Immune Complex Assays Research Articles

Diagnosis of tuberculous meningitis: A comparative analysis of three different antibody immunoassays, an immune complex assay, and polymerase chain reaction

Diagnosis of tuberculous meningitis: A comparative analysis of three different antibody immunoassays, an immune complex assay, and polymerase chain reaction

Activation of protein tyrosine kinase p72syk by Fc epsilon RI aggregation in rat basophilic leukemia cells. p72syk is a minor component but the major protein tyrosine kinase of pp72

Aggregation of the high affinity IgE receptors (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in protein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of approximately 72-kDa proteins (pp72) and that the tyrosine kinase p72syk is one component of pp72. Here we studied further the relationship of p72syk to pp72. The aggregation of Fc epsilon RI induced the activation of p72syk which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72syk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72syk was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phosphorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was p72syk. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated protens including 71-80-kDa proteins that associate with p53/56lyn. A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75HS1 (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72syk is activated after Fc epsilon RI aggregation.

Serologic Evaluation of Renal Status

Mechanisms of immunologic injury causing glomerulonephritis are based on immune complex or autoantibody deposition with activation of complement. Circulating immune complex disease can be diagnosed and monitored with improved assays for immune complexes, DNA antibodies, complement levels, and complement activation products. Etiologic auto-antibodies for the Goodpasture antigen, neutrophil enzymes, and ones directly promoting complement activation are now quantitated for more accurate serologic diagnosis. The spectrum of postinfectious glomerulonephritis also presents several new measurements for assessing clinical status. Diagnosis and monitoring of glomerulonephritis now entails panel testing for these humoral abnormalities that are useful to establish the etiology and treatment regimens.

N-terminal mutations modulate yeast SNF1 protein kinase function.

The SNF1 protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNF1 and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNF1-G53R, that was isolated as a suppressor of the requirement for SNF4. The mutant SNF1-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activity is wild type in a protein blot assay. Deletion of the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-delta N kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNF1-G53R and SNF1-delta N protein showed different elution profiles, consistent with differential formation of high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNF1 kinase and may be involved in interaction with a positive effector other than SNF4. We also showed that the conserved threonine residue 210 in subdomain VIII, which is a phosphorylation site in other kinases, is essential for SNF1 activity. Finally, we present evidence that when the C terminus is deleted, overexpression of the SNF1 kinase domain is deleterious to the cell.

Sequential activation of MAP kinase activator, MAP kinases, and S6 peptide kinase in intact rat liver following insulin injection.

An insulin-stimulated phosphorylation cascade was examined in rat liver after insulin injection via a portal vein by the use of immune complex kinase assays specific to the mitogen-activated protein (MAP) kinase and S6 kinase II homologue (rsk) kinase. We have prepared an antibody against the peptide consisting of a carboxyl-terminal portion of the extracellular signal-regulated kinase 1 (alpha C92), one of the MAP kinases, and an antibody against the peptide consisting of the carboxyl terminus of the mouse S6 kinase II homologue (alpha rsk(m)C). In alpha C92 immune complex assay, maximal activation of rat liver MAP kinases (approximately 4.3-fold) were observed 4.5 min after insulin injection. We also observed an insulin-stimulated MAP kinase activity (approximately 3-fold) in liver extracts from insulin-treated rat in fractions eluted from phenyl-Sepharose with 30-50% ethylene glycol. Kinase assay in myelin basic protein (MBP)-containing gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by denaturation with 6 M guanidine HCl, and renaturation revealed that insulin injection stimulated the kinase activity of the 42- and 44-kDa proteins, which corresponded to the two distinct MAP kinases. In alpha rsk(m)C immune complex assay, maximal stimulation (approximately 5-fold) of the S6 peptide (Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala) kinase activity was observed 7.5 min after insulin injection. In addition, MAP kinases purified from insulin-treated rat liver were able to activate S6 peptide kinase activity in vitro in alpha rsk(m)C immunoprecipitates from untreated rat liver, accompanied by the appearance of several phosphorylated bands including a major band at 88 kDa. We also examined whether insulin injection stimulates the MAP kinase activator (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) in rat liver. Using recombinant Xenopus MAP kinase, fractions of Q-Sepharose eluted early in the NaCl gradient were found to have MAP kinase activator activity accompanied by the phosphorylation of 42-kDa recombinant Xenopus MAP kinase. From these data, we demonstrate three tiers of a cascade composed of the MAP kinase activator, MAP kinases, and an S6 peptide kinase activity in rat liver under physiological conditions in the intact animal.

