Determination Of Avidity Research Articles

Experimental Determination of Antibody Affinity and Avidity: Guidance and Considerations.

Experimental Determination of Antibody Affinity and Avidity: Guidance and Considerations.

IgY Antibodies from Birds: A Review on Affinity and Avidity.

IgY antibodies are found in the blood and yolk of eggs. Several studies show the feasibility of utilising IgY for immunotherapy and immunodiagnosis. These antibodies have been studied because they fulfil the current needs for reducing, replacing, and improving the use of animals. Affinity and avidity represent the strength of the antigen-antibody interaction and directly influence antibody action. The aim of this review was to examine the factors that influence the affinity and avidity of IgY antibodies and the methodologies used to determine these variables. In birds, there are few studies on the maturation of antibody affinity and avidity, and these studies suggest that the use of an adjuvant-type of antigen, the animal lineage, the number of immunisations, and the time interfered with the affinity and avidity of IgY antibodies. Regarding the methodologies, most studies use chaotropic agents to determine the avidity index. Studies involving the solution phase and equilibrium titration reactions are also described. These results demonstrate the need for the standardisation of methodologies for the determination of affinity and avidity so that further studies can be performed to optimise the production of high avidity IgY antibodies.

Development and validation of a microfluidic multiplex immunoassay for the determination of levels and avidity of serum antibodies to tetanus, diphtheria and pertussis antigens

A multiplex assay for the quantitation of immunoglobulin G (IgG) serum antibodies directed against Clostridium tetani toxin (TT), Corynebacterium diphtheriae toxoid (DTxd), and the Bordetella pertussis antigens pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (Prn) was developed on an Evalution® platform to enhance the evaluation of the specific antibody response towards protein antigens in suspected humoral immunodeficiencies. Evalution® is a microfluidic and microparticle-based platform with the possibility to analyse single samples and to perform real-time kinetic measurements of antibody binding. All individual antigens were covalently linked to the carboxylated microparticles after which samples and fluorescently labelled detection antibodies were flowed over the microparticles in the microfluidic channels of the assay cartridges of the system. The developed assay showed very good sensitivity, specificity, and intra- and inter-assay coefficients of variation (CVs for the different antigens between 1.72-3.53% and 3.54‐5.79%, respectively). Furthermore, the correlation of the Evalution pentaplex with a Luminex pentaplex using a panel of 48 human serum samples was excellent, with Spearman correlation coefficients between 0.936 for PT and 0.982 for DTxd (p < 0.0001 for all). Finally, we showed in a proof-of-concept experiment the potential of the Evalution® platform to simultaneously measure concentrations and binding kinetics (as a surrogate for avidity) of the IgG antibodies to the selected protein antigens. Overall, these findings show that this new Evalution pentaplex can accurately measure the antibody response to TT, DTxd, PT, FHA and Prn. It also has the potential to measure antibody binding and dissociation kinetics.

Study of IgG avidity and the level of specific IgA antibodies and their significance in the diagnosis of human toxocarosis

Toxocarosis is a zoonotic disease caused by migration and subsequent localization of nematode larvae of Toxocara spp. in human organs and tissues, which is manifested with development of various non-specific clinical symptoms. Main diagnostic methods are serological and consists in proving the presence of anti-Toxocara IgG antibodies in patient's sera. In humans, anti-Toxocara IgG has been shown to persist in the serum for a long time and cannot be used to distinguish between past and recent infection.Aim of the present work is to investigate the diagnostic significance of the specific IgG avidity level, determined by an immuno enzyme test developed by us, and the presence of anti-Toxocara IgA for distinguishing between acute and chronic toxocarosis.The study included 130 patients with positive results in routine serological ELISA and Western blot tests and with clinical symptoms of visceral and ocular toxocarosis. The results revealed low IgG avidity (≤40%) in nine (7.3%) and presence of anti-Toxocara IgA antibodies in 36 (26.2%) of the subjects. Low avidity of IgG antibodies was found only in the first tests, and a presence of specific IgA for up to 9 months. The results of our study give us reason to believe that determination of the IgG avidity in toxocarosis is of greater diagnostic value than the presence of IgA to establish the stage of the disease.

