TY - JOUR
A1 - Deutschmann, Claudia
A1 - Roggenbuck, Dirk
A1 - Schierack, Peter
A1 - Rödiger, Stefan
T1 - Autoantibody testing by enzyme-linked immunosorbent assay-a case in which the solid phase decides on success and failure
JF - Heliyon
N2 - Background: The enzyme-linked immunosorbent assay (ELISA) is an indispensable tool for clinical diagnostics to identify or differentiate diseases such as autoimmune illnesses, but also to monitor their progression or control the efficacy of drugs. One use case of ELISA is to differentiate between different states (e.g. healthy vs. diseased). Another goal is to quantitatively assess the biomarker in question, like autoantibodies. Thus, the ELISA technology is used for the discovery and verification of new autoantibodies, too. Of key interest, however, is the development of immunoassays for the sensitive and specific detection of such biomarkers at early disease stages. Therefore, users have to deal with many parameters, such as buffer systems or antigen-autoantibody interactions, to successfully establish an ELISA. Often, fine-tuning like testing of several blocking substances is performed to yield high signal-to-noise ratios.
Methods: We developed an ELISA to detect IgA and IgG autoantibodies against chitinase-3-like protein 1 (CHI3L1), a newly identified autoantigen in inflammatory bowel disease (IBD), in the serum of control and disease groups (n = 23, respectively). Microwell plates with different surface modifications (PolySorp and MaxiSorp coating) were tested to detect reproducibility problems.
Results: We found a significant impact of the surface properties of the microwell plates. IgA antibody reactivity was significantly lower, since it was in the range of background noise, when measured on MaxiSorp coated plates (p < 0.0001). The IgG antibody reactivity did not differ on the diverse plates, but the plate surface had a significant influence on the test result (p = 0.0005).
Conclusion: With this report, we want to draw readers' attention to the properties of solid phases and their effects on the detection of autoantibodies by ELISA. We want to sensitize the reader to the fact that the choice of the wrong plate can lead to a false negative test result, which in turn has serious consequences for the discovery of autoantibodies.
KW - biochemistry
KW - coatings
KW - surface chemistry
KW - immunology
KW - proteins
KW - laboratory medicine
KW - clinical research
KW - enzyme-linked immunosorbent
KW - assay
KW - biomarker discovery
KW - reproducibility
KW - solid-phase
KW - autoantibody
Y1 - 2020
U6 - https://doi.org/10.1016/j.heliyon.2020.e03270
SN - 2405-8440
VL - 6
IS - 1
PB - Elsevier
CY - London [u.a.]
ER -
TY - JOUR
A1 - Lang, Judith
A1 - Bohn, Patrick
A1 - Bhat, Hilal
A1 - Jastrow, Holger
A1 - Walkenfort, Bernd
A1 - Cansiz, Feyza
A1 - Fink, Julian
A1 - Bauer, Michael
A1 - Schumacher, Fabian
A1 - Kleuser, Burkhard
A1 - Lang, Karl S.
T1 - Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease
JF - Nature Communications
N2 - Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1(-/-) mice results in replication of HSV-1 and Asah1(-/-) mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.
KW - immunology
KW - infection
KW - membrane fusion
KW - phagocytosis
KW - sphingolipids
Y1 - 2020
U6 - https://doi.org/10.1038/s41467-020-15072-8
SN - 2041-1723
VL - 11
IS - 1
SP - 1
EP - 15
PB - Nature Publishing Group UK
CY - London
ER -