Immunoadsorbent assay application for studies of protein mixtures and usage of paper carriers.

Proteins immobilized on insoluble carriers or immunoadsorbents are now widely used in many areas of biological research. They are especially efficient in studies of complex protein mixtures because immunoadsorbents are very useful for isolation of individual components for further investigation. Antibodies immobilized on paper retain their specific activity and can be applied as very sensitive measurement tool.

Combinatorial events in generation of antibody diversity.

Combinatorial association of immunoglobulin gene elements is the most important process in the creation of extreme diversity of antibody molecules. The recombination of germ-line variable gene elements V, D, and J can potentially generate approximately 6000 variable genes of human heavy chains. As joining of these elements is imprecise and is occurring with nucleotide additions or deletions, the created diversity is in fact much higher. The assembled variable genes can be revised and edited resulting in a change of their affinity and even specificity. Due to somatic hypermutation, the affinity of synthesized antibody increases even more. Another variant of combinatorial recombination is joining of complete variable genes with one of the several constant genes and the formation of various immunoglobulin isotypes with different effector functions but with the same antibody specificity. Consequently, these processes not only develop the antibody repertoire but also solve some other problems of the adaptive immune response.

A quantitative approach to the determination of antigen in immune complexes.

Immune complexes are present in the circulation of healthy individuals and the formation of such complexes is part of a normal immune process. During some pathological conditions, significant amounts of immune complexes are formed and deposited in the kidney and other tissues, causing severe injury. Since the levels of immune complexes can provide valuable prognostic information, dozens of methods have been developed to detect and quantify these complexes. However, many of these methods are non-specific, not quantitative, and give false-positive results. Methods based on detecting the antigen portion of immune complexes can yield more precise information about circulating immune complexes. We have used a quantitative dot-blot assay, which permits detection of antigen even if buried, to determine the levels of antigen in circulating immune complexes. In healthy donors, significant amounts of immune complexes containing DNA and beta(2)-glycoprotein I were detected (natural immune complexes). Natural immune complexes with Lewis X antigen were also observed in the circulation of healthy persons. In experimentally induced murine systemic lupus erythematosus (SLE) and SLE patients, there was a correlation between the clinical manifestations and the levels of DNA in the circulating immune complexes. At severe SLE flares, the level of DNA in circulating immune complexes decreased, probably due to tissue deposition of immune complexes. The low levels of DNA in immune complexes circulating in SLE patients correlated with low serum concentrations of the complement component C1q. No direct correlation was found between the levels of circulating anti-dsDNA antibodies and DNA in immune complexes. Thus, quantitation of antigen levels in circulating immune complexes can be used to determine the prognosis of autoimmune diseases.

DNA levels in circulating immune complexes decrease at severe SLE flares-correlation with complement component C1q.

The objective of this study was to investigate the relationship of DNA content in circulating immune complexes with disease course and activity in SLE. The DNA content in circulating immune complexes containing anti-DNA antibodies of IgG class was determined in serial samples from 28 patients with SLE by a quantitative immunochemical assay. The patients presented various active disease manifestations over 5-55 months. Disease activity (SLEDAI-score), drug treatment and ACR-criteria were recorded. Levels of anti-dsDNA, CRP, leukocytes, complement components C3, C4 and C1q were measured. Patients with severe flares and high SLEDAI scores had low Clq levels at onset of active disease manifestations. The patients with low C1q serum levels during flare (n=13) had significantly lower amounts of DNA in immune complexes than patients with normal Clq (P=0.001). Levels of DNA in immune complexes correlated with Clq at flares (r=0.62, P<0.0001) and correlated inversely with SLEDAI scores (r=-0.47, P=0.012). In conclusion, the low levels of DNA in circulating immune complexes found in severely ill SLE patients with concomitantly low serum concentrations of Clq prior to flares might be related to tissue deposition of immune complexes.

DNA levels in immune complexes circulating in mice with induced systemic lupus erythematosus.

