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PubMed1993

Relative avidity of serum antibodies to putative periodontopathogens in periodontal disease.

ELISA was used to determine both the avidity and titre of IgG, IgA and IgM antibodies to the gram-negative anaerobe, Porphyromonas gingivalis, in twenty periodontitis patients enrolled in a longitudinal study of attachment loss and eleven non-periodontitis affected subjects. The avidity and titre of IgG antibodies to Actinobacillus Actinomycetemcomitans were also examined. A cross-sectional analysis of the longitudinal patients at baseline and non-periodontally affected controls confirmed earlier findings that IgG and IgA antibody titres to P. gingivalis were higher in periodontitis patients than in individuals who were not periodontally affected. In this cross-sectional analysis, IgG antibody avidities to P. gingivalis were not found to be significantly higher in periodontitis than in control subjects (p = 0.065). However, indications of the potential prognostic value of antibody avidity was demonstrated by the higher IgM avidities to P. gingivalis in patients who did not experience attachment loss during the three-month monitoring period than in those who did (p = 0.0005).

Adult↗
PubMed1993

[Value of the study of IgG avidity for the diagnosis of toxoplasmosis].

The usefulness of IgG avidity analysis for dating acquired toxoplasmosis was documented by a study of 145 serial sera from 39 patients with acute toxoplasmosis and 104 sera from patients with chronic toxoplasmosis. ELISA measurement of IgG avidity involved comparison, for each serum at limit dilution, of optical densities obtained with and without washing with a urea solution to disrupt antigen-antibody bonds. A significant correlation was found between time since onset of toxoplasmosis and IgG avidity. Furthermore, comparison of IgG avidity of sera from patient with chronic infection or with acute infection monitored serologically for less than 20 weeks showed that an avidity index of 0.5 or more was inconsistent with toxoplasmosis of less than 20 weeks duration.

Antibody Affinity↗
PubMed1996

A synthetic peptide derived from the tumor-associated protein mdm2 can stimulate autoreactive, high avidity cytotoxic T lymphocytes that recognize naturally processed protein.

Studies in melanoma patients have shown that unaltered self proteins can function as targets for tumor-reactive CTL. Here, we have investigated in a murine model whether autoreactive CTL can be found against the widely expressed proteins cyclin D1, mdm2, and p53, which are frequently overexpressed in transformed cells. Sixteen MHC class I binding peptides were identified in these proteins, and seven of them consistently stimulated primary CTL in vitro. Avidity measurements revealed that the avidity of peptide-induced CTL differed by >1000-fold. The highest avidity CTL were induced by a peptide derived from mdm2. These CTL recognized target cells expressing mdm2 endogenously, while CTL generated against the remaining peptides were of lower avidity and did not recognize cells expressing relevant proteins endogenously. Generation of high avidity anti-mdm2 CTL required several cycles of peptide stimulation, suggesting that the CTL precursor frequency was low. The data show the normal T cell repertoire contains small numbers of potentially autoreactive CTL. Expansion of these CTL may lead to beneficial autoimmunity against tumors, but, equally, it may be the basis of detrimental autoimmune diseases.

Animals↗
PubMed1996

Characterization of an Ig VH idiotope that results in specific homophilic binding and increased avidity for antigen.

mAb against GD3 ganglioside demonstrate homophilic binding in which soluble anti-GD3 mAb bind, through the GD3 binding site, to a VH idiotope (designated IdHOM) on solid phase anti-GD3 mAb. In this way, homophilic binding provides a mechanism for amplifying the binding of mAb to cell surface GD3. We show that serine 52a, within CDR2, is required for IdHOM expression, homophilic binding, and high avidity binding to cell surface GD3. Computer modeling based on the crystal structure of anti-GD3 mAb R24 showed serine 52a situated at the mouth of the GD3 binding pocket, but not directly involved with GD3 binding. Substitutions at position 52a predicted to maintain the GD3 binding pocket (e.g., threonine) resulted in the loss of IdHOM expression and homophilic binding and markedly decreased binding to cell surface GD3, but maintained low avidity GD3 binding as measured by ELISA. All other substitutions at position 52a were predicted to significantly distort the GD3 binding pocket and resulted in the loss of both homophilic binding and any detectable avidity for GD3. We have structurally defined IdHOM and conclude that this idiotope is not required for the GD3 binding pocket, but that the idiotope is necessary for homophilic binding, which is required for high avidity binding to cell surface GD3. We speculate that selection of certain VH genes may result in the expression of idiotopes that allow homophilic binding, and this may represent a general mechanism for increasing the avidity of Abs against T cell-independent Ags.

