Dissociation Of Immune Complexes Research Articles

Prevalence of canine heartworm infection in Queensland, Australia: comparison of diagnostic methods and investigation of factors associated with reduction in antigen detection

BackgroundThe prevalence of Dirofilaria immitis infection in dogs is increasing globally and spreading into new areas. Prevalence of dirofilariosis in the state of Queensland, Australia, was as high as 90% before the introduction of macrocyclic lactones. Limited research on prevalence of D. immitis infection in dogs in Queensland has been reported in the last 30 years. Antigen testing is the most common method for detection of dirofilariosis but its accuracy is reduced by antigen getting trapped (blocked antigen) in immune complexes (ICs). The objectives of this research were to determine the prevalence of D. immitis infection in dogs from two geographical areas (Brisbane and Townsville) in Queensland, to determine the extent to which blocked antigen affects the validity of antigen testing, and to explore whether this was associated with microfilaraemia, location, age or sex.MethodsBlood samples from Brisbane (sub-tropical climate) and Townsville (tropical climate) shelter dogs were evaluated for the presence of D. immitis antigen before (conventional antigen testing, CAT) and after dissociation of ICs by heat treatment (antigen testing after heat treatment, ATHT), using a commercially available test. Microfilariae were detected using modified Knott’s test (MKT). Test proportions were compared with McNemar’s test and the association between antigen test-discordant results (positive for antigen after dissociation of ICs) and microfilaraemia, location, sex and age was modelled using logistic regression.ResultsDirofilaria immitis prevalence in dogs from Townsville (22% by CAT, 32.1% by ATHT and 16.7% by MKT) was significantly higher than in dogs from Brisbane (1.1% by CAT and MKT and 1.7% by ATHT) (P< 0.001). Dissociation of ICs allowed detection of significantly more D. immitis infected dogs than either conventional antigen testing or microfilariae detection, or the combined antigen and microfilariae detection (P< 0.001). The odds of dogs being positive for antigen after dissociation of ICs were significantly higher for microfilaraemic, 3–4-year-old female dogs from Townsville.ConclusionsThe high prevalence of infection with D. immitis in dogs from Townsville poses a health risk for local susceptible host species, including humans. Dissociation of ICs increases antigen detection and should be considered in dogs suspected of D. immitis infection but negative on routine testing.Graphical

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C2H2 Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis.

With Intact Transition Epitope Mapping-Thermodynamic Weak-force Order (ITEM-TWO) analysis in combination with molecular modeling, the phosphorylation-dependent molecular recognition motif of the anti-HpTGEKP antibody has been investigated with binary and ternary component mixtures consisting of antibody and (phospho-) peptides. Amino acid sequences have been selected to match either the antibody's recognition motif or the cancer-related zinc finger protein mutations and phosphorylations of the respective amino acid residues. Upon electrospraying of all the components of the mixtures, that is, hexapeptides, antibody, and intact immune complexes, the produced ions were subjected to mass spectrometric mass filtering. The antibody ions as well as the immune complex ions traversed into the mass spectrometer's collision chamber, whereas paths of unbound peptide ions were blocked prior to entering the collision cell. After dissociation of the multiply charged immune complexes in the gas phase, the complex-released peptide ions were recorded after having traversed the second mass filter. Complex-released peptides were unambiguously identified by their masses using mass analysis with isotope resolution. From the results of our studies with seven (phospho-) peptides with distinct amino acid sequences, which resembled either the antibody's binding motif or mutations, we conclude the following: (i) A negatively charged phospho group, located near the peptide's N-terminus is mandatory for antibody binding when placed on the peptide surface at a precise distance to the C-terminally located positively charged ε-amino group of a lysinyl residue. (ii) A bulky amino acid residue, such as the tyrosinyl residue at the N-terminal position of the (phospho-) threoninyl residue, abolishes antibody binding. (iii) Two closely spaced phospho groups negatively interfere with the surface polarity pattern and abolish antibody binding as well. (iv) Non-phosphorylated peptides are not binding partners of the anti-HpTGEKP antibody.

