Immune Complex Assays Research Articles

Novel assay format for total anti-adeno-associated virus antibody detection with low capsid consumption and built-in specificity control.

Aim: To develop an assay format for detection of total anti-adeno-associated virus 2 (AAV2) antibodies with low capsid material consumption. Methods: An immune complex (IC) assay format was developed. The format is based on the formation of ICs in solution and their subsequent detection using an anti-AAV2 antibody for capture and an antibody against the study species IgG for detection. Results: The feasibility of the IC assay for detection of preexisting and treatment-emergent anti-AAV2 antibodies was demonstrated in cynomolgus monkey and human serum samples, including samples from a preclinical study with AAV2-based therapies. Conclusion: The presented IC assay is an easy-to-perform total anti-AAV2 antibody assay that requires a small amount of unlabeled capsid material and provides an intrinsic specificity control.

Immune complexes as a tool for strongyloidiasis immunodiagnosis in kidney and liver transplant candidate.

Strongyloidiasis is a chronic and asymptomatic infection in immunocompetent patients. Immunocompromised patients, such as organ transplant candidates, can develop severe forms of this disease, and the best way to prevent progression to these forms is early diagnosis. Serological techniques using specific IgG and immune complexes (IC) detection can help in the diagnosis of these patients. This study aimed to detect specific anti-Strongyloides IC and IgG antibodies in kidney transplant (KT) and liver transplant (LT) candidates. A total of 100 blood samples was collected from transplant candidates (50 blood samples each from KT and LT candidates). Serum was obtained and analysed using enzyme-linked immunosorbent assay for IC and IgG detections. The IC levels showed frequencies of 18% and 2% in the KT and LT groups, respectively, whereas anti-Strongyloides IgG was detected in 34% and 12% of KT and LT candidates, respectively. The correlation between IC and IgG detection is poor in KT candidates, while in LT candidates, there is a significant positive correlation. The detection of IC can be an additional tool for the diagnosis of strongyloidiasis, especially when associated with the detection of specific IgG anti-Strongyloides antibodies.

Generic immune complex assay for detection of murine anti-drug-antibodies in complex with human IgG

Rapid and versatile methods are needed for evaluation of immunogenicity in early safety studies. The present work presents a generic, simple and easy to use sandwich enzyme-linked immunosorbent assay for quasi-quantitative measurement of circulating immune complexes (CICs) formed by anti-drug antibodies (ADAs) in complex with human IgG in mouse plasma. The assay is suitable for evaluating the presence of in vivo formed CICs in mice exposed to human IgG antibodies independent of target and IgG subtype. The assay is established using commercially available antibodies, and calibrated using CIC mimics based on bis(sulfosuccinimidyl)suberate conjugated human and mouse IgG. The development and qualification process of the generic methodology is described and include acceptance criteria, stability, sensitivity, drug tolerance, spike recovery, precision and cut point determination. In order to demonstrate assay performance, its use is exemplified by quantifying CICs in mice administered with a fully human anti-TNF-α IgG1 antibody (adalimumab) or a humanized anti-trinitrophenol (TNP) IgG4 antibody. Results show a well-qualified reproducible assay set-up with adequate sensitivity, easy discrimination between positive and negatives and quasi-quantitative measurement of ADA-human IgG CICs in mice administered with each of two different human/humanized IgG antibodies.

P24 revisited: a landscape review of antigen detection for early HIV diagnosis.

