Antigen-specific ELISAs Research Articles

Antigen-Specific Detection of Autoantibodies Against Myeloperoxidase (MPO) and Proteinase 3 (PR3).

ANCA testing was introduced in many laboratories throughout the world when it was recognized that a significant subset of patients with small vessel vasculopathies presented with such antibodies. Many laboratories developed and introduced in-house testing methods for antigen-specific ANCA detection complementary to indirect immune fluorescence screening. Such in-house tests have proven their merit in diagnosing vasculitis and were important to identify critical steps in the development of antigen-specific assays with high sensitivity and specificity. In the meantime various commercial assays became available for antigen-specific ANCA testing. Because of the high diagnostic accuracy of such assays it can be anticipated that commercial, antigen-specific tests will completely replace in-house testing for MPO- and PR3-ANCA. Furthermore, such tests will replace the need for IIF in the diagnostic workup of AAV. In this light it can be foreseen that the knowledge that underlies the development of in-house ANCA testing will gradually disseminate over time. Therefore we describe the current antigen-specific ANCA ELISAs (direct and capture) with the intention to maintain the knowledge and the identification of the critical steps in the development of robust assays.

Affinity maturation of anti-lipid IgG antibodies produced via germinal center

Abstract Non-bilayer phospholipid arrangements (NPA) are lipid associations different to the bilayer and are found on cell membranes; they participate in different cellular functions and are transient, but when they are stabilized by drugs such as chlorpromazine, they become immunogenic and induce the formation of anti-NPA antibodies. Mice that receive stabilized NPA share several characteristics with patients with systemic lupus erythematosus, such as the presence of anti-cardiolipin, anti-histone and anti-nuclear antibodies, lupus band, malar exanthema and glomerulonephritis. Mice with this lupus model have been shown to develop the disease as a consequence of the production of anti-NPA antibodies; in addition, most of these antibodies are of the IgG class and are produced mainly by the germinal center pathway. In the case of protein antigens, germinal centers are the sites where class-switch recombination, somatic mutation, memory generation and affinity maturation occur. To determine if affinity maturation, a hallmark of the antibodies produced via germinal center reactions, occurs in the anti-NPA IgG antibodies produced in germinal centers, we performed antigen-specific ELISAs, both in the absence and in the presence of urea. We observed that the percentage of urea-resistant (high affinity) anti-NPA IgG antibodies in relation to the total anti-NPA IgG antibodies increases over time, and it correlates with the number of NPA-specific germinal center B cells.

Cross-reactivity of anti-PLA2R1 autoantibodies to rabbit and mouse PLA2R1 antigens and development of two novel ELISAs with different diagnostic performances in idiopathic membranous nephropathy.

About 70% of patients with idiopathic membranous nephropathy (iMN) have autoantibodies to the phospholipase A2 receptor PLA2R1. We screened sera from iMN patients for their cross-reactivity to human (h), rabbit (rb) and mouse (m) PLA2R1 by western blot (WB) and antigen-specific ELISAs. All iMN patients recognized hPLA2R1 and rbPLA2R1 by WB, and a rbPLA2R1 ELISA was as sensitive as the standardized hPLA2R1 ELISA to monitor anti-PLA2R1 in patients with active disease or in drug-induced remission. In contrast, only 51% of patients were reactive to mPLA2R1 by WB, and a maximum of 78% were weakly to highly positive in the mPLA2R1 ELISA, suggesting that iMN patients exhibit different subsets of anti-PLA2R1 autoantibodies against epitopes that are shared or not among PLA2R1 orthologs. In a cohort of 41 patients with a mean follow-up of 42 months from anti-PLA2R1 assay, the detection of anti-mPLA2R1 autoantibodies was an independent predictor of clinical outcome in multivariate analysis (p=0.009), and a ROC curve analysis identified a threshold of 605 RU/mL above which 100% of patients (12 patients) had a poor renal outcome (p<0.001). A similar threshold could not be defined in hPLA2R1 and rbPLA2R1 ELISAs. We conclude that rbPLA2R1 is an alternative antigen to hPLA2R1 to measure anti-PLA2R1 in active disease while mPLA2R1 is a unique antigen that can detect a subset of anti-PLA2R1 autoantibodies present at high levels (>605RU/mL) only in iMN patients at risk of poor prognosis, and is thus useful to predict iMN outcome.

