specific Zg antibodies caused alterations in the arrangement of mastigonemes on the flagellar surface and at Zg concentrations above 0.3 μ/ml, mastigonemes became detached from the flagellum. As a consequence of antibody binding to the mastigonemes there were concentration-dependent perturbations in zoospore swimming behaviour and anterior flagellum beat pattern. With increasing antibody concentration zoospores swam more slowly and other parameters of their swimming pattern, such as the wavelength of the swimming helix and the frequency of rotation, were also reduced. The effects of Zg antibodies were specific at two levels: control immunoglobulins or antibodies that bound to other flagellar surface components did not have an effect on motility, and Zg antibodies did not interfere with the motility of zoospores of oomycete species to which they did not bind. The effects of antibody-induced disruption of mastigoneme arrangement strongly support previous hypotheses that tubular mastigonemes are responsible for thrust reversal by the anterior flagellum, enabling it to pull the cell through the surrounding medium.

Determination of the concentration of influenza virus antihaemagglutinin antibody molecules of the IgG class and of the equilibrium constant by use of enzyme immunoassay titres determined for graded epitope concentrations.

A new technique for determining the concentration of influenza virus antihaemagglutinin antibody molecules of the IgG class (A) and of the equilibrium constant K of paratope-epitope interaction is described. The method is based on determining enzyme immunoassay (EIA) titres of antibody with graded epitope concentrations: EIA titres were defined in terms of the antibody dilution yielding a fixed amount of antibody adsorbed per ml (= 6.21 x 10(10)). Adsorption of antibody depends on the concentration of paratopes and epitopes allowed to react and on the equilibrium constant. For the use of a constant concentration of epitopes, the paratope concentration needed to yield the desired degree of antibody adsorption decreases with increasing avidity. Therefore, the EIA titres increase both with increasing avidity and increasing antibody concentration. When graded epitope concentrations are used for determining the EIA titres of a given serum, the titres are influenced in a similar manner by the antibody concentration of the serum and the increase of titres with increasing epitope concentration reflects avidity. The equilibrium constant found is subsequently used to determine the concentration of free antibody at the dilution meeting the definition of EIA titre and the product of EIA titre and the sum of free and bound antibody at this dilution gives the number of antibody molecules present in the test serum. A panel of 118 antisera was tested comparatively for A and K using the new method and by means of the guanidine titre ratio test and equilibrium filtration. The values obtained agreed well with each other. This novel technique offers the advantage that it can be easily adapted for use with other viruses.

Effect of specific antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types.

The effect of specific pseudorabies virus (PRV) antibodies on the enlargement of plaques produced by PRV were studied in monolayers of different cell types. The plaque size was used as parameter for the efficacy of the cell-associated spread (CAS) of PRV. First, the effect of anti-PRV hyperimmune serum on the plaque growth was examined in monolayers of the continuous cell lines ST, SK-6 and MDCK and monolayers of the primary cultures of porcine fibroblasts, endothelial cells and endometrial cells. A tenfold increase in the serum concentration did reduce the plaque size with 50% in both SK-cells and fibroblasts and with 40, 28 and 16% in MDCK, endothelial and endometrial cells, respectively. In ST cells, no change in size was observed with increasing antibody concentrations. Secondly, the effects of monoclonal antibodies (mAbs) directed against PRV glycoproteins gB, gC, gD and gE and polyclonal antibodies against gC were evaluated in SK-6 cells. MAbs against gB, gD and gE were able to reduce the CAS with a cumulative effect between mAbs against gD and either mAbs against gB or mAbs against gE. Monoclonal and polyclonal antibodies against gC did not change the plaque size.

Acoustic Cell Filter for High Density Perfusion Culture of Hybridoma Cells

We have developed a flow-through device which uses high frequency, low energy ultrasonic resonance fields to transiently aggregate hybridoma cells and return them by sedimentation to a perfusion bioreactor. The system retained up to 99 percent of the inflowing viable cells with no measurable effect on viability. Viable cells were selectively retained at up to 3 percent higher efficiency than nonviable cells. A stirred tank bioreactor was operated for 700 hours with acoustic cell recycle. Concentrations greater than 5 x 10(7) cells/ml were attained with a 5-fold increase in antibody concentration and a 70-fold increase in volumetric productivity compared with batch culture.

