A mechanism for the induction of immunological tolerance by antigen feeding: antigen-antibody complexes.

We have previously reported on the induction, in mice, of a systemic (splenic) immune response with IgA as the dominant antibody, as a result of a short (4 day) intragastric immunization course with foreign erythrocytes. This response was followed by a prolonged period of hyporesponsiveness to similarly administered antigen. Here it is shown that this hyporesponsiveness is also manifested towards antigen given intraperitoneally, and that one is therefore dealing with tolerance, not with failure to absorb antigen from the gut. In contrast, mice primed parenterally and then challenged intragastrically behaved as if never having any previous contact with the antigen, i.e., with a primary-type splenic response of predominant IgA character. This agrees with our former conclusion that splenic responses to enterically absorbed antigen reflect colonization of the spleen by cells sensitized locally in the gut wall, a site not readily primed by the parenteral route. Serum from intragastrically immunized mice contained a very active tolerogen. In vivo, it was capable of conferring tolerance to nonimmune recipient mice. In vitro, it paralyzed the activity of antibody-producing cells. Inhibitory sera has weak antibody activity, restricted to the IgA class, and contained immune complexes reacting with rheumatoid factor but not with C1q. Elimination of these complexes by means by insolubilized rheumatoid factor abolished the tolerogenic effect. In conclusion, the enterically induced tolerogen seems to consist of immune complexes with IgA as the antibody.

A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes.

Antigens encoded by MAGE-A3 and recognized by T cells are interesting targets for tumor immunotherapy because they are strictly tumor specific and shared by many tumors of various histological types. A number of MAGE-A3 antigenic peptides presented by HLA class I molecules have been used in clinical trials, and regressions of melanoma metastasis have been observed. We report here the identification of a MAGE-A3 epitope, TQHFVQENYLEY, presented to CD4+ T lymphocytes by HLA-DP4 molecules, which are expressed in approximately 76% of Caucasians. This new epitope may be useful both for therapeutic vaccination and for the evaluation of the immune response in cancer patients. Interest ingly, the CD4+ T cells lysed HLA-DP4 tumor cells expressing MAGE-A3, indicating that this epitope, in contrast to other class-II MAGE-A3 epitopes, is presented at the surface of tumor cells. The study of this disparity in the presentation of two epitopes from the same protein may lead to a better understanding of the endogenous class II presentation pathway.

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.

Latex agglutination assay for human anti-Brucella IgM antibodies.

We report here the development of a homogeneous, easy, precise and rapid anti-Brucella IgM antibody (Ab) latex agglutination assay based on particle counting. The interference of IgG Ab was eliminated by the addition of anti-gamma Fc and free Bru-LPS. Anti-mu Fc mAb enhanced the agglutinating activity of IgM antibodies and improved the sensitivity of the test. The possible interference of rheumatoid factor was eliminated by adding human aggregated IgG. The assay is complete in 45 min, with an interassay variation of 9%. The assay correlates well (r = 0.94) with the accurate but time consuming capture ELISA.

Particle counting immunoassay (PACIA). III. Automated determination of circulating immune complexes by inhibition of an agglutinating factor of mouse serum.

A factor capable of agglutinating human IgG coated particles (latex) has been found in mouse serum. This factor (MAG) was used in an unpurified form to detect circulating immune complexes in the particle counting immunoassay (PACIA) system, which allows measurement of agglutination with great precision. MAG did not react with monomeric IgG, nor with reduced and alkylated aggregated IgG. It was inhibited by immune complexes in small antigen excess. Among the various subclasses of IgG, IgA and IgM, only IgG1 and IgM when coupled to Sepharose beads displayed an inhibitory activity towards MAG. That the inhibitory factors detected in serum were immune complexes or aggregated Ig was suggested by the correlation obtained with the amounts of 'heavy' IgG found in the serum samples by Ultrogel chromatography and by the polydisperse distribution of the inhibitory factors in the heavy fraction of gradient ultracentrifugation.

Particle counting immunoassay (PACIA). II. Automated determination of circulating immune complexes by inhibition of the agglutinating activity of rheumatoid sera.

