Measurement of immunoglobulin G and M antibodies to type 3 pneumococcal capsular polysaccharide by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to measure immunoglobulin G (IgG) and IgM type 3 antipneumococcal capsular polysaccharide antibodies. The use of Fab2 fragments of rabbit antipneumococcal IgG antibody in the antibody-antigen sandwich increased the sensitivity for measuring IgM antibodies and decreased background activity in antigen-free cuvettes. This methodology detected type 3 IgM antibody responses in six of six subjects vaccinated with polyvalent pneumococcal vaccine and detected type 3 IgG antibody responses in three subjects. Results of the enzyme-linked immunosorbent assay and radioimmunoassay procedures were concordant, and postvaccination enzyme-linked immunosorbent assay IgM titers showed a stronger correlation with total radioimmunoassay antibody than did postvaccination ELISA IgG titers.     

Assay of antibody to group A streptococcal carbohydrate by enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay system for determination of antibody levels to the group A streptococcal cell wall carbohydrate antigen is described. Optimal conditions for antigen preparation, purification, and conjugation to poly-L-lysine for adequate adsorption to the solid phase are presented. Antibody titers of unknown sera were determined by comparison to known reference standard pool sera. A highly significant correlation (p less than 0.0001) was found between enzyme-linked immunosorbent assay antibody titers and antigen-binding capacity in a previously described radioimmunoassay. Utilizing an isotype-specific anti-immunoglobulin reagent and immunoabsorbent-purified antibody to group A streptococcal cell wall carbohydrate antigen, we were able to detect nanogram quantities of antibody by the enzyme-linked immunosorbent assay technique. This system will provide for more generalized use of group A streptococcal cell wall carbohydrate antigen antibody determinations for the study of immune responses after streptococcal infections and their complications.     

Antibody assay of rabbit sera for outer membrane vesicles ofHaemophilus influenzae by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) was applied to the detection of IgG and IgM antibodies against outer membrane vesicles (OMV) antigen ofHaemophilus influenzae type b. In this ELISA system, IgG antibody titers were about 40 fold higher than those in indirect hemagglutination assay (IHA). The IgG antibody titers by this ELISA of rabbit sera obtained after immunization were comparable with those by radioimmunoassay (RIA) of the same sera. A significant correlation was established between these two assays (r=0.973,P<0.001).Subject section: Bacteriology (infection and immunity)     

Detection of enterovirus specific IgG and IgM antibodies in humans by an indirect solid phase radioimmunoassay

The development of a solid phase radioimmunoassay which is able to detect virus-specific IgG and IgM antibodies in serum specimens from patients with enterovirus infections is described. Viral antigen partially purified from infected tissue culture fluid was adsorbed passively to individual polystyrene microtiter wells. Dilutions of sera were incubated on these antigens and bound anti-viral antibodies were monitored by the addition of 125-iodine labeled anti-human-IgG or anti-human-IgM antibody. Specificity of the assay to detect virus-specific IgM antibody was ensured by highly specific anti-IgM antibody which did not cross react with IgG and 2-mercaptoethanol sensitivity of IgM antibody titers. Changes of IgM antibody titers clearly indicated a current infection by that virus strain which was isolated as etiological agent. Advantages and restrictions of the introduced radioimmunoassay are discussed.This study was supported by the Bundesministerium für Forschung und Technologie (ZA/NT/SS 0313/6)     

A solid-phase enzyme immunoassay for anti-insulin antibody in diabetes mellitus patients

A solid-phase enzyme immunoassay for the measurement of anti-insulin antibodies in the sera of patients with diabetes mellitus was developed. Porcine insulin conjugated with bovine serum albumin was used for coating microtiter plates. This assay was as sensitive as the conventional radioimmunoassay. Anti-insulin antibody titers measured by the enzyme immunoassay and conventional radioimmunoassay correlated well and the correlation coefficient was 0.920, which was statistically significant (P less than 0.001). The enzyme immunoassay detected anti-insulin antibody in 23 out of 35 sera of patients with insulin-dependent diabetes mellitus. The present enzyme immunoassay does not require radioactive materials, is less expensive and is concluded to be practically useful.     