The variability of the serological response to SARS-corona virus-2: Potential resolution of ambiguity through determination of avidity (functional affinity).

Data on the serological response toward severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in 16 recent reports were analyzed and a high degree of variability was shown. Immunoglobulin M (IgM) responses were either found earlier than IgG, or together with IgG, later than IgG, or were missing. Therefore, clear distinctions between early, intermediate, and past infections are obviously not possible merely on the basis of IgM and IgG determinations. A review of publications on the serology of other virus groups shows that variable IgM responses can be found as well and therefore are not unique for SARS‐CoV‐2 infections. A model to explain this variability is proposed. The inclusion of avidity determination into regular diagnostic procedures has allowed to resolve such “atypical” serological constellations. The potential use of avidity determination for the diagnosis of COVID‐19, for risk assessment, epidemiological studies, analysis of cross reactions, as well as for the control of vaccination programs is suggested and discussed.

Diagnostic significance of immunoglobulin G avidity in symptomatic and asymptomatic West Nile virus infection.

West Nile virus (WNV) immunoglobulin M (IgM) antibodies have been shown to persist for up to 500 days in certain patients. To evaluate the usefulness of immunoglobulin G (IgG) avidity assessment in the diagnosis of WNV infection, we analyzed 54 WNV IgM- and/or IgG-positive serum samples from 39 patients with neuroinvasive disease and 15 asymptomatic cases tested during a seroprevalence investigation. Serological tests (WNV IgM/IgG antibody detection, IgG avidity) were performed using commercially available enzyme-linked immunosorbent assays. WNV IgM antibodies were detected in 47 (87%) samples. Acute/recent WNV infection was confirmed based on low/borderline avidity index (AI) in 44 IgM-positive samples (93.6%). In three IgM-positive samples (6.4%), high IgG AIs were detected, thus indicating persisting IgM antibodies from previous infections. All IgM-negative samples showed high AIs. Patients with WNV neuroinvasive disease tested within 30 days showed low AIs. In six patients tested 34-50 days after disease onset, AI was borderline (42%-60%), suggesting earlier WNV IgG maturation. Samples with the highest IgM values were associated with the lowest AIs (Spearman's rho coefficient -0.767, p < 0.001). Our results indicate that IgG avidity differentiates current/recent WNV infection from persistent IgM seropositivity from the previous WNV transmission season both in patients with WNV neuroinvasive disease and in asymptomatic persons. A strong negative correlation between IgM antibody levels and AI indicates that in cases with very high IgM levels, determination of IgG avidity may not be necessary. As many patients showed rapid avidity maturation, low IgG avidity is indicative of WNV infection within the previous month.

Determination of avidity of IgG against protein antigens from Streptococcus pneumoniae: assay development and preliminary application in clinical settings.

The measurement of antibody levels is a common test for the diagnosis of Streptococcus pneumoniae infection in research. However, the quality of antibody response, reflected by avidity, has not been adequately evaluated. We aimed to evaluate the role of avidity of IgG against eight pneumococcal proteins in etiologic diagnosis. Eight pneumococcal proteins (Ply, CbpA, PspA1 and 2, PcpA, PhtD, StkP-C, and PcsB-N) were used to develop a multiplex bead-based avidity immunoassay. The assay was tested for effects of the chaotropic agent, multiplexing, and repeatability. The developed assay was applied to paired samples from children with or without pneumococcal disease (n=38 for each group), determined by either serology, polymerase chain reaction (PCR), or blood culture. We found a good correlation between singleplex and multiplex assays, with r≥0.94.The assay was reproducible, with mean inter-assay variation≤9% and intra-assay variation<6%. Children with pneumococcal disease had lower median avidity indexes in the acute phase of disease for PspA1 and 2 (p=0.042), PcpA (p=0.002), PhtD (p=0.014), and StkP-C (p<0.001). When the use of IgG avidity as a diagnostic tool for pneumococcal infection was evaluated, the highest discriminative power was found for StkP-C, followed by PcpA (area under the curve [95% confidence interval, CI]: 0.868 [0.759-0.977] and 0.743 [0.607-879], respectively). The developed assay was robust and had no deleterious influence from multiplexing. Children with pneumococcal disease had lower median avidity against five pneumococcal proteins in the acute phase of disease compared to children without disease.

