Evaluation of anti-pneumococcal capsular antibodies as adjunctive therapy in experimental pneumococcal meningitis

OBJECTIVE: Bacteraemia concomitant with meningitis has been shown to greatly affect outcome. Consequently, the efficacy of serotype-specific anti-pneumococcal antiserum (APAS) was investigated in a rat model of pneumococcal meningitis. METHODS: Rats were infected with Streptococcus pneumoniae serotype 3. All rats received ceftriaxone starting 26 h post-infection. APAS was administered either at the time of infection or 26 h post-infection and effects were compared with rats treated with antibiotics only. RESULTS AND CONCLUSION: A significant clinical benefit was found when APAS was given at the time of infection whereas no effect was found when administered 26 h after infection. This work indicates that the clinical value of using APAS in pneumococcal meningitis may be limited.     

Specific IgG subclass antibodies, IgE and IgG S-TS antibodies to wheat gluten fraction B in patients with coeliac disease

Antibodies were measured in the sera of fifteen patients with untreated coeliac disease and twenty-eight patients with inflammatory bowel disease. Increased levels of specific IgG, IgG1, IgG2, and IgG4 antibody to wheat gluten fraction B, measured by an enzyme-linked immunosorbent assay, were shown in the coeliac disease group, but not in the inflammatory bowel disease group. No specific IgE antibody to fraction B was detected but 33% of the patients with coeliac disease had specific short-term sensitizing (anaphylactic) IgG antibody activity (IgG S-TS) to fraction B. There was no correlation between the IgG2 or IgG4 specific antibody and the presence of IgG S-TS activity.     

Enterovirus IgG ELISA using synthetic peptides as antigens

An indirect IgG enzyme-linked immunosorbent assay (ELISA) based on three different synthetic peptides with amino acid sequences from viral protein VP1 of enteroviruses was explored for the diagnosis of enteroviral infections. In a panel of acute and convalescent sera from 137 patients with culture confirmed infection with one of 17 different enterovirus serotypes, a combination of all three peptides, and the use of two serum dilutions, yielded significant titre rises of enterovirus specific IgG-antibodies in 73% of the patients. This broad-reactive peptide IgG ELISA is useful for serodiagnosis of enteroviral infections.     

Enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to thyroglobulin

We used a computer programmed standard IgG curve for computer-assisted quantification of assay results for autoantibodies to thyroglobulin (Tg) by quantitative enzyme-linked immunosorbent assay (ELISA). Specific antibody levels in unknowns were quantified by comparison of their optical density readings with a standard curve of absorbance vs concentration obtained with dilutions of the reference serum. Anti-Tg antibodies were detected in 80% of the patients with chronic thyroiditis and 90% of those with Graves' disease. Anti-Tg antibodies were also detected in 14.3% of the healthy controls. The titer of anti-Tg antibodies detected by tanned red cell hemagglutination correlated well with that detected by ELISA, although, the sensitivity of the ELISA was higher. By our computer-assisted conversion method, the anti-Tg antibody can be readily and reliably quantified and low titer antibodies to Tg can be detected with adequate precision.     

An autoanalyzer test to determine immunoglobulin class and IgG subclass of blood group antibodies

An automated antiglobulin test was used to characterize the immunoglobulin class and IgG subclass of red blood cell-bound antibodies. Immunoglobulin classes and IgG subclasses of some clinically significant antibodies (anti-D and anti-CD, anti-Fy, anti- Jka) as well as of clinically insignificant anti-Chido antibodies were determined. Eighteen anti-Duffy antibodies were structurally homogeneous, and were mostly composed only of IgG1. Of the 16 anti-Fya antibodies studied only three had IgG2, and four had IgM molecules. Ten anti-Jka antibodies were heterogeneous, but all samples were either IgG1 or IgG3, or both. The most prominent immunoglobulin of 12 hyperimmune anti-Rh (anti-D and anti-CD) antibodies was IgG1. Under the conditions of our test, we also detected IgG3 and very low concentrations of IgG2 and IgG4 molecules in the anti-Rh antibodies.     