Antigenic Specificity of Neutralizing Antibody to Cholera Toxin

Selected rabbit antisera to cholera toxin antigens and convalescent cholera patient sera were analyzed using the permeability factor neutralization test and two sensitive in vitro serological assays specific for cholera toxin, cholera toxin A subunit, and cholera toxin B subunit. The results indicated that antisera to cholera toxin contained toxin-neutralizing activity as well as antibodies specific for both the A subunit and B subunit. It was clearly established that antisera to B subunit, devoid of significant anti-A subunit activity, neutralized the vascular permeability activity of cholera toxin. Antisera to A subunit contained neutralizing antibodies and antibodies to both A and B subunits. Absorption with B subunit removed both the toxin-neutralizing and anti-B subunit activities, while the anti-A activity was unaffected. Neutralizing antibody titers of rabbits immunized with B subunit were also observed to be significantly higher than neutralizing antibody titers of sera from A subunit-immunized rabbits, despite the overall similarity in anti-B subunit titers as determined by passive hemagglutination and radioimmunoassay of sera from the two groups of rabbits. Anti-alpha chain sera neither neutralized cholera toxin nor possessed significant antitoxin or anti-B subunit titers as determined by passive hemagglutination and radioimmunoassay. The anti-alpha chain sera contained high levels of antibody specific for A subunit, which is consistent with the hypothesis that the alpha chain is part of the A subunit structure. In contrast, the gamma chain was not shown to be antigenic. Sera from convalescent cholera patients possessed toxin-neutralizing antibody as well as passive hemagglutination and radioimmunoassay antibody against both A and B subunits.     

Enzyme-linked immunosorbent assay for detection of capsular antibodies against Haemophilus influenzae type b: comparison with radioimmunoassay

An enzyme-linked immunosorbent assay (ELISA) is described for detection of anti-capsular antibodies against Haemophilus influenzae type b. Polyribosephosphate was covalently bonded to poly-L-lysine before adsorption to microtiter plates. ELISA immunoglobulin G and immunoglobulin M anti-polyribosephosphate antibody titers were comparable to total anti-polyribosephosphate antibody concentration determined by radioimmunoassay. The ELISA technique will be useful for further investigations of host response to infections due to H. influenzae type b but is not intended to be used as a serological method for documenting H. influenzae type b infections.     

Enzyme-linked protein A: an enzyme-linked immunosorbent assay reagent for detection of human immunoglobulin G and virus-specific antibody.

A general-purpose reagent capable of reacting with immunoglobulin G in a modified enzyme-linked immunosorbent assay technique was prepared by using protein A coupled with horseradish peroxidase. The reagent detected low levels (0.003 to 1.0 microgram/ml) of human immunoglobulin G and was also applied in an enzyme-linked immunosorbent assay for titration of antibody to human cytomegalovirus. The antibody titers to human cytomegalovirus determined by enzyme-linked immunosorbent assay and by complement fixation were compared. The correlation coefficient between the two techniques was 0.85, but the enzyme-linked immunosorbent assay was 10 times more sensitive than complement fixation in terms of antibody titers detected.     

Intestinal and serum antibody responses in mice after oral immunization with Salmonella, Escherichia coli, and Salmonella-Escherichia coli hybrid strains.

After oral feeding of mice with avirulent Salmonella, Escherichia coli, or hybrid strains, only certain bacterial strains were able to multiply and persist within the small intestinal Peyer's patches. After oral vaccination alone, or oral priming and subsequent parenteral boosting, antibody class and titers were detected, using a radioimmunoassay on serum and intestinal fluid or a plaque-forming cell assay on spleens. Only those strains that persisted in the Peyer's patches stimulated the production of serum and intestinal immunoglobulin A antibodies against their respective O antigens. Nonpersistent strains were weakly immunogenic, and antibodies, when present, were largely non-immunoglobulin A and confined to the serum.     

Serological and immunochemical characterization of anti-PP1Pk (anti-Tja) antibodies in blood group little p individuals. Blood group A type 4 recognition due to internal binding.

