Detection of proteinase K resistant proteins in the urine of patients with Creutzfeldt-Jakob and other neurodegenerative diseases

Abstract

Recent concern about the possible secondary spread of vCJD through blood transfusion and blood products has highlighted the need for a sensitive test for the identification of PrP^TSE/res in clinical specimens collected in a non-invasive way. In addition, a more accurate estimate of the prevalence of pre-clinical vCJD in the population may be possible if there were a test that could be applied to easily available material such as urine. As a step towards this goal, the detection of putative PrP^TSE/res in the urine of CJD patients has been improved, based on Proteinase K digestion of samples and Western blotting. The modified Western blot uses concentrated urine as a starting material. After proteolytic treatment followed by electrophoresis and Western blotting, membranes are incubated with an anti-PrP antibody conjugated directly with horseradish peroxidase. This study was conducted on urine samples of CJD and other neurodegenerative disease affected individuals. Proteinase K resistant high molecular weight proteins were detected, which are suggested to be a complex of urinary PrP and immunoglobulin proteins. Whether urine can be used as a diagnostic tool for the detection of PrP could not be answered in this study.

Acknowledgements

We would like to express our gratitude to German TSE platform, for providing urine samples, special thanks to the vCJD patient's relatives for kind support and Dr. Michel Doumith for the help with all experiments involving Klebsiella pneumoniae.

Figures and Tables

Figure 1 Western blot analysis of CJD, disease and healthy controls. Urine samples of patient and control samples underwent proteolytic digestion with two different PK concentration and incubation time and were probed with anti-IgG-HRP anitbody. (A) Enriched urine samples from sCJD, vCJD, disease and healthy controls were treated with proteinase K (concentration 40 µg/ml for 20 min). Samples ID labelled with letters correspond to Table 1. After electrophoresis, protein was transferred to a nylon membrane using Bio-Rad instrument and probed with anti-IgG-HRP followed with ECL Plus addition. A Kodak Imager was used for photographic documentation. (B) Same or other samples were treated with proteinase K (concentration 60 µg/ml for 30 min). Samples ID labelled with letters correspond to Table 1, healthy control samples are shown with numbers. Protein was transferred via Bio-Rad and membrane probed with anti-IgG-HRP followed ECL Plus addition. Documentation was as above. (The exposure time for documentation was 5 minutes for A and B).

Figure 2 Western blot analysis of urine proteins from CJD and disease controls. The membrane used in Figure 1A was stripped and tested with ECL Plus to verify if the previous antibody was fully removed, before processing it. (A) The membrane was probed with SAF61-HRP and documented as described before. Samples ID correspond to Table 1. (B) The membrane used in (A) was stripped and tested with ECL Plus to ensure removal of previous probed antibody, prior to probing it with SAF32-HRP. Documentation was as described before. Samples ID letters correspond to Table 1. The exposure time for documentation was 5 minutes for (A) and 45 minutes for (B). longer exposure time was necessary for (B) as it was measured with control experiment using recombinant PrP.

Figure 3 Western blot analysis of urines from CJD, disease and healthy controls. PK treatment of patient and healthy controls and western blot analysis with 3F4-HRP antibody. (A) Proteinase K (concentration 40 µg/ml for 20 min) treatment of CJD and disease control samples. Samples ID letters correspond to Table 1. Proteins were transferred with Iblot apparatus and the membrane probed with 3F4-HRP followed with ECL Plus addition. Membrane documentation was with Kodak Imager with 5 minutes exposure time. (B) Proteolytic digestion of six healthy control urines in presence (+) or absence (−) of proteinase K (concentration 40 µg/ml for 20 minutes). Healthy control samples are shown with numbers 1–6 and are from different gender and age. Protein transfer was with the Iblot apparatus and the membrane probed with 3F4-HRP, followed with ECL Plus addition. Documentation was as described above.