The levels of DNA in IgG immune complexes, which appeared in the circulation of mice after the induction of systemic lupus erythematosus (SLE), were measured by an immunochemical quantitative assay using monoclonal anti-dsDNA antibodies. The amount of DNA in immune complexes was already high at 10-12 days following the injection of a human monoclonal anti-DNA antibody bearing the major idiotype designated 16/6 in complete Freund's adjuvant, i.e. long before the appearance of clinical manifestations. The injections of these antibodies in the alum-precipitated form did not induce the formation of DNA:anti-DNA complexes as well as SLE itself. The levels of DNA in circulating immune complexes were in general high throughout the whole experimental period (up to 7 months) decreasing gradually before the first clinical manifestations appeared and thereafter, when the disease was fully developed. Such a decrease could be explained by the retention of immune complexes in kidneys. The levels of DNA in immune complexes circulating in normal mice or in mice receiving injections of complete Freund's adjuvant was very low. Treatment of experimental SLE that affected the clinical manifestations prevented the formation of high levels of DNA containing immune complexes.

Beta2 glycoprotein I containing immune-complexes in lupus patients: association with thrombocytopenia and lipoprotein (a) levels.

In this study we determined the prevalence and clinical associations of immune-complexes-containing beta-2-glycoprotein I (beta2GPI) in randomly selected SLE patients. We studied 38 consecutive SLE patients attending the Rheumatology Unit. Previous arterial or venous-thrombosis were documented by the appropriate diagnostic test. Lipid profile including total cholesterol, LDL, VLDL, HDL and Lp(a) levels were determined from the sera of the fasting patients. Antibodies to cardiolipin, oxidized LDL and beta2GPI were detected employing ELISA. Beta2GPI containing IgG immune-complexes were assayed by using a dot-blot assay. Fourteen SLE patients (36.8%) were found to be positive for the presence of IgG anti-beta2GPI antibodies. Ten of the SLE patients (26.3%) were found to have high levels of beta2GPI containing immune-complexes. There was a positive correlation between beta2GPI-IC levels and the occurrence of thrombocytopenia in the patients (P < 0.05). Furthermore, patients with SLE and venous thrombosis had higher levels of beta2GPI-IC when compared with thrombosis-free patients or with healthy controls (P < 0.05). Patients with higher Lp(a) levels (> 50 mg/dl) possessed higher levels of beta2GPI-IC as compared with patients with lower Lp(a) concentration (< 20 mg/dl) (P < 0.05). These results suggest that IC-containing beta2GPI can help in defining a subpopulation of SLE patients with increased risk of thrombocytopenia and further aid in resolving mechanisms of immune-mediated tissue damage.

Immunochemical determination of DNA in immune complexes present in the circulation of patients with systemic lupus erythematosus.

The presence of DNA antigen in circulating immune complexes formed with anti-DNA antibodies of IgG class was determined in sera of 138 SLE patients and 23 healthy donors, employing monoclonal anti-dsDNA antibodies in a quantitative dot blot assay. About half of the SLE patients had elevated amounts of DNA antigen in the immune complexes (presumably as nucleosomes). The number of patients with the SLE manifestations was not higher in the group with the high amount of DNA in immune complexes. Elevated levels of DNA in immune complexes was found only in sera of SLE patients with the active, as well as quiescent form, of the disease and not in sera of healthy donors. The presence of increased amounts of DNA antigen in circulating immune complexes could indicate the presence of SLE pathology even if no manifestations of SLE are found. The level of the circulating anti-DNA antibodies was not correlated with the amount of DNA in immune complexes.

Expressed immunoglobulin repertoire of LPS-stimulated splenocytes of unimmunized mice as studied by two-dimensional gel electrophoresis.

The repertoire of isolated immunoglobulin polypeptide chains synthesized by LPS-stimulated splenic B cells from unimmunized 6 weeks old mice was studied by two-dimensional gel electrophoresis. These B cells formed mainly mu heavy chains, while only a small amount of gamma chains was detected on two-dimensional electrophoregrams. The number and character of spots corresponding to each class and type of H and L chains were analyzed. Most of the detected 52 spots, which corresponded to L chains, were well resolved with clearly defined round boundaries. Six of them belonged to two isotypes of lambda chains and the rest to the kappa chain. About 25 clusters corresponded to mu chains. They had different appearance from those of L chains and their characteristic elliptic form with prolonged vertical axes indicated the presence of several H chain variants of slightly different length (due probably to the length variations of CDR3 and carbohydrate heterogeneity) in each cluster. The limited number of spots both of H and L chains is explained as being due to restrictions in the expressed repertoire of preimmune splenic B cells, which have no somatic mutations in the immunoglobulin genes. The concept of macrorepertoire (referring to the relatively small number of detected molecular species) and microrepertoire (describing the mutationally altered molecules) is introduced.