Anti-DNA antibodies in autoimmune disorders by ELISA using nylon as the solid phase

A rapid, simple and highly sensitive enzyme-linked immunosorbent assay (ELISA) utilizing nylon as the solid support is described for the detection of anti-DNA antibodies in autoimmune disorders. The optimal reaction conditions were established with an anti-DNA antibody-positive SLE serum. Fifty-three percent of systemic lupus erythematosus patients, 29 percent of patients with systemic lupus erythematosus with overlapping progressive systemic sclerosis and 10 percent of progressive systemic sclerosis patients were positive for anti-DNA antibodies. The sensitivity of the method was compared with passive hemagglutination and fluorometric assays, the latter using ethidium bromide as intercalating dye. The method described is specific, reproducible and convenient for use in clinical laboratories where large numbers of samples are to be screened for the detection of anti-DNA antibodies. The procedure requires small quantities of antigen or antibody and is, therefore, highly economical.     

Quantitation of red cell-associated IgG using an immunoradiometric assay

In this report, we describe a sensitive immunoradiometric assay (IRMA) for quantitating IgG on the surface of red cells. Washed red cells were prepared to a purity of greater than 99.9 percent. Varying dilutions of these cells were incubated with a fixed concentration of 125I-anti-IgG. After equilibrium was achieved, the unbound 125I-anti-IgG was measured by the addition of IgG covalently linked to agarose beads. The red cells were lysed by detergent, and the 125I-anti-IgG bound to the IgG- beads was measured. The amount of IgG on the red cells was determined by relating the concentration of test red cells causing 50 percent inhibition of binding of the 125I-anti-IgG to the IgG-beads to 50 percent inhibition of binding caused by the IgG standard. Using this assay, the red cell-associated IgG (RCA-IgG) of 20 healthy male and female controls with normal hemoglobin concentrations was 7.23 +/− 6.11 fg IgG per 10(3) cells (mean +/− 2 SD). The mean RCA-IgG on washed cells from 34 different tests performed on 19 anemic patients with clinically diagnosed autoimmune hemolytic anemia was 176.1 +/− 375.6 fg IgG per 10(3) cells. There was no correlation between the levels of RCA- IgG and the hemoglobin levels or reticulocyte counts in these patients.     

Development of a sandwich ELISA to detect IgG and IgG sub-class antibodies specific for a major antigen (Ag 7) of Aspergillus fumigatus

A sandwich ELISA has been developed, using an affinity purified monospecific antiserum as a capture antibody, to detect specific IgG and IgG sub-classes to a major antigen (Ag 7) of Aspergillus fumigatus in the sera of patients with allergic bronchopulmonary aspergillosis (ABPA). Significantly elevated levels of specific IgG to Ag 7 were detected in 97% of ABPA sera tested, as compared to control sera and to sera from A. fumigatus skin-prick test positive individuals. IgG sub-class antibody levels to Ag 7 were also determined in a similar sandwich ELISA, but using specific monoclonal antisera instead of the polyclonal anti-IgG. Both Ag 7 specific IgG1 and IgG4 levels were found to be significantly raised in the ABPA sera compared to controls. It is proposed that this antigen-specific ELISA may provide a more specific diagnostic test for IgG antibody detection in sera of ABPA patients.     

Serum IgA and IgG antibodies to α-gliadin: Comparison between two ELISA methods

Summary We have developed two ELISA methods, i.e., enzyme immunoassay (EIA) and fluorescence immunoassay (FIA), for the semiquantitative detection of specific IgA and IgG antibodies directed against α-gliadin. The tests differ only for the enzyme substrate and, when optimized, could be used in large routine screening of celiac disease. Several serum samples from patients with celiac disease and gastrointestinal disorders as well as from control subjects were tested. Both methods gave good correlation with clinical data, were easily performed and had same specificity features, while FIA proved to be more sensitive.     

Detection of IgG antibodies specific for measles virus by enzyme-linked immunosorbent assay (ELISA).

A solid-phase, enzyme-linked immunosorbent assay (ELISA) for determination of IgG antibodies against measles virus is described. The assay utilized antigen-coated polystyrene microplates. The antigen consisted of a sonicated extract of measles-infected Vero cells. Goat and anti-human IgG-peroxidase conjugate was used to detect human IgG bound to viral antigen. Sera taken from 63 healthy adults, 11 young children and 36 patients were evaluated for their IgG titer against measles virus. Comparison of results obtained by ELISA with those obtained by hemagglutination-inhibition (HI) assay or by complement fixation showed good agreement between the tests. The geometric mean titer (GMT) for healthy adults was 753 for ELISA and 32.8 for HI. If these averages are taken as a measure of comparison, then ELISA is approximately 23 times more sensitive than HI. ELISA technique is rapid to perform and could be recommended for routine diagnosis.     