Use of anti-peptide antibodies for the design of antigen-specific immune complex assays

A simple and sensitive method is described for the detection of circulating immune complexes (ICs) in an antigen-specific manner. The method is based on the use of anti-peptide antibodies as solid-phase capture reagents to bind antigen which is complexed to serum antibodies. The bound serum antibody is detected with a labelled second antibody. The method requires that the anti-peptide antibodies bind native protein efficiently, and that the anti-peptide antibodies do not compete with antibodies raised against the native protein which are involved in IC formation. Two anti-peptide antibodies specific for the hepatitis B surface antigen (HBsAg) and the hepatitis B e antigen (HBeAg), which possessed the requisite characteristics, were chosen as models for IC assay development. The solid-phase, anti-peptide based assays efficiently detected HBsAg and HBeAg-containing ICs in preformed antigen/antibody mixtures and in the serum of chronically infected hepatitis B patients.

A sensitive method for the detection of immune complexes in human gingival crevicular fluid.

An assay for immune complexes [ICs] in gingival crevicular fluid [GCF] could be potentially useful to ascertain the risk of periodontal disease activity at specific oral sites. This paper describes and ELISA-based method sufficiently sensitive to measure ICs in GCF samples at levels below 1 microgram/ml. Static GCF samples taken from 5 adults showed IC levels ranging from 5 to 166 micrograms/ml. Additional tests of 10 GCF samples from 1 adult were conducted with complement components and indicated that either C1q or rabbit anti-human C3d was suitable as a capture agent in the assay procedure. Further application of this assay may help to assess the usefulness of IC levels in GCF samples as possible diagnostic indicators of periodontal disease activity.

Immune complexes and antibody levels in blisters over human leprosy skin lesions with or without erythema nodosum leprosum

Erythema nodosum leprosum (ENL) is a serous complication of lepromatous leprosy, affecting skin and peripheral nerves in a large percentage of these patients, and is presumed to result from spontaneous immunologic changes. Its pathogenesis is poorly understood, although histopathologic features have suggested immune complex (IC)-mediated injury. Abundant circulating antibody is present but no convincing correlation has been established between circulating IC and ENL. We have examined cutaneous leprosy lesions in vivo using blisters induced by prolonged gentle suction to determine whether or not IC are demonstrable in lesions with or without ENL, using an IC assay based on monoclonal rheumatoid factor binding. We also examined whether antibodies involved in such IC are produced locally or reach the skin via the circulation. Surprisingly large quantities of IC were found in ENL lesions, and in some cases the quantities were significantly higher than in matching serum. Total IgG, IgA, and IgM in the skin were not higher than expected, however. Attempts to demonstrate increases in intracutaneous levels of specific anti- Mycobacterium leprae antibodies were unsuccessful. This is the first report of the demonstration of IC in suction blister fluid. The results indicate that large quantities of IC may be present in cutaneous leprosy lesions and are consistent with the hypothesis that they are formed in situ when circulating antibody combines with antigen in the skin. The nature of the antigen in these IC remains undefined.

Protein tyrosine kinase activities of the epidermal growth factor receptor and ErbB proteins: correlation of oncogenic activation with altered kinetics.

We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2+, ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and approximately 2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higher Km(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphorylation suggested that the ErbB proteins had lower catalytic efficiencies for the exogenous substrate than did the chEGFR. The three tested ErbB proteins had cytoplasmic domain mutations that conferred distinctive disease potentials. These mutations did not affect the kinetics for the phosphorylation of the exogenous substrate TK6. Two of the ErbB proteins contained all of the sites used for autophosphorylation. In these, a mutation that broadened oncogenic potential to endothelial cells caused an additional increase in Vmax(app) for autophosphorylation. Thus, mutations that change the EGFR into an ErbB oncogene cause multiple changes in the kinetics of protein tyrosine kinase activity.