: Despite major advances in HIV testing, early detection of infection at the point of care (PoC) remains a key challenge. Although rapid antibody PoC and laboratory-based nucleic acid amplification tests dominate the diagnostics market, the viral capsid protein p24 is recognized as an alternative early virological biomarker of infection. However, the detection of ultra-low levels of p24 at the PoC has proven challenging. Here we review the landscape of p24 diagnostics to identify knowledge gaps and barriers and help shape future research agendas. Five hundred and seventy-four research articles to May 2018 that propose or evaluate diagnostic assays for p24 were identified and reviewed. We give a brief history of diagnostic development, and the utility of p24 as a biomarker in different populations such as infants, the newly infected, those on preexposure prophylaxis and self-testers. We review the performance of commercial p24 assays and consider elements such as immune complex disruption, resource-poor settings, prevalence, and assay antibodies. Emerging and ultrasensitive assays are reviewed and show a number of promising approaches but further translation has been limited. We summarize studies on the health economic benefits of using antigen testing. Finally, we speculate on the future uses of high-performance p24 assays, particularly, if available in self-test format.

Immunogenicity testing of therapeutic antibodies in ocular fluids after intravitreal injection.

High drug concentrations in ocular fluids after intravitreal administration preclude the use of drug-sensitive immunoassays. A drug-tolerant immunoassay is therefore desirable for immunogenicity testing in ophthalmology. Immune complex (IC) antidrug antibody (ADA) assays were established for two species. The assays were compared with the bridging assay in ocular and plasma samples from two preclinical studies. The IC assays showed high drug tolerance, which enabled a reliable ADA detection in ocular fluids after intravitreal administration. The IC assays were superior to the bridging assay in the analysis of ocular fluids with high drug concentrations. The IC assay allows a reliable ADA detection in matrices with high drug concentrations, such as ocular fluids.

Novel drug and soluble target tolerant antidrug antibody assay for therapeutic antibodies bearing the P329G mutation.

Bridging immunoassays for detection of antidrug antibodies (ADAs) are typically susceptible to high concentrations of residual drug. Sensitive drug-tolerant assays are, therefore, needed. An immune complex assay to detect ADAs against therapeutic antibodies bearing Pro329Gly mutation was established. The assay uses antibodies specific for the Pro329Gly mutation for capture and human soluble Fcγ receptor for detection. When compared with a bridging assay, the new assay showed similar precision, high sensitivity to IgG1 ADA and dramatically improved drug tolerance. However, it was not able to detect early (IgM-based) immune responses. Applied in combination with a bridging assay, the novel assay serves as orthogonal assay for immunogenicity assessment and allows further characterization of ADA responses.

免疫複合体検出法 (Clq-microplate ELISA) における Clq と可溶性熱会合 IgG との結合への単体 IgG の阻害作用

免疫複合体検出法 (Clq-microplate ELISA) における Clq と可溶性熱会合 IgG との結合への単体 IgG の阻害作用

Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection.

Here we demonstrate a novel homogeneous one-step immunoassay, utilizing a pair of recombinant antibody antigen-binding fragments (Fab), that is specific for HT-2 toxin and has a positive readout. Advantages over the conventional competitive immunoassay formats such as enzyme-linked immunosorbent assay (ELISA) are the specificity, speed, and simplicity of the assay. Recombinant antibody HT2-10 Fab recognizing both HT-2 and T-2 toxins was developed from a phage display antibody library containing 6 × 10(7) different antibody clones. Specificity of the immunoassay was introduced by an anti-immune complex (IC) antibody binding the primary antibody-HT-2 toxin complex. When the noncompetitive immune complex assay was compared to the traditional competitive assay, an over 10-fold improvement in sensitivity was observed. Although the HT2-10 antibody has 100% cross-reactivity for HT-2 and T-2 toxins, the immune complex assay is highly specific for HT-2 alone. The assay performance with real samples was evaluated using naturally contaminated wheat reference material. The half-maximal effective concentration (EC50) value of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay was 9.6 ng/mL, and the limit of detection (LOD) was 0.38 ng/mL (19 μg/kg). The labeled antibodies can be predried to the assay vials, e.g., microtiter plate wells, and readout is ready in 10 min after the sample application.