Amino Acid Sequence↗
PubMed1996

Differential binding avidities of human IgM for staphylococcal protein A derive from specific germ-line VH3 gene usage.

Human IgM that express the variable region genes of the VH3 family bind staphylococcal protein A (SPA). We previously reported that the SPA-binding IgM can be divided into two groups based on the differential binding avidities for solid-phase SPA. To study the molecular basis for these differences, we cloned B cells from human blood by EBV transformation. The nucleotide sequences of the expressed Ig heavy chain genes were determined on 20 B cell clones that produce SPA-binding IgM. The germ-line VH3 gene usage in IgM with high avidities for SPA were distinct from the germ-line VH3 genes used in IgM with low avidities for SPA. There was no correlation in the usage of D or JH genes or in the usage of light chains in IgM according to the SPA binding avidity. These results suggest that the differential binding avidities for SPA are at least partly due to specific germ-line VH3 gene usage. An investigation of direct binding of SPA to the synthetic peptides corresponding to the portions of the variable regions of SPA-binding and non-SPA-binding IgM showed that the peptides corresponding to the VH3 family specific framework region 3 sequences had significant SPA binding capacities, while the peptides corresponding to the other subdomains and those corresponding to framework region 3 of the reported VH3 sequences from non-SPA-binding IgM showed little or no binding. It is of interest that the Ig-framework region 3 subdomain corresponds to the fourth hypervariable region, which in the TCR-beta chain has been implicated as a critical site for T cell superantigen binding.

Adult↗
PubMed2001

Diverse expansion potential and heterogeneous avidity in tumor-associated antigen-specific T lymphocytes from primary melanoma patients.

While tumor-associated antigen (TAA)-specific CD8(+) T lymphocytes have been detected in metastatic melanoma patients, immune response in early disease phases has not yet been carefully evaluated. We looked for circulating cytotoxic T lymphocytes (CTL) directed against Melan-A / MART1, tyrosinase, gp100 and MAGE-3 antigens in patients with a diagnosis of primary cutaneous melanoma by using fluorescent HLA-A2 tetramers. In five out of six cases high numbers of CD8(+)/tetramer(+) cells could be detected by flow cytometry, and in four patients lymphocyte populations specific for two different melanoma antigens (Melan-A/MART1 and tyrosinase) were contemporaneously present. The TAA-specific cells could represent as much as 1/220 T lymphocytes in the circulating CD8(+) population. When tetramers were used to monitor the in vitro expansion of TAA-specific CTL precursors upon antigen-specific stimulation, a diverse expansion potential was evidenced in CTL from the different donors and, more strikingly, in CTL specific for the different TAA. Melan-A/MART1-specific CTL clones derived from two patients exhibited a broad range of avidity. Only the highest avidity clones, representing about 50 % of the cases analyzed, were tumor specific. By correlating tetramer staining with clone avidity, we found that tetramer fluorescence intensity could represent a good indicator of TCR affinity, but not of overall clone avidity.

Adult↗
PubMed2003

Low-avidity recognition by CD4+ T cells directed to self-antigens.

Self-reactive T cells populate the peripheral immune system, and likely form the reservoir from which autoreactive cells are derived. We analyzed a panel of self and non-self peptides presented by HLA-DR4, a class II molecule associated with autoimmunity, by immunization of mice transgenic for HLA-DR4. Significant structural avidity for T cell recognition, as measured by MHC class II tetramer binding to CD4(+) T cells was only observed in mice immunized with the non-self antigens. T cell hybridomas were generated from mice immunized with the naturally processed self-peptide hGAD65 (552-572) and also from mice immunized with an influenza-derived non-self epitope (HA 306-318). T cells specific for the self peptide failed to bind tetramers and exhibited low functional avidity as measured by the peptide concentration required to reach half-maximum proliferation values. In contrast, T cells specific for the non-self HA (306-318) peptide exhibited high structural and functional avidity profiles. As recently described in studies of murine CD8(+) T cell function, the predominance of low avidity recognition of self-peptide epitopes may be a characteristic feature of CD4(+) T cells responding to autoantigens.