Serum samples from 13 blood group little p individuals were tested by radioimmunoassay for their IgG antibody subclass distribution against the P, P1 and Pk antigens. There was no uniform subclass distribution pattern, although all but one had IgG3 antibodies against all the P system antigens tested. Studies were performed adsorbing anti-Tja serum sequentially to columns with synthetic carbohydrate antigenic determinants within the P system coupled to silica beads (SynsorbsR). The effect on agglutinin and indirect antiglobulin titers was determined after adsorption to SynsorbsR with different P-system antigens (P1, Pk, P). Adsorption to all the three SynsorbsR was needed to eliminate or strongly reduce antibody titers. The effect on IgM, IgG, IgA as well as IgG subclass antibody binding to P, P1 and Pk antigens was also determined by radioimmunoassay and chromatogram binding assay. Anti-PP1Pk antibodies from a little p woman with repeated abortions were shown to bind to glycosphingolipid antigens prepared from one of the aborted placentae using a chromatogram binding assay. This binding was eliminated by serum adsorption to SynsorbsR with P1, Pk and P carbohydrates. Anti-PP1Pk antibodies were also shown to bind to extended structures in the globoseries, i.e. globopentaosylceramide, globohexaosylceramide (globo-H) and globoheptaosylceramide (globo-A). This binding is most probably due to antibodies recognizing internal sequences in the carbohydrate chain. Attempts were made to visualize the binding epitope of the antibodies by computer molecular modelling.     

Characteristics of different solid-phase immunoassay formats for the measurement of BK virus immunoglobulin M in sera of patients on renal dialysis or with kidney allografts.

Solid-phase immunoglobulin M (IgM) antigen capture enzyme immunoassay (AgCEIA) and antibody capture enzyme immunoassay (AbCEIA) were developed for the diagnosis of BK virus (BKV) infections. Of 37 serum samples from renal allograft recipients, 15 were positive for BKV IgM antibody by either AgCEIA, AbCEIA, or antigen capture radioimmunoassay. False-positive IgM results were observed in the AgCEIA in the presence of high levels of BKV IgG antibody (titers greater than or equal to 1:51,200), when rheumatoid factor (RF) titers were greater than or equal to 1:20, or in the presence of high levels of RF (titers greater than or equal to 1:10,240) when BKV hemagglutination inhibition titers exceeded 1:40. False-positives due to RF could be eliminated by treatment of sera with anti-human IgG antisera or IgG-coated latex particles. The presence of RF did not, however, produce false-positive results in the AbCEIA. Both AgCEIA and AbCEIA were specific for BKV IgM antibody, as 14 serum samples containing either JC papovavirus, cytomegalovirus, rubella virus, hepatitis A virus, or hepatitis B virus core IgM antibody were negative in both EIAs. Comparison of results obtained for 37 serum samples revealed 14 positive by radioimmunoassay and 11 positive by both AgCEIA and AbCEIA. Both EIAs detected BKV IgM antibody in sera of renal allograft patients and patients on renal dialysis who had reactivated BKV infections persisting for several months after transplantation.     

Radioimmunoprecipitation Assay for Quantitation of Serum Antibody to the Hemagglutinin of Type A Influenza Virus

A double-antibody radioimmunoprecipitation (RIP) assay has been developed to provide a sensitive and specific measure of antibody to hemagglutinins of H3N2 influenza viruses. Chloramine T was used to radiolabel purified hemagglutinins to high specific activity without loss of antigenicity. The purity of the labeled hemagglutinin was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which also established that both the HA(1) and HA(2) polypeptides were iodinated. Radiolabeled hemagglutinins with a specific activity that did not exceed 12 muCi/mug of protein could be maintained for up to 30 days at -70 degrees C in the presence of supplemental protein. The RIP assay was compared with conventional methods, hemagglutination inhibition and viral neutralization tests, using H3N equine 1 hybrid viruses for determining serum antihemagglutinin antibody titers. The geometric mean titers for human convalescent sera after infection with A/England/72 virus were 118, 161, and 18,822 for hemagglutination inhibition, viral neutralization, and RIP tests, respectively, and the three tests demonstrated significant rises in antihemagglutinin antibody titers with equal efficiency. In general, a positive correlation existed between antihemagglutinin antibody titers determined by these three procedures; however, the antibody level determined by RIP assay for each individual could not be related to hemagglutination inhibition or viral neutralization titers by a constant factor. A similar lack of a constant relationship was found by using hyperimmune guinea pig antisera, which suggests that the RIP assay can detect antibody populations that exhibit differing efficiencies for inhibition of viral hemagglutination and replication.     