Avidity of antineurocytoskeletal antibodies in Alzheimer's disease patients.

To optimise the ELISA method for the avidity of IgG antibodies against neurofilament heavy chain (NfH) and to determine the levels and avidity of anti-NfH antibodies in patients with Alzheimer's disease (AD) and a healthy control group. Various dilutions of sera and concentrations of urea and sodium chloride as chaotropic reagents were tested in the process of the ELISA optimisation. The levels and avidity of anti-NfH antibodies were determined in 30 patients with Alzheimer's disease and 30 age-matched cognitively normal elderly adults. Sera dilution 1:200 and urea as a chaotrope in a concentration 6 mol/L were chosen to be the most suitable for the avidity assay of anti-NfH antibodies by ELISA. The results showed no differences in either level or avidity of IgG anti-NfH antibodies between AD patients and cognitively normal persons. The levels of anti-NfH IgG antibodies inversely correlated with their avidities. We optimised the ELISA method for the determination of anti-NfH antibody avidity determination which is suitable for research of anti-NfH antibody avidity in patients with neurological diseases associated with neurocytoskeletal defects. The determination of serum anti-NfH antibody avidity in AD patients seems to have limited diagnostic significance.

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.

Current diagnosis of mixed herpesvirus infection in children with viral encephalitis

Sixty-five children aged 1 month to 15 years with a diagnosis of viral encephalitis, who had been admitted to the Clinic of the Pediatric Research and Clinical Center for Infectious Diseases, Federal Biomedical Agency, in 2013-2016 were examined. Objective: to establish the frequency of mixed herpesvirus infection in children with viral encephalitis, by using the current diagnostic methods to determine the role of reactivation of herpesvirus infection on the development of viral encephalitis. The investigators identified anti-herpesvirus antibodies and their avidity using an enzyme immunoassay and anti-cytomegalovirus antibodies specific for antigens by immunoblotting applying the kits. Herpesvirus DNA was detected in the biological material (blood, cerebrospinal fluid) by real-time polymerase chain reaction (PCR) and by electrophoresis using the PCR kits (Central Research Institute of Epidemiology, Russian Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Moscow). Young children were shown to develop the disease as a result of primary infection more commonly as monoinfection in the old age groups during the persistence of one or two herpesviruses in the body. In encephalitis caused by herpes simplex virus type 1 and type 2, reactivation of cytomegalovirus that could be a cofactor in the development of brain inflammation was proven by the current diagnostic technique immunoblotting. The comprehensive laboratory approach to the etiological diagnosis of viral encephalitis, which encompassesthe determination of antibody classes, avidity, and antibodiesto individual protein determinants of viral particles, makes it possible to specify the activity of different herpesviruses in mixed infection and to determine the stage of the infectious process, which is important for the choice of therapeutic policy.

Determination of IgG avidity in BALB/c mice experimentally infected with Toxocara canis.