Calcium-dependent and calcium-independent signals in the conglutinin-binding assay (KgBa) for immune complexes: Influence of anti-collagen-antibodies

A solid phase ELISA conglutinin-binding assay (KgBa) was evaluated for the detection of circulating immune complexes. ELISA wells were coated with purified bovine conglutinin and incubated with test sera. Bound IgG was detected with enzyme labelled anti-immunoglobulin. Heat aggregated IgG which had been “solubilized” (i.e., complement treated by incubation with serum) was employed as a reference. The binding of the complement-reacted IgG to solid phase conglutinin was found to be calcium-dependent and inhibitable with N- acetyl- D- glucosamine (GlcNAc). Prolonged incubation (4 days) of aggregated IgG with serum at 37°C abolished the binding to conglutinin, a finding consistent with the complete degradation of deposited C3b to C3c and C3d. The solubilized IgG that bound to solid phase conglutinin was found by gel chromatography to be of high molecular weight (>600 kDa). Binding of IgG to solid phase bovine conglutinin was also observed to a variable degree in normal and pathological sera. However, in this situation the IgG binding was largely calcium-independent, was not inhibited by GlcNAc and did not decrease after prolonged incubation of the serum at 37°C. The reactive IgG eluted on gel chromatography at the position of monomeric IgG suggesting binding via the antigen binding sites. Binding of this IgG was inhibited by both collagen type II and purified conglutinin. These observations suggest that the assay detects cross-reacting autoantibodies against collagen epitopes, or, alternatively, antibodies against the dietary antigen, bovine conglutinin.

Partial purification and characterization of the lck protein-tyrosine kinase from bovine thymus

Soluble extracts prepared from bovine thymus contain an angiotensin-I-phosphorylating activity that is activated several-fold by high concentrations of NaCl. Fractionation of this protein-tyrosine kinase activity by chromatography on DEAE-cellulose yields a major diffuse peak of activity. The enzymes responsible for this activity are found at much higher levels in extracts from bovine thymus than from bovine spleen. The peak of activity from the DEAE-cellulose column can be further separated into two major peaks by chromatography on heparin-agarose. The second peak to elute from the heparin-agarose column was previously purified through several chromatographic steps to yield a 40 kDa protein-tyrosine kinase (p40). We have now partially purified the early-eluting peak of kinase activity by chromatography on columns of butyl-agarose, protamine-agarose and Sephacryl S200. The enzyme was identified following covalent modification with 5'-fluorosulphonylbenzoyladenosine (FSBA) by reactivity with anti-FSBA antibodies. This procedure labelled a series of 52-56 kDa proteins. These proteins were also recognized by polyclonal anti-peptide antibodies raised against the C-terminal 33 amino acids of p56lck, a major T lymphocyte protein-tyrosine kinase. Peptide maps of the partially purified enzyme were identical to maps generated from p56lck obtained from LSTRA cells. These data suggest that bovine thymus p56lck is responsible for the activity found in the early-eluting peak from heparin-agarose. Antibodies raised against a peptide corresponding to amino acids 39-64 of p56lck, a sequence found near the N-terminus, recognized the slower-migrating, but not the faster-migrating, form of the enzyme, indicating that a fraction of the protein had been proteolysed near the N-terminus during purification. The partially purified bovine enzyme exhibited a restricted substrate specificity in vitro and did not readily phosphorylate human erythrocyte band 3, casein or histone, but was able to phosphorylate acid-treated enolase. The dilute enzyme present in fractions eluting from chromatography columns was unable to catalyse an autophosphorylation reaction. Autophosphorylation could be detected in more concentrated enzyme samples and was readily observed in immune-complex assays. The phosphorylation of angiotensin I by bovine thymus p56lck was weakly activated by polyionic compounds such as heparin and polylysine, and was strongly activated by high concentrations of NaCl.

