Microparticle-enhanced nephelometric immunoassay of anti-thyroid peroxidase autoantibodies in thyroid disorders.

Crude thyroid peroxidase extracted from human thyroid microsomes was covalently bound onto polyacrylic and polyfunctional copolymerized microparticles. We observed agglutination of the thyroid peroxidase-microparticle conjugate with 13 monoclonal antibodies (mAbs) specific for epitopes on four different antigenic domains of human thyroid peroxidase (TPO; EC 1.11.1.7), after addition of anti-mouse immunoglobulins. We quantified agglutination by measuring with a specially designed nephelometer the light scattered by the conjugates. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for human anti-TPO autoantibodies (aAbs) with defined epitopic specificity, based on the ability of aAbs to inhibit mAb-induced agglutination. Applied to patients with autoimmune thyroid diseases, this assay confirmed the polyclonality of anti-TPO aAbs and their preferential reactivity toward epitopes located on the A and B antigenic domains of the TPO molecule. The same specificities seem to be present in patients with Hashimoto thyroiditis or Graves disease.

Interactions of complement fraction C1q, fibronectin, and immunoglobulin G with polyacrylic microparticles used as solid-phase in immunoassay.

A microparticle-enhanced nephelometric immunoassay was recently described, where polyacrylic, hydrophilic, and polyfunctional microparticles are used as the solid phase. It is a one-step immunoassay based on the nephelometric quantification of microparticle agglutination. In such assays, the measurement of analytes at low concentration may be impaired by the need of using undiluted biological samples. This leads to work with high concentrations of several proteins liable to interfere with the agglutination process. In this paper, we report on a study performed with human serum and purified proteins, which were assayed by classical analytical methods. This work identified three major components of human serum specifically involved in yielding polyacrylic microparticle instability: complement fraction C1q, fibronectin, and immunoglobulins G. In this order of importance, they all showed a marked ability to be adsorbed on the microparticle's surface. Pretreatment of human serum with microparticles decreased the concentrations in C1q (82%), fibronectin (16%), and immunoglobulin G (4%) very unequally. However, it allowed the elimination of microparticle instability, consequently providing the possible use of such polyacrylic microparticles in a one-step nephelometric immunoassay of analytes at low concentration in biological samples, without washes or phase separation.

Use of microsphere-antibody conjugates in microparticle-enhanced nephelometric immunoassay.

A new microparticle-enhanced nephelometric immunoassay has been recently described as a sensitive, accurate, and easy-to-perform competitive immunoassay for various analytes. As initially described, this test is based on the nephelometric quantification of the inhibition, by the antigen to be assayed, of immunoagglutination of microparticle-antigen conjugates. Its applicability as a competitive immunoassay is thus limited by the necessary availability of pure antigens to prepare microparticle-antigen conjugates. In this paper, we report an adaptation of this initial test, where microparticles are coated by monoclonal antibodies, eliminating the need for purified antigens. The new configurations of particle agglutination-based immunoassays described include use of these microparticle-antibody conjugates with microparticle-antigen conjugates, free antigen, and anti-mouse immunoglobulins antiserum. The feasibility of such configurations is studied with human chorionic gonadotropin hormone, human thyroid stimulating hormone, and human myoglobin as antigens. Capture of the analyte by microparticle-antibody conjugates is evidenced by inhibition of their agglutination with microparticle-antigen conjugates and by agglutination in a sandwich assay with a complementary monoclonal antibody or polyclonal antiserum. The use of a second xenogenic antibody enhances the agglutination process and increases the assay sensitivity. Microparticle-antibody conjugates may extend the applications of microparticle-enhanced nephelometric immunoassays to unavailable analytes.

Detection of antithyroglobulin autoantibodies with defined epitopic specificity by a microparticle-enhanced nephelometric immunoassay.