Antigen--antibody complexes were detected in patients' sera by inhibition of the agglutinating activity of rheumatoid sera toward IgG coated particles (latex). Precision, sensitivity (1--10 microgram/ml equivalents of heat-aggregated IgG), and reproducibility (maximum coefficient of variation of 11%) were obtained by measuring agglutination with an instrument counting the residual free particles. Automation allowed testing of 20 to 40 samples per hour. The inhibitory activity of spontaneously agglutinating sera was determined after inactivating endogenous rheumatoid factor by reduction with dithiothreitol. Non-aggregated IgG did not significantly interfere. The agglutinating activity of 6 rheumatoid sera was tested after incubation with the various immunoglobulin classes and subclasses polymerized by coupling to agarose, and all were found to be readily absorbed by IgG1, but poorly by IgG3, IgA1 and IgM. Reactivity with IgG2, IgG4 and IgA2 clearly differed for different rheumatoid sera. Among 70 sera from blood donors, 7 had abnormally high inhibitory activity. Six of these had also an abnormal protein profile, suggesting existence of latent disease. High inhibitory activity in 15 sera out of 18 from patients with systemic lupus erythematosus, and in 23 sera out of 46 from patients with breast cancer suggested that the rheumatoid factor inhibition test has a discriminatory capacity comparable with that of more sophisticated techniques requiring radioisotopes and/or cellular material.

Immunological detection of mycobacterial antigens in infected fluids, cells and tissues by latex agglutination. Animal model and clinical application.

We devised an immunoassay for the detection of mycobacterial antigens in cell lysates and in tissue extracts which is based on the agglutination of latex particles coated with anti-Mycobacterium bovis F(ab')2, followed by counting of non-agglutinated particles. Mycobacterium bovis cell lysates were tested and a reference curve was established, having a lower limit of detection of 15-20 Mycobacteria. We were able to detect mycobacterial antigens in cell lysates from bronchoalveolar washings and in spleen and liver lysates obtained from experimentally infected rabbits. Antigens were also detected in ten out of 11 samples obtained from patients with proven tuberculous infection. These samples were readily distinguished from 32 negative control samples after pepsin treatment. In contrast, periodate treatment of samples to destroy carbohydrate, abolished all reactivity. Following gel filtration chromatography we identified three peaks with antigenic properties in samples of all types. The detection of mycobacterial carbohydrate antigens by latex agglutination and particle counting should be a useful adjunct in the diagnosis of tuberculosis.

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.

Particle counting immunoassay. IV. The use of F(ab')2 fragments and N epsilon-chloroacetyl lysine N-carboxyanhydride for their coupling to polystyrene latex particles.

Improved performance with the latex agglutination immunoassay, PACIA, is possible when the F(ab')2 fragments of antibody are chemically coupled at their hinge region to a protein-coated latex using a new coupling reagent, N epsilon-chloroacetyl lysine N-carboxyanhydride. This reagent, whether by orientating the complete antibody molecule or its F(ab')2 fragment or by preventing antibody denaturation, increased the sensitivity of the PACIA method. The use of F(ab')2 fragments eliminated most serum interference with the test.

Homogeneous latex immunoassay for thyroid hormone testing. Determination of thyroxine and triiodothyronine.

We describe here two latex immunoassays for total thyroxine (T4) and total triiodothyronine (T3). These homogeneous 60 min assays are quantified by optically counting the monomeric particles remaining after agglutination. When precision is assessed, both methods display coefficients of variation of 3-7% for within-run assays and 4-10% for between-run assays. The accuracy of the methods, as tested by dilution and spike recovery experiments, was found to be satisfactory. Two correlation studies were carried out to compare the present method with leading commercial methods. The coefficients obtained were: r = 0.92 and r = 0.93 with 150 sera for T3, and r = 0.95 (150 sera) and r = 0.93 (108 sera) for T4.

Particle counting assay for anti-toxoplasma IgG antibodies. Comparison with four automated commercial enzyme-linked immunoassays.