Association of autoimmunity with IgG2 and IgG4 subclass deficiency in a growth hormone-deficient child

An association between humoral immune deficiency and childhood autoimmune disease has been previously established. We describe a 7-year-old male with severe autoimmune disease, recurrent infections, a marked deficiency of IgG2 and IgG4, and an inability to respond to polysaccharide antigens. This child was also found to have isolated growth hormone (GH) deficiency. Laboratory results included a positive anti-smooth muscle antibody, a positive Raji-cell assay for immune complexes, and normal levels of IgG, IgM, and IgA. IgG subclasses revealed an IgG1 of 1225 (normal for age, 280-1120 mg/dl), IgG2 of less than 10 (30-630 mg/dl), IgG3 of 36 (40-250 mg/dl), and IgG4 of less than 4 (11-620 mg/dl). No increase in antibody titer was noted to either Pneumovax or unconjugated Haemophilus influenzae vaccine. Numbers of circulating B cells (CD19) were markedly diminished (less than 0.5%). Liver biopsies have shown chronic active hepatitis. Somatomedin C was 0.28 U/ml (normal for age, 0.5-2.06 U/ml). Challenge with either L-dopa or clonidine produced a peak GH response of 2.3 ng/ml (normals = greater than 7 ng/ml). Children with autoimmune disorders should be evaluated for IgG subclass deficiencies and ability to make antibody in response to antigen challenge regardless of the serum immunoglobulin levels. Growth failure in immune-deficient children should not be assumed to be due to chronic illness or recurrent infections. Other etiologies for growth failure should be sought.

Evaluation of the efficiency of two IgA immune complex assays

Two IgA immune complex assays, namely the anti-IgA inhibition of binding assay (a-IgA-InhBA) and the IgA polyethylene glycol assay (IgA-PEG assay) were evaluated using IgA aggregates (AIgA) of well defined sizes. AIgA was prepared by heat treatment. After ultracentrifugation the size of AIgA was found to be in a range between 7 S and 64 S. Five AIgA pools (> 64 S, 44–64 S, 24–43 S, 20–24 S and 9–19 S) and a pool of monomeric IgA were prepared to test the efficiency of the two assays. The IgA-PEG assay exclusively detected very large sized AIgA (> 64 S), whereas the a-IgA-InhBA detected also AIgA of intermediate size (9–19 S). The sensitivity of the latter assay was found to be size dependent. After ultracentrifugation of serum samples from patients with IgA nephropathy, I gA of large and intermediate macromolecular size was detected. It is concluded that the a-IgA-InhBA is useful for the detection of circulating IgA-containing immune complexes in IgA nephropathy.

Circulating immune complexes containing chlamydial lipopolysaccharide in acute myocardial infarction

The presence of circulating immune complexes (IC) was studied using two detection methods specific for chlamydial lipopolysaccharide (LPS) in paired serum samples of 44 patients (30 men and 14 women) with acute myocardial infarction (AMI). Forty-four random controls were individually matched for locality, age and sex with the AMI patients. As specificity controls for the IC assays single serum samples from 29 patients with diseases characterized by the presence of circulating IC were used. Fifty-seven per cent of AMI patients, 12% of their random controls and 10% of the patient controls were shown to have chlamydial LPS-specific immune complexes in their sera ( P < 0.0001, AMI versus random and patient controls). This finding provides further evidence of the possible association of chronic chlamydial infection with AMI.

Identification of Mitogen-Responsive Ribosomal Protein S6 Kinase pp90rsk, a Homolog of Xenopus S6 Kinase II, in Chicken Embryo Fibroblasts

Antiserum raised against recombinant Xenopus ribosomal protein S6 kinase (rsk) was used to identify a 90,000-Mr ribosomal S6 kinase, pp90rsk, in chicken embryo fibroblasts. Adding serum to cells stimulated the phosphorylation of pp90rsk on serine and threonine residues and increased the activity of S6 kinase measured in immune complex assays. Xenopus S6 kinase II and chicken embryo fibroblast pp90rsk had nearly identical phosphopeptide maps.

Identification of mitogen-responsive ribosomal protein S6 kinase pp90rsk, a homolog of Xenopus S6 kinase II, in chicken embryo fibroblasts.

