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Background: To prevent unwanted complement activation and subsequent damage, complement activation must be tightly regulated on healthy host cells. Dysregulation of the complement system contributes to the pathology of diseases like Paroxysmal Nocturnal Hemoglobinuria and atypical Hemolytic Uremic Syndrome. To investigate complement regulator deficiencies, primary patient cells may be used, but access to patient cells may be limited and cells are heterogeneous between different patients. To inhibit regulator function on healthy host cells, blocking antibodies can be used, though it may be difficult to exclude antibody-mediated effects. To circumvent these issues, we created single and combined complement regulator human knockout cells to be able to in vitro investigate complement activation and regulation on human cells., Methods: CRISPR/Cas9 was used to knockout (KO) complement regulatory proteins CD46, CD55 and/or CD59 in human HAP1 cells. Single cell derived cell lines were profiled by Sanger sequencing and flow cytometry. To confirm the lack of complement regulatory function, the cells were exposed to complement in normal human serum and subsequently C3 and C4 deposition on the cell surface were detected by using flow cytometry., Results: We created single KO cell lines that completely lacked CD46, CD55 or CD59. We additionally generated double CD46/CD55, CD46/CD59 and CD55/CD59 KOs and triple CD46/CD55/CD59 KOs. Upon classical pathway activation, deletion of CD46 resulted in increased C3 and C4 deposition, while deleting CD55 mainly resulted to increased C3 deposition, confirming their reported function in complement regulation. Upon alternative pathway activation, C3 deposition was only observed on the triple CD46/CD55/CD59 KO cells and not on any of the other cell lines, suggesting that human cells are resistant to spontaneous complement activation and suggesting a role for CD59 in C3 regulation., Conclusions: The generation of complement regulator KO cell lines provides a relevant tool for future in vitro investigations of complement activation and regulation on human cells. Furthermore, these cell lines may also be helpful to evaluate therapeutic complement inhibitors and may shed light on novel roles of complement regulatory proteins as we here observed for CD59., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Neutralizing autoantibodies (NAbs) against plasma serpin C1-inhibitor (C1-inh) are implicated in the rare disorder, acquired angioedema (AAE). There is insufficient understanding of the process of antibody formation and its correlation with disease progression and severity. We have developed an ELISA for detecting neutralizing capacity of anti-C1-inh positive plasma samples that can be used to study changes in NAb repertoire in patient plasma over the course of disease., Methods: The ELISA is based on the specific interaction of active C1-inh with its target protease C1s. Decrease in the amount of C1s bound to immobilized C1-inh in the presence of test samples is proportional to the neutralizing capacity of the sample. Assay specificity, intra- and inter-assay variation and assay cut-off are determined using anti-C1-inh antibodies. Assay capability is demonstrated using plasma samples from AAE patients., Results: The assay is specific to a neutralizing anti-C1-inh antibody and shows no interference by a non-neutralizing anti-C1-inh antibody or by the plasma matrix. Intra-assay and inter-assay variations are determined as 17 and 18% respectively. Neutralizing capacity of antibody positive AAE patient plasma samples (n=16) with IgG or IgM type antibodies is readily determined. All samples show positive neutralizing capacity., Conclusion: We have developed a robust, specific and semi-quantitative assay to detect the neutralizing capacity of plasma samples containing anti-C1-inh antibodies. This assay can be an important tool for the study of clinical implications of anti-C1-inh NAbs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Rheumatoid factors are antibodies directed against IgG that may confound immunogenicity testing for therapeutic monoclonal antibodies. We developed antigen-binding assays to monitor anti-drug-antibody (ADA) responses against infliximab and adalimumab using F(ab')2 fragments of the drug. This avoids possible detection of rheumatoid factor activity. During development of these assays, a number of sera from patients before treatment as well as several healthy control sera were tested positive. None of these sera contained antibodies specific for the therapeutic mAb. Instead, they were found to contain anti-hinge antibodies. We demonstrate that this aspecific antibody binding can be inhibited by adding F(ab')2 of intravenous immunoglobulin (IVIG), which consists of pooled polyclonal IgG derived from plasma. Using this protocol, anti-infliximab antibodies can be measured specifically without interference by anti-hinge antibodies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The presence of anti-drug antibodies (ADA) in adalimumab-treated patients is associated with reduced serum adalimumab levels and a lower clinical response. Currently, there is no standard for measurement of anti-drug antibodies and many factors influence the results. Consequently, the incidence of ADA as reported in different studies varies considerably. Here we investigated the differential effect of drug interference in two common types of assays