A putative enzyme from various secretions specifically inhibits antibody-antigen interactions

Poethke, Reinhild; Mäder, Michael; Zedler, Inga; Beuche, Wolfgang; Felgenhauer, K.; Kolbus, Norbert

doi:10.1016/0022-1759(96)00012-9

Cited by 6 publications

(4 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Lf concentration at which half of the maximum immune complex is formed is 262.4 ± 0.9 nM, which represents a low binding affinity in the assay conditions when compared to ten previously characterized monoclonal antibodies to human Lf which had dissociation constants ranging from 0.5 nM to 18 nM . When compared to measurements in the presence of saliva and serum, tear fluid has been shown to dramatically reduce the binding efficiency of immunoglobulins, especially in alkaline pH conditions . Therefore, this low measured binding affinity may be due to the contribution of weak electrostatic binding interactions between the alkaline target and antibody, some denaturation of antibody or antigen in the high pH conditions, and/or the presence of these unidentified factors present in tear fluid which inhibit the binding efficiency of immunoglobulins .…”

Section: Resultsmentioning

confidence: 82%

“…35 When compared to measurements in the presence of saliva and serum, tear fluid has been shown to dramatically reduce the binding efficiency of immunoglobulins, especially in alkaline pH conditions. 8 Therefore, this low measured binding affinity may be due to the contribution of weak electrostatic binding interactions between the alkaline target and antibody, some denaturation of antibody or antigen in the high pH conditions, and/or the presence of these unidentified factors present in tear fluid which inhibit the binding efficiency of immunoglobulins. 8 Nevertheless, as demonstrated in the immunoassays presented here, the immune complex formation is sufficient for Lf quantitation on the time scales of the assay.…”

Section: ' Results and Discussionmentioning

confidence: 99%

“…8 Therefore, this low measured binding affinity may be due to the contribution of weak electrostatic binding interactions between the alkaline target and antibody, some denaturation of antibody or antigen in the high pH conditions, and/or the presence of these unidentified factors present in tear fluid which inhibit the binding efficiency of immunoglobulins. 8 Nevertheless, as demonstrated in the immunoassays presented here, the immune complex formation is sufficient for Lf quantitation on the time scales of the assay. Assay specificity was assessed by Lf depletion of healthy and SS tear samples, yielding tear matrix with Lf concentrations below the lower LOD of the assay (3.18 nM Lf remaining in healthy sample, 1.2 nM Lf remaining in SS sample as measured by ELISA) and comparing Ab* spiked in Lf-depleted tears (LfÀ) to that in non-depleted (Lf+) tears.…”

Section: ' Results and Discussionmentioning

confidence: 99%

“…Alkaline proteins, such as lactoferrin, are known to rapidly associate with other analytes in solution . Further, interfering factors in tear fluid decrease antibody binding efficiency (i.e., through selective cleaving of the Fab region or steric hindrance of the epitope , ), block the interaction of proteins with surfaces (e.g., ELISA), , and contribute to a myriad of other assay artifacts (e.g., high background signals, blooming effects, matrix effects, and cross-talk between capture and probe antibodies in microwell arrays and immunoassays). , Many of the tear-specific components responsible for these interfering effects have not yet been identified, and given these confounding factors and the high variability of tear film fluid, the reliability of any tear assay is dependent on the volume and type of tear sample, individual donor, and assay employed. Consequently, obtaining quantitative and specific tear protein measurements is difficult .…”

mentioning

confidence: 99%

See 3 more Smart Citations

Human Tear Protein Analysis Enabled by an Alkaline Microfluidic Homogeneous Immunoassay

Karns

Herr

2011

Anal. Chem.

View full text Add to dashboard Cite

The ability to probe the protein content of human tear fluid has enormous potential for deepening our understanding of ocular and systemic disease pathology and enabling novel noninvasive tear-based diagnostic technologies. To overcome current challenges in tear proteomic measurements, we report on the first microfluidic homogeneous immunoassay capable of making rapid, quantitative, and specific measurements of endogenous tear protein biomarkers in human tear fluid. Lactoferrin (Lf) is a tear-specific biomarker for Sjögren's syndrome (SS), a serious systemic autoimmune disease currently diagnosed through rudimentary volumetric and surface chemistry measurements and an invasive lip biopsy. We detail optimization of a homogeneous electrophoretic immunoassay for Lf in <1 μL of tear fluid at clinically relevant concentrations. In particular, we present assay development details and a final assay that enables quantification of Lf in <5 s in a clinically relevant range for SS diagnostics. Characterization suggests the on-chip assay is accurate to within 15% of ELISA, specific (<15% nonspecific signal), and with a lower limit of detection of 3 ± 2 nM Lf in human tear matrix. Additionally, we develop and characterize a protocol for eluting proteins from nitrocellulose Schirmer strips, the clinical de facto standard for tear collection and storage. We relate on-chip measured Lf concentrations back to ocular surface concentrations for the first time to our knowledge. Taken in sum, this work details important steps toward (1) expanding the set of proteins quantified by electrophoretic immunoassays to encompass a wider range of isoelectric points than has been reported, (2) creating a first-in-kind translatable assay with clinical relevance to SS diagnostics, and (3) expanding the analytical toolkit available for rapid tear protein measurements, as is relevant to the advancement of basic research and clinical medicine.

show abstract

Section: Resultsmentioning

confidence: 82%