Toxocariasis is a zoonotic disease in that IgM titers can remain high for long periods making difficult to determine the stage of the disease. The aim of this study is to investigate the applicability of indirect ELISA, associated with urea, to discriminate between the acute and chronic toxocariasis. IgG avidity was evaluated in 25 BALB/c mice experimentally infected with 1000 Toxocara canis eggs. Blood samples were collected, and sera treated with 6 M urea and assayed by ELISA every two weeks. The percent IgG avidity was determined using the mean absorbance of sera treated with urea, divided by the mean absorbance of untreated sera. In the first 15 days post-inoculation, was observed a low percentage, between 7.25 and 27.5%, IgG avidity, characteristic of an acute infection. After 60 days of infection, all the mice showed between 31.4 and 58% IgG avidity, indicating a chronic infection.

Clinical evaluation of the Roche Elecsys® CMV IgG Avidity assay

Congenital cytomegalovirus (CMV) infection has potentially severe consequences in newborns. The testing of pregnant women for CMV-specific antibodies may be useful for the identification of women at risk of transmitting the infection to the fetus. The determination of CMV IgG avidity helps to establish the timing of infection as IgG avidity matures during the course of infection. This study examines the performance of the Elecsys® CMV IgG Avidity assay using preselected samples from patients at different phases of CMV infection. The Elecsys® CMV IgG Avidity assay was tested at three sites using sequential samples from patients with recent primary CMV infection, as well as single samples from patients with recent primary or past CMV infection. The Elecsys® assay discriminated well between early (low avidity) and late (high avidity) phases of infection in sequential serum samples. Overall, 98.8 % of low-avidity samples corresponded to infection onset <180 days before sampling and 77.8 % of all high-avidity results corresponded to infection onset >90 days before sampling. The assay’s sensitivity was 90–97 %, with specificity ranging from 89 to 100 %, depending on the consideration of gray-zone avidity values. Single samples from recent primary or past infection showed similar distributions of avidity results. The Elecsys® CMV IgG Avidity assay results are in agreement with preselected samples from patients with primary or past CMV infection, showing that the test is an adequate predictor of the phase of infection.

Avidity of anticardiolipin antibodies—A factor that could be important for their detection by ELISA methods

Avidity is an important feature of antibodies associated with their pathogenic effects. Anticardiolipin antibodies (aCLs) are the most commonly examined antiphospholipid antibodies, however, their avidity has been investigated only marginally. The aim of the study was to compare the avidity of the antibodies specifically bound to the cardiolipin-coated microtitrate wells with those bound to the wells bearing no cardiolipin in various conditions. We analysed 22 serum samples with high, medium and low levels of aCL IgG. The avidity of aCL IgG was determined in serially diluted sera by the ELISA method in the presence of increasing urea concentrations (from 2 to 8mol/L) as a chaotropic agent. The serum dilution 1:50 and 1:100 and the concentrations of urea 6mol/L and 8mol/L seemed to be suitable for the determination of aCL avidity. The avidity of antibodies specifically bound to the cardiolipin and those bound to the cardiolipin-free wells significantly differed in their avidity. The simultaneous determination of higher-avidity aCL and lower-avidity polyspecific antibodies, whose presence complicates the ELISA methods by an increase of the background signal, might limit the shortcomings of some ELISA methods.

Recent infection testing algorithm (RITA) applied to new HIV diagnoses in England, Wales and Northern Ireland, 2009 to 2011

In 2009, Public Health England (PHE) introduced the routine application of a recent infection testing algorithm (RITA) to new HIV diagnoses, where a positive RITA result indicates likely acquisition of infection in the previous six months. Laboratories submit serum specimens to PHE for testing using the HIV 1/2gO AxSYM assay modified for the determination of HIV antibody avidity. Results are classified according to avidity index and data on CD₄ count, antiretroviral treatment and the presence of an AIDS-defining illness. Between 2009 and 2011, 38.4% (6,966/18,134) of new HIV diagnoses in England, Wales and Northern Ireland were tested. Demographic characteristics of those tested were similar to all persons with diagnosed HIV. Overall, recent infection was 14.7% (1,022/6,966) and higher among men who have sex with men (MSM) (22.3%, 720/3,223) compared with heterosexual men and women (7.8%, 247/3,164). Higher proportions were among persons aged 15-24 years compared with those ≥50 years (MSM 31.2% (139/445) vs 13.6% (42/308); heterosexual men and women 17.3% (43/249) vs 6.2% (31/501)). Among heterosexual men and women, black Africans were least likely to have recent infection compared with whites (4.8%, 90/1,892 vs 13.3%, 97/728; adjusted odds ratio: 0.6; 95% CI: 0.4-0.9). Our results indicate evidence of ongoing HIV transmission during the study period, particularly among MSM.