Detection of antigens in immune complexes by a dot blot assay.

A simple assay is described for detection of antigens as a part of immune complexes. The complexes together with free IgG molecules are absorbed on protein A (or G)-Sepharose. The Sepharose beads are transferred to nitrocellulose membrane where all IgG including their complexes with antigens are eluted by acidic buffer directly on the surface of the membrane. The antigens immobilized on the membrane after elution are detected by corresponding labeled antibodies. The validity of the procedure was assessed by experiments with complexes of avidin and rabbit antibodies. The assay was used also for detection of DNA in immune complexes in mice with experimental systemic lupus erythematosus (SLE).

Detection of the C3a complement component in commercial gamma globulins by dot blotting.

The C3a complement component (anaphylatoxin) was found in all studied samples of commercial gamma globulins. The corresponding monoclonal antibodies were used to detect C3a. It was shown that each gram of gamma globulin contained about 2-4 mg of bound C3a.

Proteins separated from human IgG molecules.

Immunoglobulin G binding proteins were separated from human IgG molecules using 1 N acetic acid followed by 5 M guanidinium chloride in 0.1 M acetic acid. The proteins thus obtained were heterogeneous as demonstrated by SDS-PAGE and reverse-phase HPLC. The isolated proteins consisted of two types: the C3a and C4a complement fragments (anaphylatoxins) and immunoglobulin peptide chain fragments V kappa I and C gamma 3. Both anaphylatoxins immobilized on cellulose nitrate membranes could reassociate with intact IgG molecules. The ubiquitous presence of C3a in IgG preparations was demonstrated using monoclonal antibodies specific for C3a. Nearly all of the bound anaphylatoxin molecules were found in the Fab fragment. These findings suggest that IgG molecules can eliminate anaphylatoxins from the circulation, and thus prevent harmful effects due to these active complement components.

Complexes of IgG molecules and C3a and C4a complement components in human serum.

A heterogeneous group of proteins was separated from human serum IgG using 5 M guanidine hydrochloride solution in 0.1 M acetic acid. The protein mixture was fractionated by reverse-phase high-performance liquid chromatography and two proteins were isolated and identified as the C3a and C4a complement components (anaphylatoxins) according to their molecular masses and N-terminal sequences. Using a chemical cross-linking technique, the capacity of C3a and C4a to reassociate with the heavy and the light chains of IgG was shown. On the basis of the molecular masses of reconstituted complexes one molecule of C3a (or C4a) was bound to one heavy or one light chain of IgG.

[Intramolecular mobility of human major histocompatibility complex protein. A spin-label study]. / Vnutrimolekuliarnaia podvizhnost' belkov glavnogo kompleksa gistosovmestimosti cheloveka. Isuchenie metodom spinovoi metki.

Membranes of human splenocytes were hydrolyzed by papain and extracellular portions of class I and class II HLA antigen molecules were isolated by monoclonal antibodies fixed on Sepharose 4B. The isolated proteins were spin-labeled by TEMPO-dichlorotriazine and the values of rotational correlation times (tau) of labeled proteins were found using dependencies of ESR spectra parameters vs viscosity at constant temperature. The tau-values were equal to 8 ns for class I molecules and 14 ns for class II molecules. These values are 2-3 times lower than predicted for a rigid ellipsoid with mol wt. 50 kDa (about 20 ns). This fact suggests the existence of flexibility of HLA molecules which seems to be important for their biological activity. In this respect extracellular portions of HLA antigen molecules resemble flexible Fc fragments (tau = 12 ns) and differ from rigid Fab fragments (tau = 20 ns) of immunoglobulins G. The values of tau of spin-labeled proteins adsorbed from membrane hydrolysates on IgG-column was equal to 6.5 ns. The proteins adsorbed on lentil lectin column (after isolation of HLA proteins) have the tau-values equal to 9 ns.