To hydrophilic, polyfunctional spherical microparticles of predetermined diameter, produced by copolymerization of acrylic monomers, we covalently bound human thyroglobulin. The thyroglobulin-microsphere conjugate was agglutinated, in the presence of antimouse immunoglobulins antiserum, by four monoclonal antibodies, each recognizing a different antigenic domain on the thyroglobulin molecule. These agglutinations were quantified by measuring with a specially designed nephelometer the light scattered by clusters of the conjugates. Agglutination with the monoclonal antibody recognizing antigenic domain II of the thyroglobulin molecule was specifically inhibited by some human sera that contained antithyroglobulin autoantibodies. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for these autoantibodies with defined epitopic specificity. Using this assay, we detected and quantified antithyroglobulin autoantibodies in serum samples from all eight patients examined with Hashimoto disease and from most (75%) patients with untreated Graves disease.

Synthesis of new hydrophilic microspheres: optimized carriers for microparticle-enhanced nephelometric immunoassays.

To optimize antigen-antibody reactions, we have synthesized chemically well-defined hydrophilic microspheres. Proteins or haptens were covalently linked to these carriers. When the microsphere conjugates were agglutinated by the corresponding antiserum, the size of the complex artificially increased during the immunological reaction. After optimizing various parameters such as the hydrophilic character, repulsion charges, and amount of antigen coupled to the microspheres, we developed a rapid and sensitive immunoassay based on laser light scattering by the complexes.

Measurement of eleven serum proteins by microparticle-enhanced nephelometric immunoassay.

Eleven proteins (immunoglobulins IgG, IgA, IgM, orosomucoid, alpha 1-antiproteinase, haptoglobin, ceruloplasmin, C-reactive protein, transferrin, prealbumin and alpha 2-macroglobulin) in human serum were quantitated by a new microparticle-enhanced nephelometric immunoassay. This is a one step competitive assay, based on the nephelometric measurement of light scattered by clusters of protein-coated microparticles specially synthesized for that use. Statistical evaluation (precision, recovery and method comparison) shows that the determination of serum proteins is reliable and accurate for wide ranges of concentration and that the method is quite adequate for strongly increased concentrations. This microparticle-enhanced nephelometric immunoassay appears to offer an alternative method for routine measurement of a great variety of serum proteins at high and intermediate concentrations, which are usually quantified by radial immunodiffusion or conventional immunonephelometry. On account of its sensitivity, it can also be used for the determination of relatively low concentrations of analytes.

Microparticle-enhanced nephelometric immunoassay with microsphere-antigen conjugates.

gamma-Irradiation of acrolein and other acrylic monomers allowed the synthesis of spherical polyfunctional hydrophilic microparticles in the size range of 50 to 300 nm, on which antigens (immunoglobulins G, chorionic gonadotropin hormone, prealbumin) could be covalently bound. Microsphere-antigen conjugates clustered together in the presence of specific antiserum or monoclonal antibodies and their agglutination was quantified by light-scattering measurement performed with a specially designed nephelometer. Essential factors concerning the conjugate agglutination and its quantitation (size of microsphere, amount of antigen bound on microsphere, concentration of conjugate, concentration of agglutinating reagent, angle of light-scattering observation) were successively studied. A microparticle-enhanced nephelometric immunoassay for prealbumin was finally developed as an example of application. It was based on the inhibition of the immunoagglutination of microspheres-prealbumin conjugate by free prealbumin. This prealbumin immunoassay was easy to perform (one-step assay without washing or phase separation), fast (30 min), reliable (variation coefficients ranged from 3.6% to 7.5% for within- and between-assay determination), and sensitive (1 microgram/L detected). It was correlated with conventional immunonephelometry and radial immunodiffusion (correlation coefficients, 0.98). Microparticle-enhanced nephelometric immunoassay offered many advantages over the last two methods. Its better sensitivity allowed a lower reagent consumption and a larger sample dilution (contrary to the conventional immunonephelometry, sample pretreatment and sample blank measurement were unnecessary). Its inhibition mode induced a total accuracy for sample with high analyte concentration (a risk of underevaluation in antigen excess conditions existed in all method based on a noncompetitive antigen-antibody reaction) and provided the possibility to quantify haptens.(ABSTRACT TRUNCATED AT 250 WORDS)

Microparticle-enhanced nephelometric immunoassay for human C-reactive protein.