Human antibody titers to Epstein–Barr Virus (EBV) gp350 correlate with neutralization of infectivity better than antibody titers to EBV gp42 using a rapid flow cytometry-based EBV neutralization assay

Measurement of neutralizing antibodies to Epstein–Barr virus (EBV) is important for evaluation of candidate vaccines. The current neutralization assay is based on antibody inhibition of EBV transformation of B cells and requires 6 weeks to perform. We developed a rapid, quantitative flow cytometry assay and show that neutralizing antibody titers measured by the new assay strongly correlate with antibody titers in the standard transformation-based assay. Antibodies to EBV gp350 and gp42 have been shown to block infection of B cells by EBV. Using new assays to quantify antibodies to these glycoproteins, we show for the first time that human plasma contains high titers of antibody to gp42; these titers correlate with neutralization of EBV infectivity or transformation. Furthermore, we show that antibody titers to EBV gp350 correlate more strongly with neutralization than antibody titers to gp42. These assays should be useful in accessing antibody responses to candidate EBV vaccines.     

An immunofluorescence assay for the detection of parvovirus B19 IgG and IgM antibodies based on recombinant viral antigen

An indirect immunofluorescence assay for serum IgG and IgM antibodies to human parvovirus B19 was established using recombinant B19 viral antigen, the capsid protein VP1, which had been produced in a baculovirus expression system. This protein gives a strong and characteristic signal in the immunofluorescence assay, making it a suitable candidate for this test system. The test results showed a good correlation with results obtained with a solid-phase capture radioimmunoassay (Cohen et al., 1983). 76% of sera from a random selection of blood donors were positive for B19 IgG which agrees with previous findings. The course of the IgM and IgG antibody response to B19 infection could be followed with the immunofluorescence assay by determining the titers of series of sera taken after a recent B19 infection. Investigation of 24 sera containing rubella-specific IgM showed no cross-reactivity with the recombinant B19 VP1 used in this test system. The test described here has the advantage of being based on a renewable source of antigen and will be further evaluated for routine diagnostic use in comparison with radioimmunoassay.     

Detection by radioimmunoassay of antibodies in human smallpox patients and vaccinees.

A radioimmunoassay procedure was developed for determining smallpox and vaccinia antibodies in human sera. The test detected and measured both primary and secondary immune responses in persons infected with variola virus or vaccinia virus. The antibody titers obtained by complement fixation, hemagglutination inhibition, plaque reduction neutralization, and radioimmunoassay methods were compared. In sequential serum specimens, the radioimmunoassay test indicated fourfold or greater increases in all of the smallpox patients and in six of eight vaccinated persons. Both the complement fixation and the hemagglutination inhibition tests were less effective. In persons who had been vaccinated, radioimmunoassay and plaque reduction neutralization tests appeared to measure the same immune response. However, in smallpox patients the immune response was readily detected by radioimmunoassay, whereas an immune response was not detected by the plaque reduction neutralization test when vaccinia virus was the antigen in the test system. Radioimmunoassay is an operationally simple procedure which provides objective and quantitative end-point titers in serological determinations.     

Detection of virus-specific immunoglobulins using a doubly labeled fluorescein-125I antibody.

Commercially prepared fluorescein-labeled antihuman antibodies were labed with 125I and used to compre specific herpes simplex virus antibody titers as determined by indirect fluorescent antibody and radioimmunoassay techniques. Total virus-specific immunoglobulin and virus-specific immunoglobulin G titers did not vary by more than one twofold dilution when compared by the two methods.     

Recombinant parvovirus B19 capsids as a new substrate for detection of B19-specific IgG and IgM antibodies by an enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay for the detection of B19-specific IgG and IgM antibodies was established using B19 capsids synthesized in a baculovirus expression system. These B19 capsids, consisting of either coat protein VP2 alone or of both VP1 and VP2, have been shown to be similar to native virus in size and appearance. The results obtained for the detection of B19-specific antibodies showed good correlations with a radioimmunoassay which uses native B19 virus and an immunofluorescence assay based on insect cells expressing coat protein VP1. The course of the antibody response could be followed by determining the titers of sequential serum samples taken after a recent B19 infection. Both types of recombinant capsids form an excellent source of antigen for the detection of both B19 IgG and IgM antibodies and are a very promising substitute for native virus.     