Interferometric detection of chloramphenicol via its immunochemical recognition at polymer-coated nano-corrugated surfaces

Three types of polymer-coated glass slides were prepared by acylation of their γ–aminopropyl derivatives with succinic anhydride or poly(p-nitrophenyl acylate) (PNPA) at various reaction times. The variations in duration of chemical adsorption of PNPA allowed control over nano-corrugation of the reactive surfaces employed for biosensing. The visually smooth and optically clear polymer-coated slides were used for immobilization of a conjugate of bovine serum albumin with chloramphenicol (CAP) and label-free measurement of concentration of this antibiotic. The sensor chips prepared in this way provided strong biosensing response due to fast biomolecular interactions and efficient competition of the target analyte during indirect immunoassay. The kinetics of formation and dissociation of immune complexes “conjugate-antibody” on the sensor chip surface was registered by the label-free methods of spectral-correlation and spectral-phase interferometry. The average thickness of immune complex adsorption layers was in the range of 5–12 nm. The highest value of kinetic association constant (kon = 5 × 104 M−1s−1) and the lowest equilibrium dissociation constant (Kdiss = 2 × 10-8 M−1) were achieved with the chips originated in the PNPA-coated slides, which were produced under short (2 min) polymer chemisorption and exhibited nano-corrugation almost unchanged with respect to the parent glass, according to the atomic-force microscopy. The detection limit for CAP detection was 80 pg/mL, and the dynamic range was 5 orders. The proposed methods for fabrication of sensor chips with nano-corrugated surface, the results of their characterization, as well as the developed assay can be employed for high-throughput recording of multiple molecular interactions by spectral interferometry and in a wide range of biosensing platforms for registration of biologically active compounds in medicine, veterinary, food control and ecological monitoring.

Comparison of different enzyme-linked immunosorbent assay methods for avidity determination of antiphospholipid antibodies.

Avidity of antiphospholipid antibodies may be clinically useful as a valuable additional characteristic. The aim of this study was to compare several ELISA modifications with different chaotropic agents and calculation of avidity indices for the determination of anticardiolipin antibody (aCL) avidity. We examined 28 serum samples with positive IgG aCL by adapted ELISA using various concentrations of urea and sodium chloride as chaotropic agents and different dilution of sera. We tested these conditions of ELISA-a single diluted serum sample with fixed concentration of a chaotrope and a serially diluted serum in the constant concentration of a chaotropic agent. We demonstrated that ELISA method for avidity determination based on a single dilution of serum in the presence of fixed concentration of chaotrope is convenient for determination of IgG aCL antibody avidity. Concentrations 6 and 8mol/L of urea or 1 and 2mol/L of NaCl were suitable for sufficient dissociation of immune complexes during ELISA procedure. This way was in good agreement with more demanding procedures. Both urea and sodium chloride may be used as chaotropic agents. Reference values of avidity indices essential for interpretation of patients' results must be established individually for distinct assay conditions.

Levels of Circulating Tumor Necrosis Factor-α in Children with Symptomatic Dengue Evaluated by ELISA and Bead-Based Assays.

Tumor necrosis factor (TNF)-α is a key cytokine in the pathogenesis of dengue virus infection, and its accurate detection in several types of human samples is critical. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for the detection of TNF-α, but multiplexed bead-based assays such as cytometric bead array (CBA) are now frequently used. Here, using ELISA and two CBAs commercially available, we measured TNF-α concentrations in plasma and serum from children with acute dengue virus infection and healthy controls. To evaluate the detection efficiency and factors affecting it, spiked recovery and immune complex dissociation assays were also performed. The levels of TNF-α evaluated by ELISA in paired serum and plasma samples from children with dengue positively correlated (rho = 0.99, p < 0.0001). Children with dengue had higher levels of plasma TNF-α than those of healthy children (p = 0.004). The ELISA detected TNF-α in a higher number of plasma samples than the CBA (p < 0.0001), and both methods only correlated when TNF-α was evaluated in buffer-based solutions but not in plasma, indicating the presence of a factor interfering with the detection of TNF-α in plasma. The recovery of several types of human recombinant TNF-α was dramatically decreased in plasma but not in tissue culture media (p ≤ 0.01), and this effect was similar in the plasma obtained from the children with dengue or the healthy controls. The dissociation of immune complexes did not improve TNF-α recovery. Dilution of the plasma samples increased the recovery of TNF-α, but at high concentrations of the cytokine. In short, plasma affects the efficiency of TNF-α detection, and this effect should be considered in the measurement of this cytokine.