Extracellular portions of HLA antigens are not compact globulae.

Membranes of human spleen cells were hydrolyzed by papain and the extracellular portions of HLA antigen molecules isolated by monoclonal antibodies fixed on Sepharose. The isolated proteins were spin-labeled by TEMPO-dichlorotriazine. The values of rotational correlation times (tau) of spin-labeled proteins were calculated using dependencies of magnetic parameters found from ESR spectra vs viscosity at constant temperature. The tau-values were equal to 8 nsec for class I molecules and 14 nsec for class II molecules. These values were significantly lower than those predicted for a rigid sphere with dimensions equal to the extracellular portions of HLA molecules (20 nsec). This fact suggests the existence of flexibility in poly-functional HLA molecules, which seems to be important for their biological activity. In this respect, extracellular portions of HLA molecules resemble flexible Fc fragments (tau = 12 nsec) and differ from rigid Fab fragments (tau = 20 nsec) of immunoglobulins G. The rotation of the oligosaccharide chains attached to HLA molecules is restricted.

[Determining the motility of glycoprotein oligosaccharides from lymphocyte membranes using the spin label method]. / Opredelenie podvizhnosti oligosakharidov glikoproteinov iz membran limfotsitov metodom spinovoi metki.

Splenocyte glycoproteins solubilized by papain were purified on lectyl-lectin Sepharose 4B. Glycoproteins eluted from lectin were spin-labeled at carbohydrates. Quantitative evaluation of ESR spectra of spin-labeled glycoproteins pointed to strong restriction of reorientation of the spin label bound to oligosaccharides. Calculated correlation time of relaxing volume tagged with the spin label was equal to the molecular weight about 6000-7000 dalton. This value suggests the existence of flexibility of the glycoproteins studied.

[Segmental motility of the COOH-terminal fragment of immunoglobulin G heavy peptide chains in a solution]. / Segmental'naia podvizhnost' COOH-kontsevogo fragmenta tiazhelykh peptidnykh tsepei immunoglobulina G v rastvore.

The rotational correlation time of pFc' fragment of IgG1 determined by spin-label method was found to be equal to 5.2 nsec. This value points to the significant segmental mobility of Ch3 domains built fragment as well as to the structural lability of Ch3 domains themselves.

[Structural studies of human IgA1 and IgA2 immunoglobulins labeled with two different spin labels]. / Strukturnye issledovaniia IgA1 i IgA2 immunoglobulinov cheloveka, mechennykh dvumia razlichnymi spin-metkami.

The spin-label method was used for structural study of different subclasses of human immunoglobulin A. The spin label was incorporated into the protein part, as well as into carbohydrates of the IgA molecules. Well resolved outer wide extrema were characteristic of the ESR spectra of IgA spin-labeled at the protein moiety. ESR spectra of IgA tagged at carbohydrates reflected moderately immobilized rotation of the spin label. Dependencies of the parameters of ESR spectra of spin-labeled IgA1 and IgA2 upon viscosity at constant temperature have been investigated and a quantitative analysis of the isotherms was carried out. Spin-labeled oligosaccharide chains of IgA2 possessed great freedom of rotation. At least some of IgA1 oligosaccharides were closely attached to the protein moiety. Both proteins under study have shown flexible structure. The Fc fragment of IgA1 molecule appeared to have a rigid structure.

Spin labeling of immunoglobulin M and E carbohydrates.

The analysis of ESR spectra of spin-labeled human myeloma immunoglobulins M and E has shown the rotation of spin labels bound to carbohydrates to be restricted most probably due to close attachment of oligosaccharide chains to protein parts of molecules. The values of rotational correlation times for IgM, IgMs and Fc5, spin-labeled at carbohydrates, were found to be equal to 7,7 and 6 ns, respectively. These data pointed to the existence of internal lability of the Fc5 fragment. ESR spectra of IgE spin-labeled at carbohydrates also reflected restricted rotation of the spin label, but the presence of a more than one wide extremum spoke in favour of differences in the degree of immobilization between different oligosaccharide chains. The value of rotational correlation times calculated for IgE, spin-labeled at protein and carbohydrate moieties, were similar (60 and 66 ns). These data confirmed the previously found rigidity of IgE molecule as compared with IgG molecule.