Generic anti-drug antibody assay with drug tolerance in serum samples from mice exposed to human antibodies

Knowledge of the anti-drug antibody (ADA) status is necessary in early research studies. Because specific assay materials are sparse and time is pressing, a generic assay format with drug tolerance for detection of ADAs in serum samples from mice exposed to immunoglobulin G (IgG) or antigen-binding fragments (Fabs) is highly desirable. This article describes a generic immune complex assay in the sandwich enzyme-linked immunosorbent assay (ELISA) format based on (i) transformation of free ADAs to immune complexes by preincubation with excess drug, (ii) the use of a murine anti-human Fab constant domain Fab as capture reagent, (iii) detection of the immune complexes by a peroxidase-labeled rabbit anti-murine Fc antibody, and (iv) ADA-positive control conjugates consisting of human Fab and murine IgG. Results of the experiments suggest that the generic immune complex assay for mouse serum samples was at least equivalent to specific ADA immune assays and even superior regarding drug tolerance. The generic immune complex assay confers versatility as it detects ADAs in complex with full-length IgG as well as with Fabs independent of the target specificity in mouse serum samples. These features help to save the sparse amounts of specific antibodies available in early research and development and speed up drug candidate selection.

Characterization of hsp27 kinases activated by elevated aortic pressure in heart

Chronic hemodynamic overload results in left ventricular hypertrophy, fibroblast proliferation, and interstitial fibrosis. The small heat shock protein hsp27 has been shown to be cardioprotective and this requires a phosphorylatable form of this protein. To further understand the regulation of hsp27 in heart in response to stress, we investigated the ability of elevated aortic pressure to activate hsp27-kinase activities. Isolated hearts were subjected to retrograde perfusion and then snap frozen. Hsp27-kinase activity was measured in vitro as hsp27 phosphorylation. Immune complex assays revealed that MK2 activity was low in non-perfused hearts and increased following crystalline perfusion at 60 or 120 mmHg. Hsp27-kinase activities were further studied following ion-exchange chromatography. Anion exchange chromatography on Mono Q revealed 2 peaks (b and c) of hsp27-kinase activity. A third peak a was detected upon chromatography of the Mono Q flow-through fractions on the cation exchange resin, Mono S. The hsp27-kinase activity underlying peaks a and c increased as perfusion pressure was increased from 40 to 120 mmHg. In contrast, peak b increased over pressures 60-100 mmHg but was decreased at 120 mmHg. Peaks a, b, and c contained MK2 immunoreactivity, whereas MK3 and MK5 immunoreactivity was detected in peak a. p38 MAPK and phospho-p38 MAPK were also detected in peaks b and c but absent from peak a. Hsp27-kinase activity in peaks b and c (120 mmHg) eluted from a Superose 12 gel filtration column with an apparent molecular mass of 50 kDa. Hence, peaks b and c were not a result of MK2 forming complexes. In-gel hsp27-kinase assays revealed a single 49-kDa renaturable hsp27-kinase activity in peaks b and c at 60 mmHg, whereas several hsp27-kinases (p43, p49, p54, p66) were detected in peaks b and c from hearts perfused at 120 mmHg. Thus, multiple hsp27-kinases were activated in response to elevated aortic pressure in isolated, perfused rat hearts and hence may be implicated in regulating the cardioprotective effects of hsp27 and thus may represent targets for cardioprotective therapy.

Src mediates cytokine-stimulated gene expression in airway myocytes through ERK MAPK