Polyfunctional hydrophilic microspheres of 125-nm diameter can be produced by copolymerization of acrylic monomers. Purified c-reactive protein (CRP) was covalently bound to these new micropheres, and the conjugate obtained was used as reagent in a microparticle-enhanced nephelometric immunoassay for human CRP. This assay was based on the measure, with a specially designed nephelometer, of the light scattered by aggregates formed during the immunoagglutination of the conjugate with anti-CRP antiserum. Sensitive inhibition of this agglutination by free CRP (6 ng/ml) allowed CRP quantitation in highly diluted serum samples (1/500-1/2,000), excluding any interference or sample pretreatment. The CRP assay was easy to perform (no washing or phase separation), reliable (coefficients of variation ranged from 1.3% to 9.3% for within-run and between-run determinations), and accurate (mean percentage of recovery: 104%; correlation coefficients with accepted analytical methods greater than or equal to 0.97) over a large range of concentrations. The inhibition mode excluded errors in the antigen excess zone and provided total security at high concentrations.

Development of a rapid microparticle-enhanced nephelometric immunoassay for serum myoglobin in acute myocardial infarction.

A microparticle-enhanced nephelometric immunoassay was developed for myoglobin quantitation in human serum. It uses rabbit antimyoglobin serum and hydrophilic polyacrylic microparticles covalently coated with baboon myoglobin in a competitive immunoagglutination system. The level of microparticle agglutination is assessed with a specially designed nephelometer. This sensitive (45 ng/ml of myoglobin detected in serum) and accurate (coefficients of variation from 3.0% to 8.2% in precision study and linear recovery of myoglobin in overloaded sera) immunoassay was evaluated with human sera from patients suffering from acute myocardial infarction. Myoglobin levels in patient's serum on admission appeared to be correlated with clinical and biological parameters assessed in emergency wards and later. This new, rapid, and easy microparticle-enhanced nephelometric immunoassay could thus be useful in emergency conditions for the early quantitation of serum myoglobin.

Polyacrylic microspheres as a solid phase for microparticle enhanced nephelometric immunoassay (NEPHELIA (R)) of transferrin.

Polyfunctional hydrophilic microspheres (MS) can be produced by copolymerisation with gamma-irradiation of acrylic monomers. Transferrin (TRF) can be covalently bound to these MS by reaction between aldehyde groups of the MS and primary amino groups of the protein. MS-TRF conjugates thus obtained are agglutinated by specific antiserum and this agglutination is inhibited by free TRF. Agglutination and inhibition are quantified by measurement of the light scattered by MS-TRF conjugate clusters with a specially designed nephelometer, a process designated as microparticle enhanced nephelometric immunoassay (NEPHELIA (R)) for TRF. Recovery, correlation and reproducibility studies, simultaneously performed in three different laboratories, show that this TRF immunoassay is accurate for a large concentration range. NEPHELIA (R) may appear as an alternative method for a large variety of molecules.

A microparticle enhanced nephelometric immunoassay (Nephelia) applied to thymulin measurement.

This article describes a microparticle enhanced nephelometric immunoassay (Nephelia) applied to the quantification of the thymic peptide hormone thymulin. Nephelia uses antibody recognition by the antithymulin antiserum in a competitive reaction between free thymulin and thymulin bound to the microspheres. The binding between microsphere and thymulin is achieved with the aid of a protein carrier. The sensitivity of the competitive reaction varied with the protein carrier and the antiserum sample. The most efficient reaction was obtained with the thymulin-metallothionein-microsphere conjugates; as little as 5 pg/ml of thymulin could be detected. In adult human serum or in its ultra-filtrate, spiked synthetic thymulin was totally recovered. Measurement of thymulin in serum or ultra-filtrate samples demonstrated the presence of interference from molecules in the serum. Nephelia led to the same conclusions as those reported with other methods and performs as well, and is more simple to use than radio- or enzymo-immunoassays for thymulin measurement.