Amino Acid Sequence↗
PubMed2006

A correlation between function and selected measures of T cell avidity in influenza virus-specific CD8+ T cell responses.

Activation of mature CD8+ T cells requires recognition, via the T cell receptor (TCR), of peptide + MHC (pMHC) complexes with an avidity that exceeds a designated threshold. Multiple indicators of T cell avidity have been described that provide unique information on the characteristics of T cell interactions. However, these indicators are routinely used in isolation, and, consequently, little is known about correlations between these measures or which measure, if any, correlates with the quality of the T cell response. Following influenza virus infection of C57BL/6J mice, we analyzed the relative avidities of five epitope-specific CD8+ T cell populations using five different measures. We demonstrated that the quality of CD8+ T cell responses, in terms of cytokine profiles, correlates with TCR dissociation rate and CD8 dependence, but not with the sensitivity to tetramer binding or peptide stimulation. Thus, we propose that, despite significant differences in TCR dissociation rate, the stimulation threshold of influenza-specific CD8+ T cell populations may be equivalent due to compensatory mechanisms largely provided by the CD8 coreceptor. Furthermore, this study shows that different indicators of avidity do not necessarily provide similar information and should be used in combination to obtain an overall picture of the characteristics of TCR binding.

Animals↗
PubMed1992

VLA-4 integrin on sarcoma cell lines recognizes endothelial VCAM-1. Differential regulation of the VLA-4 avidity on various sarcoma cell lines.

Osteosarcomas and rhabdomyosarcomas are vigorously invading tumors. Before they can extravasate to the parenchymal organs and form metastases, they have to adhere to the endothelial cells lining the blood vessels and then penetrate through the endothelium. We show that several human sarcoma cell lines, osteosarcomas HOS, MG-63, U2-OS, and a rhabdomyosarcoma RD, express VLA-4 molecule on their surface and bind to the VCAM-I-expressing activated endothelial cell line Ea.hy 926. The increased sarcoma-cell adhesion could be abolished by treating the sarcoma cells with monoclonal antibodies (MAbs) VLA4 (both alpha- and beta-chain, HP2/1 and 4B4 respectively) or treating endothelial cells with VCAM-I antibody (4B9). Furthermore, we show that sarcoma cells adhere to recombinant soluble VCAM-I protein. On the other hand, these sarcoma cell lines do not express marked amounts of other ligands (such as CDII/18 or sialyl-Lex) for other endothelial adhesion molecules (ICAM-I, ICAM-2, E- and P-selectin) indicating that the VLA-4-VCAM-I dependent pathway might be of major importance in sarcoma extravasation. VLA-4 is not always in an avid form and therefore the expression of VLA-4 does not directly predict adherence to VCAM-I. The avidity of VLA-4 (measured by adherence to soluble VCAM-I) of MG-63 and U2-OS cells could be increased by a 30-min PMA treatment, whereas the avidity of VLA-4 on HOS cells increased only after 48 hr of PMA induction. Our results show that sarcoma cell lines (HOS, MG-63, U2-OS and RD) adhere to stimulated endothelium via VLA-4-VCAM-I adhesion molecules and that VLA-4 avidity on sarcoma cells can be differentially modulated by PMA.

Base Sequence↗
PubMed1993

Slow maturation of IgG1 avidity and persistence of specific IgM in congenital rubella: implications for diagnosis and immunopathology.

Without appropriately timed specimens, serological confirmation of congenital rubella infection may be a problem. We have compared the persistence of specific IgM and low avidity specific IgG1 in 141 sera from 120 cases of serologically confirmed congenital rubella infection with the known time scales for postnatal primary rubella. The results demonstrate that the maturation of the immune response to the rubella virus is abnormally slow in congenital rubella cases both in terms of the isotype switch and especially the development of high avidity specific IgG1. Thus avidity studies may permit serological confirmation of congenital rubella for longer than is possible with tests currently in use. The pathological implications of prolonged low avidity antibody production are discussed.