Antiserum raised against recombinant Xenopus ribosomal protein S6 kinase (rsk) was used to identify a 90,000-Mr ribosomal S6 kinase, pp90rsk, in chicken embryo fibroblasts. Adding serum to cells stimulated the phosphorylation of pp90rsk on serine and threonine residues and increased the activity of S6 kinase measured in immune complex assays. Xenopus S6 kinase II and chicken embryo fibroblast pp90rsk had nearly identical phosphopeptide maps.

Efficacy of Alternative Tests for Delayed‐Cyclic Food Hypersensitivity

With the oral challenge food test (OCFT) used as the standard for delayed-cyclic food hypersensitivity diagnosis, blinded comparison studies were accomplished with 175 in vitro food specific IgE and 180 IgG radioallergosorbent tests, 180 food enzyme-linked immune complex assays, and 155 in vivo Multi-Test prick tests. The study was multi-centered, eight physicians and 37 patients participating. All of the compared tests were shown to be approximately 50% efficient when compared with the OCFT results, and, thus, nonefficacious.

An enzyme-linked immunosorbent assay for immune complex of HTLV-I

A sandwich enzyme-linked immunosorbent assay (ELISA) for immune complexes of human T cell leukemia virus type I (HTLV-I) was developed using monoclonal antibody (MoAb) 3G1 which recognizes a different epitope on HTLV-I to that with which natural human anti-HTLV-I antibody binds. The assay was capable of titrating artificial immune complexes not only at antigen-antibody equivalence but also at antibody excess. Although the antigen-antibody ratios could not be determined in the individual sera from patients with overt ATL, the level of immune complexes in three out of four sera was estimated to be 250 ± 36 ng/ml. Immune complexes of HTLV-I could not be identified in sera obtained from one patient with overt ATL, three healthy HTLV-I carriers and three normal human controls.

Antibodies to the collagen-like region of C1q in sera of patients with autoimmune rheumatic diseases.

Antibodies to the collagen-like region of C1q have recently been observed in sera of patients with systemic lupus erythematosus (SLE). In this study, we documented that these antibodies were present in 47.3% of SLE patient sera, whereas they were uncommon in sera from patients with rheumatoid arthritis (2.8%) and Sjögren's syndrome (12.8%), as well as in normal sera (6.4%). Markedly elevated antibody levels (greater than 4 SD above the normal mean) were observed almost exclusively in sera of patients with SLE. Levels of antibodies to the collagen-like region correlated highly with levels of solid-phase C1q-binding IgG when analyzed by the C1q solid-phase assay for immune complexes (r = 0.87). We previously found that, after sucrose density gradient ultracentrifugation, a predominance of the solid-phase C1q-binding IgG in SLE sera sediments as monomeric IgG. These findings, together with the present data, indicate that reactivity of SLE patients' sera in the C1q solid-phase assay reflects primarily the presence of antibodies to the collagen-like region, and not the presence of immune complexes.

Enzyme immunoassay detection of circulating immune complexes by monoclonal antibodies to C3 neoepitopes with special reference to IgG concentration and to interfering anti-immunoglobulin antibodies

A sensitive solid-phase anti-C3 enzyme immunoassay for detection of circulating immune complexes (CIC) is described. A mixture of the monoclonal antibodies (MoAbs) bH6 and Clone 9 specific for neoepitopes on C3 activation products was used as capture reagent. MoAb bH6 recognized C3b, iC3b and C3c, and Clone 9 recognized iC3b and C3dg. Detection antibody was a polyclonal peroxidase-conjugated rabbit anti-human Ig antiserum. A quantitative assay was constructed using serum incubated with heat aggregated IgG (HAG) as standard. The lower detection limit was 5 μg/ml of HAG. Interassay and intra-assay coefficient of variation was 15% and 5%, respectively. Anti-animal immunoglobulin antibodies were detected both in normal and pathological sera. This activity was efficiently absorbed by nonimmune immunoglobulins added to the samples. The present assay was compared with a polyethylene glycol precipitation assay for CIC determination. The latter assay was strongly influenced by the IgG concentration ( r s = 0.78; P = 0.006), whereas no such correlation was seen for the anti-C3 immune complex assay ( r s = −0.30; P = 0.20).