A MAGE-1 peptide recognized on HLA-DR15 by CD4(+) T cells.

Antigens encoded by MAGE genes and recognized by T cells are of interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Several MAGE-1 peptide that are recognized by CD8(+) cytolytic T lymphocytes have been used in therapeutic vaccination trials. To obtain anti-tumor immune response, vaccines combining peptides recognized by CD8(+) and peptides recognized by CD4(+) T cells might be optimal. We focused therefore on the identification of MAGE peptides recognized by CD4(+) T cells. We report here the identification of MAGE-1 epitope EYVIKVSARVRF, which is presented to CD4(+) T lymphocytes by HLA-DR15. This HLA allele is present in 29 % of Asians and 17 % of Caucasians.

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.

Particle counting immunoassay for urinary cotinine. Comparison with chromatography, enzyme-linked immunoassay and fluorescence polarization immunoassay.

Urinary cotinine was measured according to its inhibitory activity on the agglutination of cotinine-coated latex particles by anti-cotinine antibodies, the agglutination being measured by optical counting of the remaining non-agglutinated particles (particle counting, PaC). The detection limit was 0.03 microgram/ml and the practical range extended from 0.03 to 3.9 micrograms/ml. The correlation results of 320 urine samples with those of high pressure liquid chromatography, enzyme-linked (Coti-Tracq EIA, Serex Inc., Maywood, NJ, USA), and fluorescence polarization immunoassay (TDX instrument, Abbott, Abbott Park, IL, USA) were r = 0.90, r = 0.69, r = 0.87, respectively, whereas the correlation coefficients between the assays other than particle counting ranged from 0.62 to 0.88. PaC does not require any separation step and can thus be easily automated.

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.

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.

Detection of mycobacterial antigens present in short-term culture media using particle counting immunoassay.

Particle counting immunoassay (PACIA) was compared with the BACTEC system for detecting mycobacterial growth after short-term culture and was used to identify M. tuberculosis. The latex particles were coated with polyclonal anti-BCG or with specific 2A1-2 monoclonal antibodies. Bottles containing nonradioactive Middlebrook 7H9 liquid medium and BACTEC 12B vials were inoculated with equal amounts of mycobacteria from four reference strains (M. tuberculosis, M. kansasii, M. avium, and M. xenopi). Using anti-BCG, PACIA detected mycobacterial antigens 3 to 6 days before the BACTEC system. M. tuberculosis was differentiated from the other mycobacteria using 2A1-2. Seventeen clinical samples were also studied. In the same 10, the two techniques detected mycobacteria, PACIA with anti-BCG after 9 days and BACTEC 1 to 5 days later. For 9 of the 10 samples, PACIA with 2A1-2 detected M. tuberculosis after 20 days, a result confirmed with the AccuProbe system. M. xenopi was biochemically identified in Specimen 10. Nonmycobacterial diseases were diagnosed in the 7 remaining unreactive specimens. We conclude that PACIA detects mycobacterial growth earlier than BACTEC and that M. tuberculosis can be distinguished from other mycobacteria in PACIA performed with specific monoclonal antibodies.

An interspecies idiotope associated with the anti-cholera toxin response detected by a monoclonal auto-anti-idiotypic antibody.

We have produced an auto-anti-idiotypic (auto-anti-id) monoclonal antibody (mAb) which reacted with syngenic mouse polyclonal anti-cholera toxin (anti-CT) IgG antibody (Ab) and six/eight different anti-CT IgG mAb, but not with normal mouse BALB/c IgG. The binding of this auto-anti-id mAb on one anti-CT mAb was significantly inhibited by polyclonal anti-CT sera of rats, rabbits and mice. Thus the idiotope on anti-CT Ab recognized by this auto-anti-id mAb was public and expressed in different species. Because of the absence of competition between CT and this auto-anti-id mAb for the binding to the six anti-CT mAb, the anti-id mAb was classified as an Ab2 alpha. The efficiency of this auto-anti-id mAb to induce anti-CT Ab3 was tested with success in rabbits and rats. Auto-anti-id mAb-immunized rats were significantly protected against an intraintestinal CT challenge.

Characterization of an anti-idiotypic MoAb bearing an internal image of the receptor-binding epitope of cholera toxin.

A mouse anti-cholera toxin (CT) MoAb, mAb1, specific for the GM1-binding epitope of CT, was used to raise a syngenic anti-idiotypic MoAb, mAb2. Purified mAb2 was specific for mAb1 as shown by latex particle counting immunoassay and ELISA. Several experiments of competition between mAb2 and CT for binding to mAb1 demonstrated that mAb2 bore an internal image of the GM1-binding epitope of CT. Binding of mAb2 to GM1 unambiguously corroborated the mAb1-paratopic specificity of mAb2. Furthermore, mAb2 acted as a CT-surrogate antigen: rabbits injected with mAb2 produced some anti-CT antibodies, Ab3, which resembled mAb1 in specificity as expected. The potential use of this mAb2 as vaccine or as prophylactic agent to prevent CT from binding to its cellular receptor is discussed.

Latex agglutination assay of human immunoglobulin M antitoxoplasma antibodies which uses enzymatically treated antigen-coated particles.