The p38 and extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) participate in cytokine-stimulated inflammatory gene expression in airway smooth muscle cells. The following study was undertaken to determine whether Src tyrosine kinases are signaling intermediaries upstream of cytokine-stimulated MAPK activation and gene expression. Treating human airway myocytes with interleukin (IL)-1β, tumor necrosis factor (TNF) α and interferon (IFN) γ caused a rapid 1.8-fold increase in Src family tyrosine kinase activity within 1 minute that remained 2.3 to 2.7 fold above basal conditions for 15 minutes. This activity was blocked by addition of 30 μM PP1, a pyrimidine inhibitor specific for Src family tyrosine kinases, in immune-complex assays to confirm that this stimulus activates Src tyrosine kinase. Addition of PP1 also blocked cytokine-stimulated expression of IL-1β, IL-6 and IL-8, while decreasing phosphorylation of ERK, but not p38 MAPK. Since this inflammatory stimulus may activate additional inflammatory signaling pathways downstream of Src, we tested the effects of PP1 on phosphorylation of signal transducers and activators of transcription (STAT). PP1 had no effect on cytokine-stimulated STAT 1 or STAT 3 phosphorylation. These results demonstrate that Src tyrosine kinases participate in the regulation of IL-1β, IL-6 and IL-8 expression and that these effects of Src are mediated through activation of ERK MAPK and not p38 MAPK or STAT1/STAT3 phosphorylation.

Non-specific diagnosis of bacterial pneumonia in children

To the Editor: From the late 1990s onwards, antigen, antibody, and immune complex assays have been applied to the diagnosis of pneumococcal pneumonia. The findings have shown a poor correlation with non-specific inflammatory markers like procalcitonin (PCT), and no combination of PCT, C-reactive protein (CRP), and white blood cells (WBC) has been able to screen bacterial from viral pneumonia [1]. Schutzle et al. published in the journal a pragmatic retrospective analysis on PCT in 327 children with lower respiratory infection (ARI) to clarify if PCT could distinguish bacterial cases requiring antibiotics from viral ARI [1]. Viruses and atypical bacteria were studied by polymerase chain reaction in nasopharyngeal aspirates. However, no methods were available for pneumococci. PCTwas <0.1 ng/ml in 132(40.3%) cases, and 94(71%) of them were successfully treated without antibiotics. Thirty-eight patients with PCT <0.1 ng/ml received antibiotics, and were analyzed further [2]. After exclusions, only 12 children (four viral, four atypical bacterial, and four unknown infections) were checked, and none of them had any findings suggestive for typical bacterial infection [1]. The authors concluded that PCT <0.1 ng/ml identified children with ARI in whom antibiotics can be withheld [2]. However, the critical patients for any conclusions are these 26/132(19.7%) ARI cases with low PCT, treated with antibiotics and excluded from the analyses. Due to increasing antibiotic resistance, antibiotic use for children’s respiratory infections should be lessened in the community. WBC (especially neutrophils) rise more rapidly than PCT and CRP in invasive bacterial infections, being helpful for diagnosing pneumococcal bacteremia in young children [3]. Thus, the combined use of the markers sounds rationale in children with pneumonia. Rapid, sufficiently reliable bedside tests are currently available for PCT, CRP, and WBC, and their combined use should be studied in children with pneumonia. Children with other respiratory infections rarely benefit from any laboratory tests.

Redox- and Calmodulin-dependent S-Nitrosylation of the KCNQ1 Channel

Nitric oxide (NO) is a gaseous signal mediator showing numerous important biological effects. NO has been shown in many instances to exhibit its action via the protein S-nitrosylation mechanism, in which binding of NO to Cys residues regulate protein function independently of activation of soluble guanylate cyclase. The direct link between protein S-nitrosylation and functional modulation, however, has been demonstrated only in limited examples. Furthermore, although most proteins have more than one Cys residue, the mechanism by which a certain Cys becomes a specific target residue of S-nitrosylation is poorly understood. We have previously reported that NO regulates currents through the cardiac slowly activating delayed rectifier potassium channel (I(Ks)) irrespective of soluble guanylate cyclase activation. Here we demonstrate using a biotin-switch assay that NO induced S-nitrosylation of the alpha-subunit of the I(Ks) channel, KCNQ1, at Cys(445) in the C terminus. A redox motif flanking Cys(445) and the interaction of KCNQ1 with calmodulin are required for preferential S-nitrosylation of Cys(445). A patch clamp experiment shows that S-nitrosylation of Cys(445) modulates the KCNQ1/KCNE1 channel function. Our data provide a molecular basis of NO-mediated regulation of the I(Ks) channel. This novel regulatory mechanism of the I(Ks) channel may play a role in previously demonstrated NO-mediated phenomenon in cardiac electrophysiology, including shortening in action potential duration in response to intracellular Ca(2+) or sex hormones.