Aging↗
PubMed2002

Occurrence of hepatitis E virus IgM, low avidity IgG serum antibodies, and viremia in sporadic cases of non-A, -B, and -C acute hepatitis.

Serum samples were taken from 57 patients with sporadic non-A, -B, and -C (Non A, B, C) acute hepatitis at different times after onset of the disease and tested for the presence of the hepatitis E virus (HEV) RNA, IgM, and low avidity IgG antibodies. The viral antibodies were detected using two ELISA. One assay (GL) was produced using a mixture of recombinant peptides specified by ORF2 and ORF3 of the viral genome. The other was produced with an ORF2 specified peptide, pE2. The latter occurs naturally as homodimer, it is recognized strongly in its dimeric form by human sera and, in the primate model, it confers protection against experimental HEV infection. Nineteen samples were positive for one or more of these acute markers of HEV infection, 14 of which were acute sera with elevated ALT levels and 5 were convalescent sera with normal ALT level. The results showed that icteric phase of sporadic hepatitis lasts for about 17 days and it coincides with a period when viremia is subsiding as HEV antibodies are developing. Viremia was intermittent and all but one of the 5 instances were confined to the icteric phase with elevated ALT levels. On two of these occasions, viremia preceded detection of HEV antibody, on another 2 occasions it was concurrent with the detection of pE2 specific IgM and/or low avidity IgG and only in one case of protracted viremia was the viral genome detected concurrently with avid pE2 IgG antibody. Ten (71%) of the 14 acute sera were reactive for pE2 IgM, eight (57%) were reactive for low avidity pE2 IgG, and six (43%) for the GL IgM. The sensitivity for the diagnosis of acute hepatitis E may be increased to 87% by combining pE2 IgM and viremia. GL IgM was detected later, but persisted for a longer period of time than the pE2 antibodies, and it was the only acute antibody detected in the convalescent sera.

Acute Disease↗
PubMed2000

Clinical and diagnostic relevance of the Toxoplasma IgG avidity test in the serological surveillance of pregnant women in Austria.

For evaluation of the medical relevance of a Toxoplasma IgG avidity test within the Austrian program for screening of pregnant women, 23 sera from women with seroconversions (group 1) and with proven latent Toxoplasma infections (group 3), respectively, as well as 92 sera from women suspected of having a primary infection (group 2) were tested by the indirect immunofluorescence test (IFAT), Sabin-Feldman's dye test (SFT), IgM enzyme-linked immunofluorescence assay (ELFA-IgM), IgA microparticle enzyme immunoassay, and the IgG avidity test. Group 1 sera (seroconversions) revealed a median avidity index (AI) of 0.25, whereas the median AI of group 3 sera (latent infections) was 0.66. In 31 (33.7%) of 92 cases suspected of involving a primary Toxoplasma infection, low (<0.41) or borderline AIs (0.41-0.50) were assessed, and in 61 cases (66.3%) the AIs exceeded 0.50. Finally, a recent infection could be excluded due to the results of the IgG avidity test in 59 cases; in at least 34 IgM-positive cases an unnecessary and, thus, unjustified treatment could be avoided.

Animals↗
PubMed2006

Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis.

The precise diagnosis of an acute and recent Toxoplasma infection in pregnant women and the newborn child is important before treatment. This study describes a new Toxoplasma gondii IgG avidity test based on a combination of recombinant GRA1, GRA7 and SAG1 antigens and shows that this test is useful for diagnostic purposes and may replace the lysed, whole-cell antigens. Although more sera need to be tested, the results obtained here suggest that the IgG avidity test performed with rec-antigens correlated more with the stage of a T. gondii infection than the IgG avidity results obtained with the lysed, whole-cell antigen test, the VIDAS Toxo IgG avidity (bioMérieux).

Animals↗
PubMed1979

Low avidity as a cause of prozone phenomena.