A monoclonal antibody-based radioimmunoassay for the in vitro production of IgE by lymphocyte cultures

Very sensitive radioimmunoassay systems have been described for the measurement of IgE produced in cultures of human peripheral blood mononuclear cells. However, differing results have been reported when cultures from non-atopic donors are stimulated with pokeweed mitogen, which may be due to cross-reactivity of anti-IgE antibodies with IgG. A monoclonal antibody specific for the Fc region of human IgE, and two polyclonal affinity-purified antibodies to IgE were tested for binding to 125I-labelled IgE myeloma proteins and polyclonal IgG in a sensitive double antibody precipitation assay. The monoclonal antibody and one of the polyclonal antibodies bound only IgE, whereas the other polyclonal antibody bound a significant proportion of labelled IgG. A solid phase radioimmunoassay was developed which combined the specificity of the monoclonal antibody with the sensitivity of the first polyclonal antibody as radioactive tracer. A second assay system was also tested using the cross-reacting antibody as tracer. Supernatants of pokeweed mitogen-stimulated peripheral blood mononuclear cell cultures from non-atopic donors were examined for IgE synthesis using both assays. The assay based on the monoclonal antibody did not detect IgE synthesis, whilst the second assay, based on the cross-reacting antibody indicated that spurious IgE had been produced in the same cultures. This study shows that protein-binding assays provide a simple means for checking the specificity of antibodies in solid phase radioimmunoassays, and confirms that pokeweed mitogen does not stimulate IgE production by cells from non-atopic donors when measured by a specific radioimmunoassay.     

Development of a novel in vitro assay (ALS assay) for evaluation of vaccine-induced antibody secretion from circulating mucosal lymphocytes

We describe here a novel method for measuring in vitro antibody secretion from the tissue culture of human B lymphocytes in peripheral blood mononuclear cells (PBMC) after oral vaccination with a killed cholera vaccine. Enzyme-linked immunosorbent assay (ELISA) titers of the antibody secreted in the cell supernatant were determined. The validation results demonstrated that human PBMC remained viable and continued to secrete antibodies (total immunoglobulin A [IgA] and IgG) for up to 4 days of incubation at 37 degrees C with 5% CO(2) in cell cultures. The secreted antibody concentration correlated positively with the PBMC concentration and incubation time in the tissue culture and correlated negatively with the storage time of the whole blood at room temperature. In vitro assay of secreting antibody in the lymphocyte supernatant (i.e., the ALS assay) is capable of the detecting specific antibody response after oral vaccination with a killed whole-cell-plus-B-subunit cholera vaccine (WC-B) in healthy adults in a phase I clinical trial. Postimmunization PBMC secreted antibodies to cholera toxin in the cell supernatants. Antibody production did not require any in vitro antigen stimulation. In the ALS assay, antigen-specific antibody titers of prevaccination samples were barely detectable, whereas serum antitoxin ELISA titers in background of prevaccine samples were significantly higher than the ALS titers. We conclude that, without any in vitro antigen stimulation after vaccination, PBMC secrete antibodies into the supernatants in the ALS assay. This assay can quantitatively measure the antigen-specific antibody production from the PBMC culture in postvaccination blood samples.     

Enzyme-like immunosorbent assay (ELISA) for immunoglobulin G antibodies against insect venoms

IgG "blocking" antibodies were measured in patients receiving insect venom immunotherapy. The enzyme-linked immunosorbent assay (ELISA) described herein was found to be sensitive and reproducible. Results with ELISA correlated well with values obtained with a radioimmunoassay and with inhibition of the release of histamine from sensitive basophils. Also, specific antibody titers against phospholipase A and whole bee venom were correlated. Serial determinations of venom-specific IgG antibodies were made in 17 patients receiving Polistes wasp or bee venom immunotherapy. The majority of patients showed a rise in IgG antibodies, which peaked after administration of approximately 500 micrograms of venom. Only one out of 13 of these venom-treated patients had allergic symptoms after an insect sting while on maintenance therapy.