Angiogenic cytokines are antibody targets during graft-versus-leukemia reactions.

The graft-versus-leukemia (GVL) reaction is an important example of immune-mediated tumor destruction. A coordinated humoral and cellular response accomplishes leukemia cell killing, but the specific targets remain largely uncharacterized. To learn more about the antigens that elicit antibodies during GVL reactions, we analyzed patients with advanced myelodysplasia (MDS) and acute myelogenous leukemia (AML) who received an autologous, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell vaccine early after allogeneic hematopoietic stem cell transplantation (HSCT). A combination of tumor-derived cDNA expression library screening, protein microarrays, and antigen-specific ELISAs were used to characterize sera obtained longitudinally from 15 patients with AML/MDS who were vaccinated early after allogeneic HSCT. A broad, therapy-induced antibody response was uncovered, which primarily targeted intracellular proteins that function in growth, transcription/translation, metabolism, and homeostasis. Unexpectedly, antibodies were also elicited against eight secreted angiogenic cytokines that play critical roles in leukemogenesis. Antibodies to the angiogenic cytokines were evident early after therapy, and in some patients manifested a diversification in reactivity over time. Patients that developed antibodies to multiple angiogenic cytokines showed prolonged remission and survival. These results reveal a potent humoral response during GVL reactions induced with vaccination early after allogeneic HSCT and raise the possibility that antibodies, in conjunction with natural killer cells and T lymphocytes, may contribute to immune-mediated control of myeloid leukemias.

Maintenance therapy using rituximab-induced continuous B cell depletion for ANCA vasculitis

Propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The aim of this study was to compare the IgG subclass distribution and avidity of MPO-ANCA in sera from patients with primary ANCA-associated vasculitis (AASV) and PTU-induced vasculitis.Nineteen patients with primary AASV with MPO-ANCA and thirteen patients with PTU-induced vasculitis were enrolled in the current study. Sera in both active phase and remission were collected. Anti-MPO IgG subclasses were detected by antigen specific ELISAs using specific monoclonal antibodies as second antibodies, and MPO-ANCA avidity was assessed by antigen-inhibition ELISAs.In primary AASV, all four anti-MPO IgG subclasses could be detected in active phase with IgG1 (100%), IgG2 (73.7%), IgG3 (63.2%) and IgG4 (94.7%), and in remission, IgG1 and IgG4 subclasses in most patients remained positive. However, in PTU-induced vasculitis, anti-MPO IgG3 subclass could not be detected, the anti-MPO IgG subclasses in active phase were IgG1 (100%), IgG2 (61.5%) and IgG4 (46.2%). Furthermore, five out of the six patients (88.8%) with PTU-induced vasculitis with positive IgG4 subclass in active phase turned to negative in remission, however, only eight out of the fourteen patients (57.1%) with primary AASV turned to negative. The median avidity constant of MPO-ANCA was 56 (8.96 to >140) × 107 mol/l for patients with primary AASV and 0.7 (<0.28 to >140) × 107 mol/l for patients with PTU-induced vasculitis respectively. Furthermore, the relative levels of MPO-ANCA avidity were associated with elevation of ESR in primary AASV and were associated with BVAS scores in patients with PTU-induced vasculitis, respectively.MPO-ANCA IgG subclass distribution and avidity were different between patients with primary AASV and PTU-induced vasculitis. It was suggested that the mechanism of ANCA production in PTU-induced vasculitis was different from that in primary AASV, and the avidity of MPO-ANCA might be associated with disease activity.

Comparative evaluation of novel African swine fever virus (ASF) antibody detection techniques derived from specific ASF viral genotypes with the OIE internationally prescribed serological tests

The presence of antibodies against African swine fever (ASF), a complex fatal notifiable OIE disease of swine, is always indicative of previous infection, since there is no vaccine that is currently used in the field. The early appearance and subsequent long-term persistence of antibodies combined with cost-effectiveness make antibody detection techniques essential in control programmes. Recent reports appear to indicate that the serological tests recommended by the OIE for ASF monitoring are much less effective in East and Southern Africa where viral genetic and antigenic diversity is the greatest. We report herein an extensive analysis including more than 1000 field and experimental infection sera, in which the OIE recommended tests are compared with antigen-specific ELISAs and immuno-peroxidase staining of cells (IPT). The antibody detection results generated using new antigen-specific tests, developed in this study, which are based on production of antigen fractions generated by infection and virus purification from COS-1 cells, showed strong concordance with the OIE tests. We therefore conclude that the lack of success is not attributable to antigenic polymorphism and may be related to the specific characteristics of the local breeds African pigs.