Immunopathology of desialylation: human plasma lipoprotein(a) and circulating anti-carbohydrate antibodies form immune complexes that recognize host cells.

Human plasma lipoprotein(a) [Lp(a)], the dominant lipoprotein in atherosclerotic plaques, contains an apo(a) subunit of variable size linked to the apoB subunit of a low-density lipoprotein (LDL) molecule. Circulating lipoprotein immune complexes (ICs) assayed by ELISA using microplate-coated anti-apo(a) or anti-apoB antibody for capture and peroxidase-labelled anti-human immunoglobulins as probe consisted mostly of Lp(a) despite several-fold excess of LDL over Lp(a) in plasma. Microplate coating of plasma lipoprotein IC and probing with antibodies to apo(a) and apoB also revealed negligible presence of LDL compared to Lp(a). Peanut agglutinin specific to desialylated O-glycans bound significantly more to Lp(a) recovered after urea dissociation of IC than to free Lp(a). Plasma lipoproteins separated by ultracentrifugation and desialylated by neuraminidase formed IC with naturally occurring antibodies in normal plasma. These de novo ICs agglutinated desialylated but not normal human RBC in proportion to the polyagglutinin antibody titre of plasma used, suggesting availability of multiple unoccupied binding sites on the participating antibodies even after IC formation. Agglutination was inhibitable by galactosides and decreased 4-8 fold if precursor lipoprotein was selectively depleted of Lp(a), showing agglutinating ICs were contributed mainly by desialylated Lp(a) and galactose-specific antibodies. IC was 2 fold more agglutinating if lipoproteins used contained smaller rather than larger Lp(a) molecules of the same number. Small size/high plasma concentration Lp(a) phenotype and neuraminidase-releasing diseases including diabetes are risk factors for vascular disorders. Results suggest a possible route of Lp(a) attachment to vascular cells that offer terminal galactose on surface glycans following desialylation.

The effect of polyamines on the binding of anti-DNA antibodies from patients with SLE and normal human subjects.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE). To elucidate specificity further, the effect of polyamines on the binding of anti-DNA antibodies from patients with lupus was tested by ELISA to calf thymus (CT) DNA; we also assessed the binding of plasmas of patients and normal human subjects (NHS) to Micrococcus luteus (MC) DNA. As these studies showed, spermine can dose-dependently inhibit SLE anti-DNA binding to CT DNA and can promote dissociation of preformed immune complexes. With MC DNA as antigen, spermine failed to inhibit the NHS anti-DNA binding. Studies using plasmas adsorbed to a CT DNA cellulose affinity indicated that SLE plasmas are mixtures of anti-DNA that differ in inhibition by spermine and binding to conserved and non-conserved determinants. Together, these studies demonstrate that spermine can influence the binding of anti-DNA autoantibodies and may contribute to the antigenicity of DNA.

A simple immune complex dissociation ELISA for leishmaniasis: Standardization of the assay in experimental models and preliminary results in canine and human samples

Visceral leishmaniasis, caused by Leishmania (Leishmania) chagasi, is a chronic parasitic disease of humans and dogs. Confirmation of the protozoal agent in bone marrow, lymph node or spleen aspirate is diagnostic, while specific-IgG serology is used mainly for epidemiology despite the general presence of high levels of serum immunoglobulin. Anecdotal reports of false-negative serology in active disease cases are known and are ascribed to the formation of immune complexes. Because dissociation of immune complexes can be accomplished by acid treatment, we devised a simple, routine enzyme immunoassay (ELISA) for the dissociation of immune complexes in serum samples using acid treatment in wells adsorbed with Leishmania antigen (dELISA). Confirmatory acid dot-blot was also developed for antigen detection by anti-Leishmania rabbit antiserum. In experimental L. chagasi hamster models, immune complexes interfered with ELISA mostly in the 30 and 60 days postinfection, according to both dELISA and antigen dot-blot results. In larger samples from endemic areas, dELISA was positive in 10% of seronegative dog samples (7/70) and 3.5% in negative human samples (3/88), showing that dELISA could be used in the serodiagnosis of visceral leishmaniasis. Moreover, dELISA could be used as an alternative approach to screening asymptomatic visceral leishmaniasis patients, instead of invasive confirmatory testing.