Autochtonous hepatitis E, an emerging infection: case report

Hepatitis E is rare in France but its increasing frequency makes it an emerging infection. Autochtonous hepatitis E is prevalent, largely confined to older men and currently caused by gentotype 3f. Patients with unexplained hepatitis should be tested by hepatitis E, even in the absence of travel from endemic areas. The diagnosis is based on serological testing (including detection of specific antibodies IgM and IgG, and sometimes by determination of antibody avidity) and nucleic acid amplification techniques which might used first.

Determination of serological profiles and avidity of specific antibodies in the sera of patients with potential Epstein-Barr virus (EBV) infection

Epstein-Barr Virus, frequently referred to as EBV, is a member of the herpesvirus family and one of the most common human viruses. The virus occurs worldwide, and most people become infected with EBV sometime during their lives. The purpose of this study was to investigate the serological profiles of specific antibodies among the sera of suspected EBV infection patients along with VCA-IgG avidity. A total of 522 patient's sera were sent to The Clinical Microbiology Laboratory for EBV specific antibody detection and were studied by IFA method during a two year period. The serum samples were tested for EBV specific VCA IgG, VCA IgM, EA, EBNA antibodies and VCA IgG aviditity. Among 33 patients those who had low avidity for VCA IgG, 27 (81.8 %) of them had a serologic profile as follows; positive VCA IgG, negative VCA IgM, negative EA and negative EBNA. While this profile is considered as a primary infection, the frequency of the coexistence of VCA IgG low avidity with this profile is interpreted that avidity may lead to detect primary infection (Tab. 2, Ref. 25)

Assessment of IgG avidity against pertussis toxin and filamentous hemagglutinin via an adapted enzyme-linked immunosorbent assay (ELISA) using ammonium thiocyanate

Antibody avidity, defined as the strength of binding between antibody and antigen, represents a functional measure of affinity maturation of antibodies. Determination of the antibody avidity is usually performed separating high and low avidity antibodies by dissociating agents, but measurement of the antibody avidity in humans is rather complicated, due to the heterogeneity of the antibodies produced in response to complex antigens, e.g. after vaccinations.The purpose of the present study was to evaluate the experimental determinants of the assessment of avidities of IgG antibodies directed against pertussis toxin (IgG-anti-PT) and filamentous hemagglutinin (IgG-anti-FHA) produced after pertussis vaccination using an adapted ELISA and ammonium thiocyanate (NH4SCN) as dissociating agent.Our experiments revealed that the results of avidity testing depend very much on experimental conditions and may over- or underestimate the relative avidity of IgG-anti-PT and IgG-anti-FHA antibodies. Whereas in our findings avidity seems to be independent from the initial antibody concentration in a wide range of measures, RAI depends on NH4SCN concentration, time of incubation and temperature of the reaction.The presented method allows an accurate measurement of the IgG antibody avidity against both Bordetella pertussis antigens PT and FHA, using NH4SCN as chaotropic agent in concentrations lower than 3.0M for 20min time of incubation at 37°C. Different experimental conditions in testing pertussis-specific IgG antibody avidity should be considered in interpretation and comparability of data of different studies.