Association of serum antibodies to heat-shock protein 65 with carotid atherosclerosis

Arteriosclerotic lesions can be induced in normocholesterolaemic rabbits by immunisation with heat-shock protein (hsp) 65, a stress protein expressed in high concentrations in human atherosclerotic lesions. If an immune reaction to hsp65 also plays a part in human atherogenesis, it should be possible to detect anti-hsp65 antibodies in patients with atherosclerotic lesions. To study the possible relation between immune reaction to hsp65 and atherosclerosis, 867 normal inhabitants of South Tyrol, aged 40-79 years, were selected randomly for determination of serum antibodies against hsp65, simultaneous sonographic assessment of carotid atherosclerotic lesions, and evaluation of established risk factors—ie, blood cholesterol, hypertension, smoking, diabetes mellitus, and obesity. Autoantibodies to nuclear antigens, thyroid antigens, and rheumatoid factors were also measured. Serum anti-hsp65 antibodies were significantly (p < 0·05) increased in subjects aged 60-79 years with carotid atherosclerosis compared with those without lesions, and increased antibody concentration was independent of age, sex, and other established risk factors. On the other hand, the incidence and titres of autoantibodies did not correlate with carotid atherosclerotic lesions. Our data provide the first evidence of a strong correlation between anti-hsp65 antibodies and carotid atherosclerosis, suggesting that hsp65 might be involved in the pathogenesis of atherosclerosis. Lancet 1993; 341: 255-59.

Immunochemical and clinical effects of immunosuppressive treatment in monoclonal IgM neuropathy.

A pathogenic role of the M protein in monoclonal IgM neuropathy has been suggested. This is based among other things on a close relation between immunosuppressive treatment, lowered concentration of M protein, and clinical effect. We studied five patients with monoclonal IgM and antibodies to peripheral nerve myelin. The immunosuppressive treatment was beneficial in three of the patients. In three patients there was a relationship between antibody concentration and clinical effect (in one there was no change in antibody concentrations and correspondingly no change in clinical status, and in two patients clinical improvement corresponded to decreased antibody concentrations). In two patients, however, there was no clear correlation, since one patient improved despite increasing antibody concentrations and one patient did not improve despite a lowered antibody concentration. It is therefore possible that other mechanisms may contribute to the effect of treatment.

Antibodies to the trypsin cleavage peptide VP8 neutralize rotavirus by inhibiting binding of virions to target cells in culture

Two distinct patterns of neutralization were identified by comparing the neutralization curves of monoclonal antibodies (MAbs) directed at the two surface proteins, VP4 and VP7, of rhesus rotavirus. VP7-specific MAbs were able to neutralize virus efficiently, and slight increases in antibody concentration resulted in a sharp decline in infectivity. On the other hand, MAbs to VP4 proved much less efficient at neutralizing rhesus rotavirus, and the fraction of infectious virus decreased gradually throughout a wide range of antibody concentrations. MAbs directed at VP8*, the smaller trypsin cleavage fragment of VP4, were shown to efficiently prevent binding of radiolabeled virions to MA104 cell monolayers, to an extent and at concentrations comparable to those required for neutralization of infectivity. Conversely, MAbs recognizing VP7 or the larger VP4 trypsin cleavage product, VP5*, showed little or no inhibitory effect on virus binding to cells. All MAbs studied were able to neutralize rotavirus that was already bound to the surface of cells. The MAbs directed at VP8*, but not those recognizing VP5* or VP7, were shown to mediate release of radiolabeled virus from the surface of the cells. With MAbs directed at VP7, papain digestion of virus-bound antibody molecules led to an almost complete recovery of infectivity. Neutralization could be fully restored by incubation of virus-Fab complexes with anti-mouse immunoglobulin G antiserum. Neutralization with MAbs directed at VP8* proved insensitive to digestion with papain as well as to the addition of anti-immunoglobulin antibodies.