An assay for anti-toxoplasma IgG antibodies based on agglutination of latex particles was set up and compared with commercial immunoassays. The reaction was measured by instrumental counting of particles remaining unagglutinated. The running time was 45 min. This test (PaC) was compared using 243 serum samples with four automated commercial immunoassays: the Enzymum test Toxo IgG (ES300, Boehringer), the Vidas Toxo IgG (Biomérieux), the IMX Toxo IgG (Abbott), the Magia Toxoplasma gondii IgG (Merck). The mean values (+/- SD) obtained by IMX (25 IU +/- 68) and ES300 (45 IU +/- 142) were significantly lower than the values obtained by Vidas (73 IU +/- 237, p < 10(-4) and p = 0.006, respectively), by Magia (80 IU +/- 300, p < 10(-4) and p = 0.0005) and by PaC (70 IU +/- 260, p < 10(-4) and p = 0.0126). The correlations between PaC and Toxo IgG Boehringer, Biomérieux, Abbott, Merck were r = 0.97, r = 0.98, r = 0.94, r = 0.98, respectively. The correlation coefficients between the enzyme-immunoassays ranged from 0.96 to 0.99. All positive samples by PaC were found to be positive by enzyme-immunoassays except for eight sera which were doubtful positives by the Enzymum test ToxoIgG from Boehringer. No negative sample by PaC was found positive by any of the enzyme-immunoassays. In PaC, when two latex preparations coated with different antigen were compared, the correlation was rather weak (r = 0.93) suggesting that the selection of the antigen can be critical. In conclusion, the four automated commercial immunoassays now available gave similar results. However, the discrepancies observed in this study underlined the importance of clinical and biological follow-up of the patients and the necessity to confirm the result. The introduction of a new technique such as PaC, which is now available for a large variety of assays in Clinical Chemistry and Microbiology, is justified by its intrinsic advantage of homogeneity. Therefore, automation is easy as well as the control of possible interference.

Immune complexes in cerebrospinal fluid and serum of neurological patients. Possible intrathecal formation in bacterial meningitis and herpetic encephalitis.

We assayed immune complexes (IC) by Particle Counting ImmunoAssay in the serum and cerebrospinal fluid (CSF) of patients with various neurological disorders. In pyogenic meningitis, the levels of IC sharply increased 4-8 days after onset with a fall before the 10th day of the disease. In herpetic encephalitis the IC and antibody levels started to increase about 12 days after onset. The IC persisted at high values for 3-4 weeks, whereas the high antibody titers persisted for several months during the follow-up. In these 2 groups of patients IC were probably locally produced as indicated by the lack of correlation with the IC levels in the serum. We did not detect any significant increase of IC in the serum and CSF of patients with multiple sclerosis (N = 48) or with acute idiopathic polyradiculoneuritis (N = 11). Using another technique based on the determination of IgG and C4 in polyethylene glycol precipitates we also failed to detect any significant increase of IC in multiple sclerosis sera.

Receptor binding-like specificity of two anti-cholera toxin monoclonal antibodies detected by latex agglutination immunoassay.

Six mouse monoclonal IgG antibodies (mAbs) reacting with the cholera toxin B-subunit (CT-B) were obtained from the spleen cells of mice immunized with CT. They were divided into two groups according to their different reactivities with CT in latex (Lx) agglutination. One group of two mAbs was called "non-repetitive" (NR) because they apparently recognized a single epitope on CT, which was unable to agglutinate Lx coated with these NRmAbs, in contrast with the other group of four mAbs that we called "repetitive" (RmAbs). The two NRmAbs, but not the four RmAbs, failed to react with CT when CT was bound first to its GM1-receptor. Thus, the NRmAbs seemed directed at an epitope close to the GM1-binding site of CT-B. These NRmAbs could represent valuable immunogenic candidates for anti-idiotypic immunization against CT and related enterotoxins.

Longitudinal study of brucellosis in mice by immunoassay of lipopolysaccharide-related antigens in blood and urine.