The incidence of prozones was studied, under various conditions, in a model indirect haemagglutination system and in a radioimmunoassay--both assays depending on the use of anti-immunoglobulin (anti-Ig). It was found that prozones were correlated with the presence of low avidity antibodies, and that these could compete with high avidity antibodies for the limited amount of anti-Ig available. It is proposed that the relatively rapid dissociation of low avidity antibodies allows them to form immune aggregates with the anti-Ig. With increasing size these aggregates would become more susceptible to being washed off. In this way low avidity antibodies could occupy the anti-Ig, and yet be relatively ineffective, either for haemagglutination or for the binding of radioactively labelled (or fluorescein-labelled) anti-Ig to the antigen.

Animals↗
PubMed1987

Estimating the relative avidity of mucosal IgA for antigen.

This report concerns the application of a method to estimate the relative avidity of mucosal IgA for casein and beta-lactoglobulin. The chaotropic ion, thiocyanate, was used to disrupt antigen-antibody binding in an enzyme immunoassay. The resultant proportional decrease in optical density of the enzyme immunoassay was directly related to the proportion of IgA eluted from the solid phase-bound antigen. The relative avidity was expressed as the molarity of KSCN resulting in a particular reduction in optical density. The measurement of avidity, the avidity index, was independent of the concentration of mucosal IgA. For anti-casein IgA the interassay coefficient of variation of the index was 14-18%, and the intra-assay coefficient of variation was 6%. For anti-beta-lactoglobulin IgA the interassay coefficient of variation was 10-13% and the intra-assay coefficient of variation was 6%. The method is simple and free of complicated calculations.

Antibody Affinity↗
PubMed1993

Avidity of antibody and agglutinability of antibody-sensitized latex in latex agglutination test.

This paper describes the avidity of IgG antibody used for preparation of latex sensitized with IgG antibody (IgG-sensitized latex) and the agglutinability of IgG-sensitized latex in slide reversed passive latex agglutination (RPLA). Using immunodiffusion techniques, it was found that anti-canine C-reactive protein (CRP) sera from four rabbits immunized with canine CRP had the same antibody titers. However, the antibodies had different levels of avidity. When lattices were sensitized under the same condition with the IgG antibodies of different avidity levels separated from the above-mentioned antisera using Protein A and canine CRP-Sepharose 4B immunosorbent, these demonstrated different patterns of agglutinability in slide RPLA. The latex sensitized with IgG antibody of higher avidity demonstrated a stronger agglutinability.

Agglutination↗
PubMed1982

An ELISA for the estimation of high-avidity and total specific IgG and IgM antibodies to rubella virus.

High-avidity and total specific IgG and IgM antibodies to rubella virus were measured by using a curve-fitting analysis for the dose-response curves of enzyme-linked immunosorbent assay (ELISA). The analysis of follow-up samples from 14 patients with primary rubella infection revealed that these two antibody categories had different kinetics during the time after rash. The high-avidity IgG antibodies increased more slowly than total specific IgG antibodies. In the IgM response, high-avidity antibodies reached their maximum slightly earlier than total specific antibodies. After reinfection with rubella, 14-30 days after the illness, convalescent patients had higher amounts of high-avidity IgG antibodies when compared to the patients who suffered from primary rubella infection. These findings are in agreement with the results obtained by measuring antibody affinity in experimental animals after immunization.

Antibodies, Viral↗
PubMed1992

Persistence of specific IgM and low avidity specific IgG1 following primary rubella.

Persistence of specific IgM in sera following primary rubella infection was compared with the maturation of the specific IgG1 response. 206 sera, from 171 patients with primary rubella, taken 1 day to 2.5 years after onset of illness, were tested. Rubella-specific IgM was detected by M-antibody capture radioimmunoassay in 100% of sera taken 15-28 days after onset, but in only 9% taken 3-4 months after onset. However, using the diethylamine (DEA) shift value (DSV) method, low avidity specific IgG1 was detected in 91% sera taken at 3-4 months and at 5-7 months 21% of sera remained positive. Using an avidity index method, with urea in the wash buffer, none of the sera were positive for low avidity specific IgG1 beyond 3 months after onset. With DEA in the wash buffer, the number of sera positive rose to 38% at 3-4 months. Thus, the DSV method for detecting low avidity specific IgG1 is a useful additional test for confirming or refuting a diagnosis of primary rubella and is of particular value for assessing pregnant patients.

Antibodies, Viral↗