Blastomyces dermatitidis Antigen Detection by Quantitative Enzyme Immunoassay

The second-generation MVista Blastomyces antigen enzyme immunoassay was not quantitative; therefore, specimens obtained previously were tested in the same assay as new specimens to assess the change in antigen levels. Furthermore, the sensitivity in serum had not been fully evaluated. The purpose of this study was to evaluate a quantitative Blastomyces antigen assay and detection of antigen in serum. Calibrators containing known concentrations of Blastomyces galactomannan were used to quantify antigen in urine and serum from patients with proven blastomycosis and from controls. Paired current and previously obtained urine specimens were tested to determine if quantification eliminated the need for concurrent testing to assess change in antigen. Pretreatment of serum with EDTA at 104°C was evaluated to determine if dissociation of immune complexes improved detection of antigenemia. Antigenuria was detected in 89.9% of patients with culture- or histopathology-proven blastomycosis. Specificity was 99.0% in patients with nonfungal infections and healthy subjects, but cross-reactions occurred in 95.6% of patients with histoplasmosis. Change in antigen level categorized as increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in ng/ml determined from different assays. Pretreatment increased the sensitivity of detection of antigenemia from 35.7% to 57.1%. Quantification eliminated the need for concurrent testing of current and previously obtained specimens for assessment of changes in antigen concentration. Pretreatment increased the sensitivity for detection of antigenemia. Differentiation of histoplasmosis and blastomycosis is not possible by antigen detection.

Detection of microbial antigenic components of circulating immune complexes in HIV patients: Involvement in CD4 + T lymphocyte count depletion

Objective To investigate the prevalence of microbial antigenic components of circulating immune complexes amongst grades of CD4 T lymphocyte counts in HIV sero positive and sero-negative participants. Methods Polyethelene glycol (PEG-600) and buffering methods of precipitation and dissociation of immune complexes was used to generate immune solution from sera of 100 HIV sero-positive and 100 HIV sero-negative participants. These were categorized into 3 grades based on CD4 count: > 500 cell/mm 3, 200-499 cell/mm 3 and <200 cell/mm 3. The immune solutions were assayed using membrane based immunoassay and antibody titration, along side its unprocessed serum for detection of various microbial antigens and or antibodies. CD4 T cell counts were estimated using Patec Cyflow SL-3 Germany. Results Antigenic component of immune complexes of various infectious agents was detected in 99 and 70 HIV sero-positive and HIV sero-negative participants, respectively. In group A, there were 10 HIV positive participants, including 4 (40.0%) had circulating immune complexes(CIC s) due to Salmonella species only; 1(10.0%) due to Salmonella-Plasmodium falciparum (P. falciparum), Salmonella-P. falciparum-HCV and P. falciparum antigens, respectively. In group B, 45(45.4%) HIV sero-positive participants with CIC s had CD4 T lymphocyte count between 200-499 cells/mm 3. Out of these, 20(44.4%) had CIC s due to Salmonella species only; 9(20%) due to Salmonella-P. falciparum. In group C, there were 44(44.4%) HIV sero-positive participants, including 3(6.8%) due to Salmonella species only; 24(54.4%) due to Salmonella-P. falciparum; 2(4.5%) due to P. falciparum only. Conclusions In HIV sero-positive participants, presence of heterogeneity of Salmonella species- P. falciparum antigens was highly incriminated in CD4 count depletion but not homogeneity of malaria parasites antigens. Malaria parasites antigens only were incriminated in CD4 + count depletion amongst HIV sero-negative participants. Before taking any decision on the management of HIV-1-positive individuals, their malaria and Salmonella paratyphi status should be assessed, but not malaria status alone.