Pneumococcal serology in children’s respiratory infections

The role of Streptococcus pneumoniae in the etiology of respiratory tract infections has been studied serologically using microbe-specific antibody and immune complex assays. Serological methods are sensitive in the bacteremic pneumococcal pneumonia of adults. In children, however, pneumococcal pneumonia is seldom bacteremic, and, thus, in the absence of a gold standard for the detection of pneumococcal infection, serological methods are still insufficiently validated. We report here indirect evidence for the sensitivity and specificity of pneumococcal serology in children. Serological evidence of pneumococcal infection has been found in 27% to 38% of children with radiologically confirmed pneumonia, in 7% to 8% of children with viral wheezy bronchitis, and in <1% to 5% of children and young adults with viral upper respiratory infection. Serological findings for pneumococcal infection have been dependent on the study venue, whether in hospital or ambulatory subjects, and on the test panel used. Where both antibody and immune complex assays have been available, the proportion of children with pneumococcal infection has been 32% to 37% in inpatients and 27% to 28% in outpatients. The respective rates have been 10% to 18% by antibody assays alone. Pneumococcal acute otitis media, when present with pneumonia, may confound findings in pneumococcal serology, but pure nasopharyngeal carriage of S. pneumoniae has little effect. In contrast, carriage acquisition of a new serotype may induce significant antibody production. Thus, understandably, significant rises between paired sera in antibodies to pneumococcal capsular polysaccharides and pneumococcal pneumolysin have been found in <1% to 3% of non-symptomatic children and young adults. Findings from the last 20 years indirectly suggest that pneumococcal antibody and immune complex assays are sensitive and specific enough for the detection of pneumococcal infection in children. However, the methods are too complex for routine clinical practice, and, so far, serological methods for S. pneumoniae infections have only been used for research purposes.

Investigation of interleukin 1β-mediated regulation of NF-κB activation in colonic cells reveals divergence between PKB and PDK-transduced events

Recent work has highlighted a role for PDK1 in adaptive immunity, however its contribution to innate immunity has not been addressed. We have investigated the role of PKB and PDK1 in IL-1beta-induced NF-kappaB activation. Over-expression of either in HCT 116 and HEK 293T cells, effected a reproducible NF-kappaB activation. This was validated in a one-hybrid assay utilizing Gal4-RelA and Gal4-luciferase assay. N-tosyl phenylalanyl chloromethyl ketone (TPCK), wortmannin and Ly294002 inhibited IL-1beta-induced NF-kappaB activation in both systems indicating involvement of the PI3K axis in this response. p65 (Rel A) Ser536 phosphorylation was not affected by the PI3K inhibitors but was dose-dependently attenuated by TPCK. Evaluation of IKK-associated activity using GST-p65 substrate phosphorylation in immune complex assays, revealed that whilst TPCK attenuated this, neither of the PI3K inhibitors had any effect. Furthermore whilst TPCK inhibited IL-1beta-induced p65 DNA binding, this was not apparent with either of wortmannin or Ly294002. Similarly, over-expression of PDK1 but not PKB resulted in promotion of p65 DNA binding. Using a p65-S536A reporter construct, we found inhibition of only PDK1 over-expression-induced, but not PKB over-expression-induced NF-kappaB activation. This was supported using biochemical analysis in which immunoprecipitated IKKgamma from IL-1beta-activated cells was unable to phosphorylate a p65-S536A substrate, confirming this as the dominant IKK-dependent site. In further support of a dissociated response, we observed an attenuation of the Ser177/181 IKK phosphorylation by TPCK but not in response to PI3K inhibition. Our data reveals for the first time that PDK1 and PKB may differentially activate NF-kappaB, and that TPCK may subserve a useful anti-inflammatory function by inhibiting IKKbeta.