Diagnosis of recent primary rubella virus infections: Significance of glycoprotein-based IgM serology, IgG avidity and immunoblot analysis

Reliable serodiagnosis of rubella virus (RV) infections requires discrimination of specific IgM induced by primary rubella from persistent, reactivated or non-specific IgM reactivity. Sera from 130 pregnant women with recent or past RV infection/vaccination, persistent IgM or negative rubella serology, 26 patients with other acute infections and 5 patients with rheumatoid factor-positivity were analyzed for RV-specific IgM by ELISA coated with whole-virus lysate or native glycoprotein, followed by determination of IgG avidity and E2-specific IgG using lysate-coated ELISA and non-reducing immunoblot. Compared to a reference μ-capture IgM ELISA, the sensitivity for diagnosing recent rubella infection/vaccination was 90.0% and 100% for the lysate-based and glycoprotein-based IgM ELISA, respectively. With respect to women with past RV infections or negative histories of RV infection/vaccination, both assays were 97.5–100% specific, whereas for patients with other acute infections the glycoprotein substrate provided a specificity of 92.3% compared to only 80.8% using whole-virus antigen. Analyzing anti-RV IgG avidity and anti-E2 IgG reactivity allowed the time point of primary infection to be determined unambiguously in >86% of samples. In conclusion, using RV glycoprotein antigen improves the specificity of indirect IgM ELISA. In cases of RV-specific IgM reactivity, recent primary rubella infection can be confirmed or excluded efficiently by specific IgG avidity and immunoblot analysis.

Comparative evaluation of eight commercial human cytomegalovirus IgG avidity assays

Background The interpretation of a positive IgM antibody result to human cytomegalovirus (HCMV) in a pregnant woman is of major importance for the correct management of the pregnancy. Determination of HCMV-specific IgG avidity is considered an useful approach for distinguishing IgM antibody due to primary HCMV infection from IgM antibody elicited during non-primary infection. Objective Comparative evaluation of eight commercial HCMV IgG avidity assays currently available in Europe. Study design A panel of 198 sequential samples collected from 65 pregnant women at 0–90, 91–180, and >180 days after the onset of primary HCMV infection was retrospectively tested by Abbott, BioMérieux, Bio-Rad, DiaSorin, Diesse, Euroimmun, Radim, and Technogenetics HCMV IgG avidity assays according to the manufacturer's instructions. Results None of the 198 samples tested yielded identical scores by the kits under evaluation. The Euroimmun and Radim assays showed the best correlation with expected results in terms of low (0–90 days), intermediate (90–180 days) and high (>180 days) avidity results, respectively. The best accuracy in diagnosing a recent (<90 days after the onset) or non-recent (>180 days after the onset) primary HCMV infection was shown by Radim followed by Euroimmun and Diesse. The best correlation with a well established in-house developed HCMV IgG avidity assay was shown by Radim. Conclusions HCMV IgG avidity kits need to be improved and standardized. In the meantime, highly specific IgM assays are preferable for screening purposes in pregnant women.

Toxoplasmosis in pregnancy: determination of IgM, IgG and avidity in filter paper-embedded blood

To detect immunoglobulin M (IgM) anti-Toxoplasma gondii antibodies and determine immunoglobulin G (IgG) titer and avidity in filter paper-embedded blood (FPEB) samples of pregnant women. A total of 100 FPEB samples of pregnant women (30 positive and 70 negative) were analyzed for anti-T. gondii-specific IgM antibodies. Eleven and nine pairs of serum and FPEB samples were used to standardize IgG titration and avidity, respectively. Then, the correlation of avidity results was determined with 23 serum/FPEB pairs from IgG-positive cases. IgM detection in FPEB was 92% sensitive and 100% specific. The titration of IgG antibodies in FPEB correlated with that of serum (r >or=0.9). Significant difference in avidity between the acute and the undetermined/chronic cases was observed in both samples. As expected, no correlation was found between IgM levels and avidity. The FPEB is useful to infer infection phase, and thus to speed clinical decisions in congenital toxoplasmosis management.