IgG subclass distribution and relative functional affinity of thyroid microsomal antibodies in postpartum thyroiditis

An association between the development of postpartum hypothyroidism and high levels of IgG1 subclass microsomal (M) antibodies has been reported. Using an assay designed to detect reasonable levels of all the four IgG subclasses, we found no differences in the proportion of each IgG subclass in M antibodies of patients with postpartum hyperthyroidism or hypothyroidism compared with control postpartum patients with M antibodies but no thyroid dysfunction. However the total amount of M antibody of each IgG subclass was elevated above the controls in the patients with thyroid dysfunction. The relative functional affinity of M antibodies did not differ between controls and patients with hypothyroidism but declined 5 and 10-12 months after delivery compared to values at 2 months. These results do not support the suggestion that the amount of IgG1 subclass M antibodies particularly determines the course of postpartum thyroiditis. Rather, the total M antibody level, in all four subclasses, is associated with clinical outcome. Resolution of the disease, despite persisting M antibodies, may occur in part because the relative functional affinity of these antibodies declines after delivery.     

Diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by ELISA for anti-pseudomonas LPS IgG antibodies

An enzyme-linked immunosorbant assay (ELISA) with urease enzyme was developed with either a polyvalent pseudomonas smooth lipopolysaccharide (LPS) extract vaccine (PEV-02) or rough LPS (R-LPS) from P. aeruginosa rough mutant PAC605. Each ELISA was able to differentiate between sera from cystic fibrosis (CF) patients chronically colonized with P. aeruginosa and sera from non-colonized patients. Sera from non-colonized and intermittently colonized CF patients seldom reacted with any of the Pseudomonas LPS, whereas sera from chronically colonized CF patients reacted strongly with most of the sixteen smooth O-serotype vaccine components and with the PAC605 R-LPS, indicating the presence either of a number of different serotype specific IgG antibodies and/or IgG antibodies directed to a common antigenic component of LPS rough core. Absorption studies and immunoblot analysis demonstrated that in sera from CF patients who were chronically colonized with P. aeruginosa a significant component of the anti-P. aeruginosa antibodies is specific for the core of P. aeruginosa LPS and cross reactive with a number of serotypes of P. aeruginosa LPS.     

Evaluation by enzyme-linked immunosorbent assay of IgG anti-D and IgG subclass concentrations in immunoglobulin preparations

BACKGROUND: Anti-D immunoglobulin preparations are injected to prevent hemolytic disease of the newborn. The concentration of IgG anti-D in these preparations is usually determined by an automated hemagglutination technique using as a reference a calibrated preparation of anti-D, but the method requires special equipment and cannot be routinely applied to measure the IgG subclasses of anti-D in these preparations. STUDY DESIGN AND METHODS:Taking advantage of a recently described enzyme-linked immunosorbent assay (ELISA) for the determination of the anti-D concentration in sera of alloimmunized pregnant women, IgG anti-D and IgG subclass concentrations were measured in the international reference preparation (IRP) coded 68/419, 10 anti-D immunoglobulin preparations, and sera of 15 D-immunized volunteers. RESULTS: An IgG anti-D concentration of 61.5 +/- 4.8 microg per ampoule (mean +/- SD) was found by ELISA in IRP 68/419.This result was in agreement with previous determinations obtained by radioimmunoassay (60 microg/ampoule). The IgG subclass concentration of anti-D in this preparation was 48.4 microg of IgG1 (78.6%), 3.0 microg of IgG2 (4.8%), 9.7 microg of IgG3 (15.8%), and 0.4 microg of IgG4 (0.7%). The mean proportion of IgG subclasses of anti-D in 10 immunoglobulin preparations was similar (81.7% for IgG1, 5.0% for IgG2, 12.7% for IgG3, and 0.6% for IgG4). In the sera of 15 immunized volunteers, the IgG anti-D concentration varied from 3.1 to 68.4 microg per mL. The mean IgG subclass composition of anti-D was 79.3 percent for IgG1, 2.2 percent for IgG2, 18.1 percent for IgG3, and 0.4 percent for IgG4. The proportions of IgG3 anti-D in these sera were found to range between 1 percent and 87 percent, as in the sera of D-alloimmunized pregnant women. CONCLUSION: ELISA provides an alternative to the radioimmunoassay and the automated hemagglutination technique. In addition, it allows the evaluation of the absolute concentration of each IgG subclass of anti-D in immunoglobulin preparations and necessitates only the conventional equipment required for an immunoenzymatic assay.     