Immunoassays based on latex agglutination or enzyme labelling (ELISA) were devised for the detection of lipopolysaccharide (LPS) of Brucella abortus, or its degradation products, in biological fluids of infected mice. The agglutination of latex was measured by counting of the remaining non-agglutinated particles in an automated immunoassay analyser. LPS was assayed by agglutination with antibody-coated latex and by competitive inhibition of agglutination of LPS-coated latex by anti-LPS antiserum. The inhibition system was more sensitive for the detection of degradation products of LPS. Correlation between ELISA and agglutination inhibition immunoassay was excellent (r = 0.96). Degradation of LPS occurred during storage, particularly when the samples contained specific antibodies. It could be prevented by removing cells immediately after collecting blood samples and by heating or alkaline denaturation of plasma. CBA/H mice were infected with various doses [65-(65 x 10(6) cfu] of B. abortus biovar 3 cells and the course of infection followed by immunoassay of LPS-related antigens in serum and urine, and by titration of specific antibodies and non-specific circulating immune complexes. The concentration of LPS degradation products, assayed by the agglutination inhibition assay, was related to the severity of the infection, which was assessed by viable counts of B. abortus in the spleen. A close correlation was observed between the values of antigenaemia, the number of cfu (r = 0.97), and the inoculum size (r = 0.99 at day 28).

Immunoassay of hepatitis B surface antigen by particle counting after pepsin digestion.

Particle counting immunoassay is based on latex agglutination, the reaction being measured by instrument counting of the particles remaining unagglutinated. Most interference which generally affects latex agglutination can be avoided by pepsin digestion of the sample, provided the antigen (Ag) of interest resists pepsin, which is the case of the hepatitis B surface antigen (HBsAg). Pepsin treatment has the additional advantage of inactivating antibodies and so releasing the Ag from immune complexes. We have set up an assay of HBsAg, proceeding in a prototype of Impact Instrument (Acade Diagnostic Systems, Belgium) at a rate of 60 samples.h-1 and a total running time of 2 or 4 h. This assay was compared with Abbott radioimmunoassay (RIA) in 706 consecutive patients (A) and 31 selected sera for which values close to the cut-off had been obtained by RIA (B). In A, 38 sera were found positive and 668 negative by both methods. In B, RIA after neutralization classified the samples as positive (n = 14), negative (n = 14), or dubious (n = 3). Complete agreement between latex and RIA was achieved for nine positive, 12 negative, and two dubious samples. Of five RIA-positive samples, two were classified as latex-negative and three as dubious in the latex assay. One sample dubious in RIA was found latex-positive and two RIA-negative samples were found, respectively, latex-positive and dubious; when retested after pepsin digestion, the first of them became RIA-positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Assay of anti-HBs antibodies using a recombinant antigen and latex particle counting: comparison with five commercial tests.

An assay of anti-HBs antibodies based on agglutination of latex particles coated with recombinant HBs-antigen was compared with Abbott radioimmunoassay (Abbott-RIA), which uses a human plasma-derived antigen. The population examined consisted of 76 Abbott-RIA anti-HBs-negative prevaccinated subjects and 1044 serum samples anti-HBs found positive by Abbott-RIA, including 283 samples of subjects vaccinated either with a human plasma-derived vaccine (group A; n = 180) or with a recombinant vaccine (group B; n = 103). Correlation coefficients between the two techniques were respectively r = 0.89 for the whole population (n = 1044), r = 0.98 in group A and r = 0.74 in group B. Anti-HBs titres were higher with latex than with RIA in group B as shown by the regression slopes: latex = 508 + 1.11 RIA in group A and latex = -1138 + 3.97 RIA in group B, suggesting that some vaccinated subjects from group B produced antibodies against epitopes proper to the recombinant antigen. In the prevaccinated population and in group A, the latex results were compared with those of radioimmunoassays (Abbott, Sorin) and enzyme immunoassays (Behring, Roche, Pasteur). Only the Roche-EIA detected anti-HBs in the prevaccinated subjects. The correlation between the various immunoassays was r greater than 0.96 only for values higher than 100 IU/l.

Detection of rifampicin and isoniazid resistances of Mycobacterium tuberculosis strains by particle counting immunoassay (PACIA).

The particle agglutinated counting immunoassay (PACIA) was used to determine the susceptibility of Mycobacterium tuberculosis strains to the two major antimycobacterial drugs, isoniazid and rifampicin. On evaluating 12 M. tuberculosis strains with different sensitivities, our results were in complete accordance with those obtained using the well-known BACTEC system. The PACIA technique is automated and quite inexpensive. Interpretation of the test may be achieved in as little as five days.

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.