[In vitro study of the cytocompatibility of 3 biological glues]. / Etude in vitro de la cytocompatibilité de trois colles biologiques.

Human derived fibrin glues are being used with increasing frequency in periodontal surgery. Three different fibrin glues were tested in an in vitro system for gingival fibroblast proliferation. The results show a cytocompatibility of these products which justifies their employment in periodontal therapy.

Thymulin (facteur thymique serique) and zinc contents of the thymus glands of malnourished children.

Protein-energy malnutrition (PEM) leads to an immune deficiency, which is now well documented. Some investigators have suggested that the associated zinc deficiency is important in thymic involution and changes in cellular immunity. To evaluate the respective roles of nutritional deficiency, infection, and zinc in the alteration of thymic function, we measured the amounts of thymulin (facteur thymic serique, or FTS) and of Zn in the thymus glands of 58 Senegalese children who died in various stages of malnutrition. In the severe forms (marasmus, kwashiorkor, and marasmic kwashiorkor) the thymus was tiny and contained very little thymulin. The Zn content of the thymus was high whatever the nutritional state of the subject and was related significantly only to the presence of infections. In Senegalese children thymic atrophy and depleted thymulin content are associated with severe PEM but not systemic infection or depleted thymic Zn content.

[Current pathogenetic data on mesangial glomerulonephritis with immunoglobulin A deposits]. / Données pathogéniques actuelles sur les glomérulonéphrites mésangiales à dépôts d'immunoglobulines A.

The pathogenesis of mesangial immunoglobulin A nephropathies was elucidated through the analysis of their clinical features and investigation of their biological characteristics. An alteration of the mucosal immune system is currently considered chiefly responsible for these diseases. A dysregulation of other compartments of the immune system is likely to enhance this abnormality and/or contribute to the persistence of immune complexes. Little is known, however, about the etiology of these renal diseases, and their treatment remains palliative or preventive.

Histoimmunological discrepancies in primary IgA nephropathy and anaphylactoid purpura sustain relationships between mucosa and kidney.

Immunohistomorphometric analysis of the tonsils from a series of patients with anaphylactoid purpura or Berger's disease showed abnormal proportions of gamma- and alpha-isotype-producing cells. Conversely, renal immunofluorescence (IF) performed on kidney biopsies from 2 patients with hematuria and clinical symptoms suggestive of one or the other of these conditions failed to confirm the suspected diagnosis. Immunohistomorphometric analysis of these patient's tonsils showed normal proportions of gamma- and alpha-isotype-producing cells. These negative results suggest that strong relationships exist between mucosal abnormalities and renal deposition of IgA in hematuria.

Allogeneic bone marrow transplantation in man: in vitro activity of FTS-Zn on T-cell markers and functions.

Thymulin (FTS-Zn) is a synthetic metallo-nonapeptide similar to the serum factor of thymic origin FTS, which induces the maturation of lymphoid cells. The activity of this compound on peripheral blood mononuclear cells from 12 bone marrow recipients was studied in vitro. It was demonstrated that thymulin was able to induce or modulate the expression of T-cell membrane markers, to enhance the proliferative responsiveness of lymphocytes to mitogens or allogeneic cells, and to increase mononuclear cells' natural killer activity. This in vitro responsiveness was contemporary to a transient decrease of FTS levels in the patients' serum, documented by sequential assays. These results suggest that thymulin could be of interest as a prophylactic therapy to speed up the immunological reconstitution of bone marrow recipients.

IgA nephropathy: dimeric IgA-secreting cells are present in episcleral infiltrate.

Recent publications have reported a frequent association between IgA nephropathy and episcleritis. In this article, the authors report on the immunohistologic study of an episcleral biopsy obtained in a female patient with Berger's disease and frequent episodes of episcleritis. Numerous dimeric-IgA-secreting cells were demonstrated in the episcleral inflammatory infiltrate. It also should be mentioned that microhematuria often occurred within a few days after the beginning of episcleritis in this patient. These results suggest the participation of ocular mucosal immunity in some cases of IgA nephropathy.