Anti-plasminogen antibodies in ANCA-associated vasculitis: An optimized anti-plasminogen assay - PubMed (original) (raw)

Anti-plasminogen antibodies in ANCA-associated vasculitis: An optimized anti-plasminogen assay

Arda Göçeroğlu et al. PLoS One. 2018.

Abstract

Anti-plasminogen antibodies (α-PLG) were previously detected in a subpopulation of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients, showing a relation to renal lesions and outcome. Several studies showed different proportions of α-PLG positive AAV patients, possibly due to differences in the assays used. We here present a new, optimized α-PLG Enzyme-Linked Immuno Sorbent Assay (ELISA) and validate the presence of α-PLG in AAV. Different ELISA set-ups were tested regarding plasminogen (PLG) antigen, concentrations, coating buffers, blocking agents, and environmental conditions. Purified lysine-PLG (lys-PLG) showed better differentiation between positive samples and negative samples than glutamic acid-PLG (glu-PLG). Therefore, lys-PLG was used as coating antigen. With the optimized α-PLG ELISA we found α-PLG in 14.3% of the myeloperoxidase (MPO)-ANCA patients, whereas all our proteinase-3 (PR3)-ANCA patients tested in our new assay were negative. Concluding, in this study we have combined important technical findings and methods from previous studies to optimize the α-PLG assay, which can be used for future research purposes and will aid in uniform reporting of α-PLG status of patients.

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Conflict of interest statement

EG, YS, JW are employees of Euro Diagnostica and their salaries are paid by Euro Diagnostica. Euro Diagnostica did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1

Fig 1. SDS-PAGE and Western blot with rabbit anti-plasminogen antibodies.

(A) SDS-PAGE showed the presence of four 4 proteins. (B) Western blot with rabbit anti-plasminogen antibodies showing strong binding for the unreduced plasminogen variants and weak binding for the reduced plasminogen variants. 1, reduced glutamic acid plasminogen; 2, reduced lysine-plasminogen; 3, unreduced glutamic acid plasminogen (88 kDa); 4, unreduced lysine-plasminogen (83 kDa). The arrows show the direction of movement of the proteins.

Fig 2

Fig 2. Spectrophotometrical results.

(A) Spectrophotometrical results using purified glutamic acid plasminogen as an antigen in the assay. Optical density was measured with spectrometry using light of 405 nanometer for the detection of anti-plasminogen antibodies in the serum samples. (B) Spectrophotometrical results using purified lysine-plasminogen as an antigen in the assay. Optical density was measured with spectrometry using light of 405 nanometer for the detection of anti-plasminogen antibodies in the serum samples. Purified lysine-plasminogen showed more spectrophotometrical differentiation between positive samples and negative samples compared to purified glutamic acid plasminogen (Fig 2A). Abbreviations: gluPLGp, purified glutamic acid plasminogen; OD, optical density; nm, nanometer; lysPLGp, purified lysine-plasminogen.

Fig 3

Fig 3. Anti-plasminogen antibody concentrations in serum samples from 175 healthy controls.

The cut-off value for anti-plasminogen antibodies positivity was set to 31 U/ml and higher based on the 97.5th percentile of the healthy controls.

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