Measuring MAP kinase activity in immune complex assays

I present an overview of published methods for measuring mitogen activated protein (MAP) kinase activity on endogenous associated substrates, exogenously added substrates as well as determination of activation loop phosphorylation as a read-out of kinase activity in vivo. Detailed procedures for these assays are given for two MAP kinases (MAPKs) Fus3 and Kss1 and compared with other published protocols, including the protocols for Hog1 and Mpk1 MAPKs. Measuring kinase activity in immune complex assays can serve as an approach for identification of potential substrates of protein kinases as well as for detecting other kinase-associated proteins.

Regulation of IκB Kinase (IKK) Complex by IKKγ-dependent Phosphorylation of the T-loop and C Terminus of IKKβ

Regulation of IκB Kinase (IKK) Complex by IKKγ-dependent Phosphorylation of the T-loop and C Terminus of IKKβ

PTPN11 (Shp2) Mutations in LEOPARD Syndrome Have Dominant Negative, Not Activating, Effects

Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinase-mediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation.

Limitations of immune complex measurements in colorectal disease.

Three techniques (Clq, Raji and L1210 binding assays) alleged to measure circulating immune complexes (ICs) were applied to the sera of 101 patients with colorectal disease (54 carcinoma; 23 inflammatory; 13 benign tumour and 11 miscellaneous) at the time of diagnostic or definitive surgery, and 58 healthy adult controls. Elevated levels in the pathological sera were observed by all 3 methods in order of sensitivity: Raji greater than Clq greater than L1210. However, none of them differentiated between benign, inflammatory and neoplastic conditions nor, in the case of colorectal carcinoma, was there any correlation with stage of disease. With the exception of Raji v. L1210 (r = 0.43, P less than 0.001), correlations between the various assays were poor and levels of serum carcinoembryonic antigen (CEA) did not correlate with ICs measured by any of the techniques. Indeed, the IC assays were even less discriminatory than CEA, which was elevated mainly in the serum of carcinoma patients and which was positively correlated with serum gamma-glutamyl transpeptidase (gamma GT) (r = 0.42, P less than 0.005). The data suggest that the lack of concordance between the IC assays is a reflection of heterogeneity among ICs, interfering factors present in pathological sera, or both. Thus the IC assays deployed here have neither diagnostic nor prognostic utility in colorectal disease at this time, and immunochemical characterization of the serum reactive material detected by the different assays is required.

Detection of Circulating CEA-IgM Complexes in Early Stage Colorectal Cancer

We have recently shown that alpha fetoprotein (AFP) and squamous cell carcinoma antigen (SCCA), biomarkers associated with hepatocellular carcinoma, may be detected in patient sera as circulating immune complexes with IgM, and that assessment of serum levels of AFP-IgM and SCCA-IgM may be used for the detection of liver cancer. In this study we measured the levels of carcinoembryonic antigen (CEA) as free form (FCEA) and complexed to IgMs (CEA-IgM) in sera of patients affected by colorectal carcinoma (CRC) at different stages as well as in healthy subjects. FCEA levels were above the 5 ng/mL cutoff in 43% of CRC patients (31/72) and CEA-IgM levels were above the 200 AU/mL cutoff in 38% of CRC patients (27/72). Serum levels of CEA-IgM immune complexes (IC) and FCEA did not overlap and 64% of patients (46/72) were positive for at least one marker without compromising the detection specificity (94%). Early detection of CRC was significantly improved by CEA-IgM IC assay. CRC patients at an early stage (stage 1) had elevated CEA-IgM levels in 29% of cases (7/24), while FCEA levels were elevated in only 8% of cases (2/24). These results indicate that CEA-IgM is a complementary serological marker to FCEA which is much more sensitive for early stage CRC, and that the combination of these biomarkers may be useful in the early detection of colorectal cancer.