Presence of anticardiolipin antibodies in cryoglobulins isolated from sera of hepatitis C virus infected patients

Most extrahepatic manifestations of the hepatitis C virus (HCV) infection are connected with influence of virus on the host's immune system. An important part of immunologic disturbances in chronic infections is the presence of circulating immune complexes. Standard serologic methods are not applicable in analysis of complexes composition. Detection of anticardiolipin antibodies (ACA) in cryoglobulins of patients with the HCV infection before and after dissociation of immune complexes. Sera and croglobulins of 27 patients (19 female, 8 male) with chronic hepatitis C and with cryoglobulinemia were tested. ACA were searched in serum and cryoprecipitate of 27 HCV infected patients with cryoglobulinemia--before and after dissociation of immune complexes. The presence of ACA was not confirmed in sera, but in 59% of cases they were detected in dissociated complexes from cryoprecipitate. The correlation between the presence of anti-HCV antibodies and ACA in cryoprecipitate before dissociation of immune complexes was 0.47 (p < 0.01) and after dissociation--0.61 (p = 0.0007). There was a statistically significant correlation between the presence of purpura (vasculitis) and the presence of ACA before (p < 0.002) and after dissociation of complexes (p < 0.05). In the tested group of patients there was a high incidence of ACA (non detectable in sera) detectable after dissociation of immune complexes from cryoglobulins. The presence of ACA in cryoprecipitate is significantly connected with clinical signs of cutaneus vasculitis.

Frequency, causes, and new challenges of indeterminate results in Western blot confirmatory testing for antibodies to human immunodeficiency virus.

More than 2 decades of developments in the field have led to a wide variety of test kits available for addressing the different needs of surveillance, diagnosis, and monitoring of human immunodeficiency virus (HIV) infection and antiretroviral therapy ([20][1], [23][2]). These test kits are based on

Anti-insulin Activity in IgG-fractions from Children with Newly-diagnosed Type 1 Diabetes and Negative for Insulin Autoantibodies

Insulin autoantibodies (IAA) are often detected as the first humoral sign of β-cell autoimmunity in prospective studies in young children with increased genetic risk of type 1 diabetes. After the appearance of IAA their level typically rise but seems to decline in many cases before the clinical presentation of type 1 diabetes. We hypothesized that the reason for the sudden drops in the levels of IAA could be the formation of immune complexes caused by binding of antibodies to free insulin in plasma.We studied whether isolation of the IgG-fraction and dissociation of immune complexes by acid treatment using protein A column results in the appearance of detectable IAA in those children with newly-diagnosed type 1 diabetes whose plasma samples test negative for IAA. IAA assay was performed in IgG-fractions and corresponding plasma samples from 17 children with type 1 diabetes and 23 unaffected children all testing negative for plasma IAA. The levels of IAA measured from IgG-fractions of diabetic children were higher than the levels of IAA measured from IgG-fractions in the control children ( p=0.004 in Mann–Whitney U-test). Forty-seven percent (8 out of 17) of newly-diagnosed patients negative for plasma IAA before IgG separation had increased levels of IAA in IgG-fractions and only 13% (3 out of 23) of controls. The levels of glutamate decarboxylase autoantibodies (GADA) did not differ between patients (n=14) and controls (n=21) negative for plasma GADA when measured in IgG-fractions. Our results suggest that formation of immune complexes results in false negative results in tests for IAA but not for GADA.

Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections.

Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot immunoassay (DBI) was compared to a commercially available DEN antigen detection kit (denKEY Blue kit; Globio Co., Beverly, Mass.) and a reverse transcription-PCR (RT-PCR) kit. Serial serum or plasma samples (n = 181) obtained from 55 acute DEN-infected patients were used. In samples obtained from 32 of these 55 DEN-infected patients, viral RNA could be detected by RT-PCR. DEN antigen was detected in only 10 of these 55 patient samples by using the denKEY kit. When these samples were treated with acid to release the immune-complex-associated NS-1 antigen for detection by DBI, 43 of these 55 patients were found to be positive for DEN NS-1 antigen. In nondissociated samples, 22 of these patients were found to be positive by the DBI. In the presence of DEN-specific immunoglobulin M antibodies, both viral RNA and DEN (NS-1) antigen could be detected. The number of positive samples identified by RT-PCR and DBI from these patients with primary DEN infections varied between 28 and 78%. In secondary DEN infections, the number of samples that tested positive by the DBI after immune-complex dissociation (DIS-DBI) was 25% higher than the number of samples that tested positive by RT-PCR and was 35% higher than that determined by nondissociated antigen (NDIS-DBI) detection. We conclude that the denKEY kit has limited diagnostic value for acute DEN infections compared to the RT-PCR and the NDIS-DBI and DIS-DBI methods. We clearly demonstrate that in secondary DEN infections the dissociation of NS-1 immune complexes is essential for early diagnosis of DEN infections.