Antigen-Specific ANCA ELISAs Have Different Sensitivities for Active and Treated Vasculitis and for Nonvasculitic Disease

This study evaluated the performance of 12 assays for antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO) in 55 active and 68 treated cases of vasculitis and in nonvasculitic disease. It examined within- and between-assay precision; binding curves, binding levels, and interassay consistency; and sensitivity, specificity, and receiver operating characteristic analysis. All assays were highly sensitive for active vasculitis (median, 94%; range, 91%-96%), but sensitivities were more varied in treated disease (median, 69%; range, 57%-82%). Binding curves and binding levels were also very variable in PR3-ANCA and MPO-ANCA assays. This has implications for studies correlating ANCA levels with disease activity and in developing ANCA-based treatment guidelines. PR3-ANCA and MPO-ANCA assays need to be standardized as a matter of urgency, but in the meantime, individual laboratories must understand the limitations of the assays used, especially with low-level ANCA in treated vasculitis and nonvasculitic disease.

The target antigens of antineutrophil cytoplasmic antibodies (ANCA) induced by propylthiouracil

ObjectiveAntineutrophil cytoplasmic antibody (ANCA) has been well documented in association with propylthiouracil (PTU), and some patients with PTU-induced ANCA also develop clinical vasculitis. The aim of the current study was to detect ANCA specificities in sera from patients with PTU-induced ANCA with and without clinical vasculitis. MethodsSera from 65 patients with PTU-induced ANCA were collected, and 27 of these patients were diagnosed with PTU-induced ANCA associated systemic vasculitis (AASV). Indirect immunofluorescence assay and antigen-specific ELISAs were used to detect ANCA and their antigen specificities. The seven known target antigens included myeloperoxidase (MPO), proteinase 3, human leukocyte elastase, lactoferrin, cathepsin G, azurocidin and bactericidal/permeability-increasing protein (BPI). ResultsIn IIF assay, P-ANCA was found in 58/65 (89.2%) sera, C-ANCA in two, both P-ANCA and C-ANCA in five, respectively. MPO (60%) and lactoferrin (63.1%) were the two most common target antigens detected in sera from all the patients. 25/27 sera from patients with PTU-induced AASV recognized multiple target antigens, which was significantly higher than those (13/38) from patients without (P<0.001). Except anti-BPI antibodies, the prevalence of antibodies against the other six target antigens was significantly higher in patients with clinical vasculitis than that in patients without (P<0.05, respectively). ConclusionAntibodies against multiple ANCA specific antigens, especially the antigens rather than MPO and PR3, might be the characteristic of PTU-induced ANCA. Patients with antibodies against more ANCA specific antigens might be at increased risk of developing overt clinical vasculitis. The mechanism of ANCA production in PTU-induced cases was different from that in primary AASV.

Anti-myeloperoxidase IgG subclass distribution and avidity in sera from patients with propylthiouracil-induced antineutrophil cytoplasmic antibodies associated vasculitis

Objective: Propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The aim of this study was to compare the IgG subclass distribution and avidity of MPO-ANCA in sera from patients with primary ANCA-associated vasculitis (AASV) and PTU-induced vasculitis. Methods: Nineteen patients with primary AASV with MPO-ANCA and thirteen patients with PTU-induced vasculitis were enrolled in the current study. Sera in both active phase and remission were collected. Anti-MPO IgG subclasses were detected by antigen specific ELISAs using specific monoclonal antibodies as second antibodies, and MPO-ANCA avidity was assessed by antigen-inhibition ELISAs. Results: In primary AASV, all four anti-MPO IgG subclasses could be detected in active phase with IgG1 (100%), IgG2 (73.7%), IgG3 (63.2%) and IgG4 (94.7%), and in remission, IgG1 and IgG4 subclasses in most patients remained positive. However, in PTU-induced vasculitis, anti-MPO IgG3 subclass could not be detected, the anti-MPO IgG subclasses in active phase were IgG1 (100%), IgG2 (61.5%) and IgG4 (46.2%). Furthermore, five out of the six patients (88.8%) with PTU-induced vasculitis with positive IgG4 subclass in active phase turned to negative in remission, however, only eight out of the fourteen patients (57.1%) with primary AASV turned to negative. The median avidity constant of MPO-ANCA was 56 (8.96 to >140) × 10 7 mol/l for patients with primary AASV and 0.7 (<0.28 to >140) × 10 7 mol/l for patients with PTU-induced vasculitis respectively. Furthermore, the relative levels of MPO-ANCA avidity were associated with elevation of ESR in primary AASV and were associated with BVAS scores in patients with PTU-induced vasculitis, respectively. Conclusion: MPO-ANCA IgG subclass distribution and avidity were different between patients with primary AASV and PTU-induced vasculitis. It was suggested that the mechanism of ANCA production in PTU-induced vasculitis was different from that in primary AASV, and the avidity of MPO-ANCA might be associated with disease activity.