Quantitation of human IgG by rocket immunoelectrophoresis at pH 5 by use of carbamylated antibodies. A routine laboratory method

Human IgG from 24 normal sera, and from 24 sera containing IgG M-components, were quantitated by rocket immunoelectrophoresis at pH 8.6 using carbamylated antigen as described by Weeke, and at pH 5.0 using carbamylated antibodies. The latter method which is introduced in this paper gives results equal to Weeke's method and hence to the single radial immunodiffusion, the single linear immunodiftusion and the rocket method performed at pH 8.6. The main advantages of utilizing carbamylated antibodies: for quantitation of IgG in routine laboratory work is (a) the method is fast (3 h), (b) it does not involve chemical modification of the antigen and (c) it can be applied to both polyclonal and monoclonal IgG.     

Serologic and IgG subclass characterization of Cartwright (Yt) and Gerbich (Ge) antibodies

Examples of anti-Yta and anti-Ge were tested for reactivity with red cells treated with proteolytic enzymes and with antisera of known IgG specificity. Eight of 14 examples of anti-Yta did not react as well with cells treated with either papain or ficin; treatment with trypsin did not reduce reactivity. Four examples of the anti-Yta were IgG4; the others tested did not react with IgG subclass antisera. Of 13 examples of anti-Ge, seven failed to react with red cells treated with trypsin, ficin, or papain. Nine examples were IgG1, one was IgG1 plus IgG3, and three did not react with IgG subclass antisera.     

Laboratory diagnosis of specific antibody deficiency to pneumococcal capsular polysaccharide antigens

BACKGROUND: Measurement of postimmunization antibody response to pneumococcal capsular polysaccharide (caps-PS) is the standard method to identify deficiency of antipolysaccharide antibody production. However, no standardized criteria have been defined for classification of patients into responders or nonresponders to caps-PS. METHODS: We vaccinated 37 healthy children and 39 healthy adults with Pneumovax and measured the anti-caps-PS antibody response to 5 serotypes. We also measured antipneumococcal antibody titers in 82 patients with increased susceptibility to airway infection. The ELISA was performed according to the 3rd-generation assay format. RESULTS: The lower 5th percentile (cutoff) concentrations for the postimmunization antibody titer in healthy individuals were 0.67 mg/L, 0.45 mg/L, 0.46 mg/L, 0.31 mg/L, and 1.04 mg/L for serotypes 3, 4, 9N, 18C, and 19F, respectively. In 96% of healthy individuals, antibody responses higher than the cutoff concentration were seen for at least 3 of the 5 serotypes. Nine of 82 patients (11%) failed to mount an adequate antibody response for at least 4 of the 5 serotypes tested, whereas only 1 control (1.3%) failed to do so. CONCLUSION: The cutoffs for antibody responses to caps-PS identified in this study appear useful for identifying individuals with an inadequate response to vaccine.     

Quantitation of expression of idiotopes recognized by a panel of monoclonal antibodies in normal serum immunoglobulin: use of a novel 4-stage ELISA assay

This paper describes the application of a sensitive ELISA assay detecting as little as 10 ng/ml of a specific idiotope. Using this assay we were able to divide monoclonal anti-idiotypic antibodies generated using a single IgG1 lambda paraprotein as the immunogen into three distinct groups. Seven antibodies detected determinants which were expressed only by the immunogen. The remaining seven antibodies all reacted with normal serum immunoglobulin. Four antibodies reacted with "public" idiotopes which were strongly expressed by serum immunoglobulin, and three antibodies reacted with "restricted public" idiotopes which were only weakly expressed by serum immunoglobulin. This last group may be of significant interest as agents for the immunotherapy of B-cell malignancies. The conservation of expression of these idiotopes in many individual sera suggests that they have a physiological role in immune regulation and would therefore be excellent targets for immunotherapy of B-cell tumours while their quantitatively low expression by circulating immunoglobulin is unlikely to interfere with tumour cell specific binding in vivo.