Mactinin: a modulator of the monocyte response to inflammation.

During inflammatory processes, monocytes leave the blood stream at increased rates and enter inflammation tissue, where they undergo phenotypic transformation to mature macrophages with enhanced phagocytic activity. alpha-Actinin, a cytoskeletal protein, is present in focal adhesion complexes and left in the microenvironment as a result of cell movement. Mactinin, a 31 kDa amino-terminal fragment of alpha-actinin, is generated by the degradation of extracellular alpha-actinin by monocyte-secreted urokinase. We have previously demonstrated that mactinin promotes monocyte/macrophage maturation. We now report that 0.5-10 nM mactinin has significant chemotactic activity for monocytes. Mactinin seems to be present in inflammatory arthritis synovial fluid, because affinity-purified antisera reacted with a protein of the expected molecular mass in various types of arthritis fluids that were immunoaffinity-purified and subjected to Western analysis. Thus, six of seven samples from patients with psoriatic arthritis, reactive arthritis, gout, or ankylosing spondylitis contained mactinin at levels that are active in vitro. Initially, mactinin was not found in affinity-purified rheumatoid arthritis samples. However, it was detectable after the dissociation of immune complexes, suggesting that it was complexed to anti-microfilament auto-antibodies. In addition, mactinin was found in the lavage fluid from the arthritic knee joints of rabbits with antigen-induced arthritis and was absent from the contralateral control knee fluids. We conclude that mactinin is present in several types of inflammatory arthritis and might modulate mononuclear phagocyte response to inflammation.

HBsAg as the antigen component of circulating immune complexes in HIV-infected patients

Seeing the same transmission pattern of HIV and HBV coinfection by these two agents is not an uncommon feature. Immunity impairment due to HIV infection can be the cause of a higher rate of HBV replication with less intensive liver damage and less effective immune response to HBV, while the pathological course in both infections involves elevated levels of circulating immune complexes (CIC). These were the reasons for us to examine the frequency of HBsAg involvement as the antigen component of circulating immune complexes formed in sera of HIV-infected patients in different stages of HIV disease. We tested 67 sera of HIV-positive patients in different stages of HIV disease for the presence of HBsAg and HIV antigen p24 (with and without acid dissociation of immune complexes), for the presence of anti-Hbc antibodies and circulating immune complexes. HBsAg was positive in 13.8% sera prior to and 33.8% after acid pretreatment. Anti-HBc antibodies were present in 76.9% serum samples tested. Fifty percent of sera were positive for both HBsAg and p24 antigen after dissociation of immune complexes. The level of CIC was elevated in 65.9% of sera. Our results suggest that HBsAg is commonly associated in immune complexes formed in the sera of HIV-infected patients and that they may simultaneously contain HIV and HBsAg in patients coinfected with both agents. This may contribute to their mutual interaction and influence the diagnosis and follow-up of patients.

Extraction of Schistosoma haematobium antigens from infected human urine and generation of potential diagnostic monoclonal antibodies to urinary antigens

Proteins in Schistosoma haematobium infected human urine were concentrated by precipitation with saturated ammonium sulphate 50% (v/v) and various fractions obtained at different stages of precipitation tested for presence of schistosome antigens (ShAgs) by dot-ELISA. The protein fraction (UP2S) obtained following two-times precipitation was found to contain high concentrations of ShAg. Fraction UP2S was dialysed against phosphate-buffered saline (pH 7.4) and further purified by Sephadex G-200 column chromatography. Two protein peaks were eluted of which the first peak UP2S(pkI) was found to contain high concentrations of ShAgs as determined by microplate-ELISA. The second peak UP2S(pkII) consisted of human urine proteins. Further analysis of UP2S(pkI) revealed that ShAgs were mainly in the form of immune complexes with human IgG, IgM, IgA, IgE and complement C3. The ShAgs in both UP2S and UP2S(pkI) were found to be active as they induced immune responses in mice which produced antibodies reactive with S. haematobium worm as well as soluble egg antigens (SEA). Pure ShAgs were obtained from UP2S following dissociation of immune complexes with a carbonate buffer (pH 11.42) and further purification on Sephadex G-200. Immunizations with UP2S led to the generation of MoAbs which could bind both SEA and UP2S.