Helicobacter pylori infection in two areas in Japan with different risks for gastric cancer.

We evaluated the relationship between Helicobacter pylori and various factors associated with gastric cancer in two areas in Japan with different risks for mortality due to gastric cancer. A total of 250 sera from Niigata and 209 from Okinawa were used. H. pylori antibody and CagA antibody were measured by antigen-specific ELISAs. Serum gastrin and pepsinogen levels were determined by RIA. Although there was no significant difference in H. pylori prevalence among the persons in Niigata (50%) and Okinawa (42%), CagA prevalence in these populations was significantly different, at 41% and 26%, respectively (OR = 1.98, 95%CI: 1.33-2.95, P < 0.01). Serum gastrin levels in Niigata were significantly lower than those in Okinawa in H. pylori-negative persons (P < 0.01). The serum pepsinogen I/II ratio in Niigata was significantly lower than that in Okinawa in H. pylori positive persons (P < 0.01), whereas there was no significant difference in H. pylori-negative persons. Among those positive for H. pylori, serum pepsinogen I/II ratio in Niigata was significantly lower than that in Okinawa in CagA-negative persons (P < 0.01), whereas no significant difference was observed in CagA-positive persons. These results suggest that the difference in the mortality ratio of gastric cancer between Niigata and Okinawa is mainly associated with the difference between areas in the prevalence of cagA-positive strains rather than that of H. pylori itself.

Screening Tests for Antinuclear Antibodies (ANA): Selective Use of Central Nuclear Antigens as a Rational Basis for Screening by ELISA

Rational, fully automated ELISA screening tests for ANA have become accessible for routine use. Full length, recombinant or purified naturally expressed antigens can be selected to deliver well defined screening tests, designed to detect ANAs of established clinical significance, while unknown specificities with no clear biological impact are omitted. By comparing results of ANA screening by immunofluorescence (IIF) on tissue section with HEp-2 cells, a correlation of 88% was revealed, while 86% of HEp-2-positive sera bound tissue sections. Eighty-nine percent of HEp-2 ANA-positive sera bound in ANA ELISA I. Seventy-five percent of ANAs detected in ELISA I also bound in ELISA II. These correlations were statistically significant. ANAs detected in ANA ELISA I were all detected upon retesting in both ANA screening ELISA systems, and also by antigen-specific ELISAs. Specific ANAs defined by ELISA, immunodiffusion or Crithidia tests were detected in both ANA ELISA tests. These data demonstrate that most ANAs detected by immunofluorescence tests contain a dominating repertoire of specificities covered by the ANA ELISA systems. The sensitivity of the different ELISA tests can easily be adjusted to internationally defined consensus levels, and screening for ANA using both ELISA systems detects expected ANA specificities in ongoing international quality assessment programs.

HPLC chromatofocusing of human immunoglobulins

A method is described for fractionation and analysis of IgA, IgM, and IgG and antibodies in human serum and/or plasma using a combination of HPLC chromatofocusing and immunoassay. A pH 9.0−3.2 gradient is utilized to separate the major proteins in the complex biological samples and monoclonal antibody based ELISAs used to determine the isotype profiles. Antigen-specific ELISAs are subsequently utilized to determine the distribution of antibody species within the chromatofocused specimens. This method is versatile since multiple simultaneous assays can easily be run on each fraction generating extensive qualitative information regarding immunoglobulin classes, and antibodies and their distribution profiles. Such spectra will prove useful for experimental kinetic analysis of the humoral immune status of humans and experimental animals.