used to measure anti-adalimumab: an antigen binding test (ABT) and a more often-used bridging elisa. We measured ADA to adalimumab in a cohort of 216 rheumatoid arthritis patients treated with adalimumab for 28 weeks. Only 15 samples (7%) were positive in the bridging elisa, compared to 29 (13%) in the ABT, despite the fact that the bridging elisa was the most sensitive assay. Furthermore, in an ABT specific for IgG4, 48 samples (22%) were found positive. The bridging elisa was found to detect only the bivalent form of (drug-specific) IgG4, resulting in an underestimation of ADA levels. However, the predominant reason for the different outcomes of these assays was a differential susceptibility to drug interference. In particular, the bridging elisa only detected ADA in the absence of detectable amounts of circulating adalimumab and is therefore not suited for measurement of ADA in complex with the drug. In summary, we showed that a bridging elisa is susceptible to drug interference and typically measures ADA only in absence of detectable drug levels., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Production of anti drug antibodies (ADA) in adalimumab treated RA patients is associated with reduced serum adalimumab levels and less clinical response. However, most current assays to measure ADA are unable to detect ADA in complex with adalimumab. Thus, ADA is only measured if antibody production exceeds drug levels in the serum, meaning that ADA formation is underestimated. The aim of this study is to develop a method to detect ADA in the presence of drug. A pH-shift-anti-idiotype Antigen binding test (PIA) was used to enable ADA measurement in the presence of adalimumab. ADA-adalimumab complexes were dissociated by acid treatment and addition of excess rabbit anti-idiotype-F(ab) before neutralization. Rabbit anti-idiotype-F(ab) blocks reformation of ADA-drug complexes by competing with patient ADA for adalimumab binding. Released ADA are measured by an antigen binding test (ABT). The PIA enabled detection of ADA in the presence of large excess of adalimumab and was used to measure ADA in 30 adalimumab treated rheumatoid arthritis (RA) patients during the first 28 weeks of treatment. It revealed ADA in 21 out of 30 tested patients, while the ABT detected ADA in only 5 patients. Indicating that an immunogenic reaction towards adalimumab is present in the majority of adalimumab treated patients., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Truncated forms and full-length forms of the amyloid-beta 40 (Abeta40) are key molecules in the pathogenesis of dementia, and are detectable in CSF. Reliable methods to detect these biomarkers in CSF are of great importance for understanding the disease mechanisms and for diagnostic purposes., Methods: VU-alpha-Abeta40, a monoclonal antibody (mAb) specifically detecting Abeta40, was generated and characterized by solid and fluid phase ELISA, surface plasmon resonance spectroscopy (SPRS), immunoprecipitation (IP), immunohistochemical and Western blot (WB) analysis. In addition, an ELISA with VU-alpha-Abeta40 as catching and 6E10 as detecting mAbs was set up and validated. This ELISA was used to measure Abeta40 in CSF of controls (N=27), patients with Alzheimer's disease (AD; N=20), frontotemporal lobe dementia (FTLD; N=14), noninflammatory (N=15) and inflammatory (N=15) neurological conditions., Results: VU-alpha-Abeta40 specifically recognizes Abeta40 with high affinity (K(A)=1.3x10(9) M(-1)) and detects Abeta40 in AD brain specimens. The developed sandwich ELISA has a detection limit of 0.21 ng/mL, a mean recovery of 90%, and an intra- and inter-assay CV of 1.4% and 7.3%. FTLD patients had a lower mean level of Abeta40 (8.8 (1.9) ng/mL) than controls (12.0 (1.7) ng/mL); p<0.01)., Conclusions: VU-alpha-Abeta40 was successfully implemented in an ELISA which enables us to measure Abeta40 accurately in human CSF. Clinical validation revealed lower levels of Abeta40 in FTLD patients. This finding opens new possibilities for early and differential diagnosis of dementia.

Classical pathway activation is often assessed by measuring circulating levels of activated C4. However, this parameter does not discriminate between activation through the classical or the lectin pathway. We hypothesized that during classical pathway activation, complexes are formed between C1q and activated C4 or C3. Using ELISA, we investigated whether such complexes constitute specific markers for classical pathway activation. In vitro, C1q-C3d/C4d complexes were generated upon incubation of normal recalcified plasma with aggregated IgG or an anti-C1q mAb that activates C1 (mAb anti-C1q-130). In contrast, during incubation with C1s or trypsin, C1q-C3d/C4d complexes were not generated, which excludes an innocent bystander effect. Additionally, C1q-C3d/C4d complexes were not generated during activation of the alternative or the lectin pathway. Repeated freezing and thawing did not influence levels of C1q-C3d/C4d complexes in recalcified plasma. To measure C1q-complement complexes in plasma samples, we separated unbound complement proteins from C1q-C3d/C4d complexes in the samples prior to testing with ELISA. In samples from patients undergoing cardiopulmonary bypass surgery or suffering from rheumatoid arthritis, we found higher levels of C1q-C4 complexes than in samples from healthy individuals. We conclude that complexes between C1q and C4 or C3 are specific markers of classical complement pathway activation.