Rapid serodiagnosis with RK39 dipstick for visceral leischmaniasis of low endemicity in northwestern china

Visceral leishmaniasis, caused by Leishmania (Leishmania) chagasi, is a chronic parasitic disease of humans and dogs. Confirmation of the protozoal agent in bone marrow, lymph node or spleen aspirate is diagnostic, while specific-IgG serology is used mainly for epidemiology despite the general presence of high levels of serum immunoglobulin. Anecdotal reports of false-negative serology in active disease cases are known and are ascribed to the formation of immune complexes. Because dissociation of immune complexes can be accomplished by acid treatment, we devised a simple, routine enzyme immunoassay (ELISA) for the dissociation of immune complexes in serum samples using acid treatment in wells adsorbed with Leishmania antigen (dELISA). Confirmatory acid dot-blot was also developed for antigen detection by anti-Leishmania rabbit antiserum. In experimental L. chagasi hamster models, immune complexes interfered with ELISA mostly in the 30 and 60 days postinfection, according to both dELISA and antigen dot-blot results. In larger samples from endemic areas, dELISA was positive in 10% of seronegative dog samples (7/70) and 3.5% in negative human samples (3/88), showing that dELISA could be used in the serodiagnosis of visceral leishmaniasis. Moreover, dELISA could be used as an alternative approach to screening asymptomatic visceral leishmaniasis patients, instead of invasive confirmatory testing.

Anti-F(ab')2 antibody in HIV type 1 infection: relationship to hypergammaglobulinemia and to antibody specific to the V3 loop region of glycoprotein 120.

As HIV infection and autoimmune disease share certain similarities, it has been suggested that HIV may disrupt control of humoral immunity by the antiidiotype network, and that this may be evident as increased IgG antibody to F(ab')2. When anti-F(ab')2 was quantified by ELISA in sera of randomly chosen HIV-infected versus uninfected donors, some HIV-infected sera did contain increased anti-F(ab')2, resulting in a median amount twofold higher than in uninfected sera. Moreover, when data were grouped by blood CD4 lymphocyte count, anti-F(ab')2 in HIV+ groups appeared to rise as CD4 lymphocytes declined. However, increased anti-F(ab')2 mirrored the elevation in serum IgG closely, and normalization of anti-F(ab')2 to serum IgG concentration equalized the groups so that no relationship to CD4 lymphocytes remained. Hypergammaglobulinemia is therefore strongly implicated as a cause of variation in anti-F(ab')2. After dissociation of immune complexes, anti-F(ab')2 activity per microgram of monomeric IgG was slightly increased over normal only in the HIV-infected group with fewest CD4 lymphocytes, without statistical significance. In contrast, the proportion of IgG antibody to the V3-neutralizing determinant in HIV-1 decreased significantly as disease advanced. The same was true for 12 HIV+ individuals studied longitudinally for 500-1300 days. The data suggest that measuring serum anti-F(ab')2 is misleading when immune complexes are present: apparent increases as disease progresses are due to increased IgG and, possibly, to related technical artifacts. During HIV infection, the proportion of antiidiotypic IgG in fact remains unaltered or falls, making this an unlikely cause of suppressed humoral immunity to HIV-1.

High-pressure-mediated dissociation of immune complexes demonstrated in model systems

The use of pressure to disrupt immune complexes was demonstrated in two model systems: prostate-specific antigen (PSA) and anti-PSA antibody; and epiglycanin, a mucin glycoprotein, and an antibody specific to that protein. Dissociation of the anti-PSA antibody from the immobilized PSA antigen was observed when pressures of 415 MPa and 550 MPa (1 MPa approximately 144 psi) were applied at room temperature (approximately 21 degrees C). Application of pressures ranging from 140 MPa to 550 MPa resulted in dissociation of antibody from epiglycanin. In both cases, the rebinding of dissociated antibody to immobilized antigen indicated that the effect of high pressure on the binding of the immune complexes was reversible. These findings suggest that application of high hydrostatic pressure has the potential to be used to significantly improve the sensitivity and specificity of clinical assays.