An assay of immunoglobulin M (IgM) antitoxoplasma antibodies which is rapid (less than 30 min), homogeneous, and reliable (interassay coefficient of variation, less than 11%) is proposed. Its principle is based on the observation that a suspension of latex particles coated with toxoplasma antigens, after treatment with proteinase K, becomes less agglutinable by IgG antibodies but more agglutinable by IgM antibodies. The difference between the activities of the two classes of antibodies is increased by the addition of a monoclonal antibody directed against the Fc region of IgM. Agglutination is measured with a special instrument which optically counts the particles that remain free after the reaction. Turbidimetric reading, although less sensitive, is also suitable. No significant interferences either by IgG antitoxoplasma antibodies or by rheumatoid factor or antinuclear antibodies were observed. The sensitivity was similar to that of the immunosorbent agglutination assay.

Protection of rat intestine against cholera toxin challenge by monoclonal anti-idiotypic antibody immunization via enteral and parenteral routes.

A mouse monoclonal anti-idiotypic (anti-id) immunoglobulin M (IgM) antibody, called MAb2, was raised against a mouse monoclonal anti-cholera toxin (anti-CT) antibody (MAb1). The MAb2 was shown, by competition with CT for MAb1, to bear the internal image of an epitope of CT. MAb2 immunization of rats was performed via the intraperitoneal, intragastric, and intrajejunal routes and compared with immunization of rats with either a control, isotype- and allotype-matched MAb or with CT via the same routes. Both serum IgG and bile IgA anti-CT Ab3's were detected by enzyme-linked immunosorbent assay in anti-id MAb2-immunized rats, although their titers were lower than those in CT-immunized rats. No anti-CT antibodies were detected in sera and bile of rats immunized with the control MAb. When tested for degree of gut protection against a CT challenge, rats immunized with MAb2 by the intrajejunal route showed a rather high degree of protection, which was only slightly lower than that of rats immunized with CT via the same route; all rats but one immunized with the control MAb were unprotected. There was, however, no correlation between serum or bile anti-CT titers and degree of gut protection in MAb2-immunized rats. Their serum anti-CT Ab3's were purified by adsorption and elution from a CT immunosorbent and resembled anti-CT MAb1 in their unique reactivity with MAb2. This constitutes to our knowledge the second report of protection against a pathogen by anti-id immunization via the enteric route.

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.

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.

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.

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.

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.

High sensitivity, homogeneous particle-based immunoassay for thyrotropin (Multipact).

We describe the first homogeneous, nonradioactive, high-sensitivity assay for human thyrotropin (TSH). The assay is based on particle immunoassay techniques, wherein 800-nm particles form the basis for the immunochemistry, delivery, and the detection technologies, respectively. Our assay also is the first to involve the use of fragmented monoclonal antibodies (to eliminate serum interferences) covalently coupled to particles without loss of their binding properties. Assays are performed in a semiautomated mode with use of a new modular system (Multipact). Equilibrium is reached in less than 2 h. Precision profile, sensitivity, and clinical studies indicate that the assay is accurate, has good precision at low concentrations, and that detection-limit characteristics compare well with those of a leading commercial high-sensitivity immunoradiometric assay (IRMA) for TSH. Dilution characteristics were satisfactory down to the assay's detection limit for a range of clinical samples. Correlation studies vs a reference IRMA method yielded the regression equation, present method = 0.976 (IRMA) + 0.002 milli-int. unit/L (r = 0.98), for 223 samples with TSH concentrations in the range 0 to 30 milli-int. units/L. For 40 samples with TSH less than or equal to 1.0 milli-int. unit/L it was: present method = 0.94 (IRMA) + 0.005 milli-int. unit/L (r = 0.96).

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 low concentrations of albumin in urine by latex particle counting.

We describe here a nonisotopic immunoassay, based on particle-counting technology, for the determination of urinary albumin. The assay takes only 35 min and has been fully automated on the IMPACT (Acade Diagnostic Systems, Brussels, Belgium) machine. The system measures albumin within a linear range between 6.25 and 50 mg/L and has a detection limit of 0.4 mg/L. Analytical recoveries at three concentrations ranged between 96% and 102%. Within-run precision ranged from 1.6% to 9.5%. The method was compared with a commercial nephelometric immunoassay system and a correlation coefficient of 0.996 was found for 216 urine samples. No antigen excess affects the shape of the curve in our system, whereas in nephelometry a 3 g/L solution of albumin starts to decrease the dose-response curve.

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)

Immunoassay of rheumatoid factor by latex particle counting.

We assayed rheumatoid factor by instrumental latex particle counting. The calibration curve ranged from 12.5 to 500 int. units/mL. Maximal within- and between-assay CVs were 5 and 11%, respectively. Analytical recoveries ranged from 92.4 to 108%, and the relation between results and dilutions was linear in the range of 15 to 400 int. units/mL. Correlation with an enzyme-linked immunoassay (ELISA, Cordia kit) was r = 0.934 (n = 58), with turbidimetry r = 0.825 (n = 100), and with the Waaler-Rose test r = 0.834 (n = 73). Of 260 blood donors, 95% gave a value less than 10 kilo-int. units/L, which was taken as the upper normal limit. In a population of patients with rheumatoid arthritis (n = 47), 87.2% had rheumatoid factor greater than 10 kilo-int. units/L.