Figure 4 Western blot analysis of CJD, disease and healthy control urines. Proteinase K treatment of enriched urine samples and comparison between anti-IgG-HRP and anti-PrP-HRP probed membranes. Samples ID correspond to Table 1. Healthy control samples are shown with numbers (Proteinase K treated healthy control samples are designated with +). (A) Proteolytic treatment of urine samples from CJD, disease control and healthy control with proteinase K (concentration 60 µg/ml for 30 min). Protein transferred with Bio-Rad instrument and membrane probed with SAF61-HRP followed with ECL Plus addition. Membrane documentation was with Kodak Imager with 5 minutes exposure time. (B) This figure is a display of Figure 2A, highlighting majority of additional bands detected only with anti-PrP antibody, shown with arrows when the figure is compared to Figure 1A. The arrows point to bands detected only when SAF61-HRP was applied, the rest of the bands were detected with anti-IgG-HRP.

Figure 5 Analysis of Kleibsiella pneumonia with two antibodies. The over night culture of Kleibsiella pneumonia was used for the extraction of outer membrane protein (OMP) and whole membrane proteins. OMPs were digested in the presence or absence of proteinase K (concentration 40 µg/ml for 20 minutes) and the membrane was probed with 3F4-HRP or SAF61-HRP. Samples ID: O4 = Total extract of bacteria − PK, O3 = Total extract of bacteria + PK, O2 = Total membrane proteins + PK, O1 = All membrane proteins − PK, p = Recombinant PrP, M = Marker. 4A: Membrane was probed with SAF61-HRP followed with ECL Plus addition. Membrane documentation was with Kodak Imager with 20 minutes exposure time. 4C: Membrane was probed with 3F4-HRP. Membrane documentation was with scanner, after it was incubated for 20 minutes in Opti-4CN (Bio-Rad) solution and rinsed subsequently in H₂O. 4B: Gel staining of the samples with Ez-Blue dye. Samples ID: 4 = Total extract of bacteria − PK, 3 = Total extract of bacteria + PK, 2 = Total membrane proteins + PK, 1 = Whole membrane proteins − PK, p = Recombinant PrP, M = Marker.

Figure 6 Western blot analysis of sCJD and vCJD urine samples after PNGase F and acetic SDS treatment. Three sCJD and one vCJD enriched urine samples were treated over night with PNGase F, followed with PK digestion (concentration 40 µg/ml for 20 minutes). Samples ID letters correspond to Table 1. The samples were subsequently supplemented with acetic SDS or left untreated and western blotted. (*) designate acetic SDS treated samples. (A) Membrane containing acetic SDS treated or untreated samples were probed with 3F4-HRP antibody, added ECL Plus and documented using Kodak imager instrument with exposure time of 5 minutes. (B) Membrane mentioned above was stripped, tested with ECL Plus to ensure removal of previous probed antibody and subsequently probed with anti-IgG-HRP. For documentation, the procedure mentioned above was followed. Exposure time for documentation was 5 minutes.

Figure 7 Western blot analysis of CJD and control urine samples after Protein-A column chromatography. Two sCJD, one vCJD and one control urine sample were loaded on Protein-A column, eluates and flow-throughs were collected, enriched and PK digested (60 µg/ml for 30 min). Samples were subsequently electrophoresed and Western blotted. Sample IDs correspond to Table 1. Eluate is displayed with a capital letter and the flow-through with and additional (*). Healthy control is indicated with ‘Ctrl’. (A and C) Proteolytic treatment of urine samples from CJD and healthy control with proteinase K (concentration 60 µg/ml for 30 min). Proteins were transferred with a Bio-Rad instrument and the membrane probed with 3F4-HRP followed with ECL Plus. Membrane documentation was with a Kodak Imager with 5 minutes exposure time. (B and D) the membrane was stripped for 35 minutes at 37°C, verified for the removal of previously used antibody. The membrane was then probed with anti-IgG-HRP and ECL Plus was added for chemiluminescence. Membrane documentation was with a Kodak Imager with 5 minutes exposure time.

Table 1 Clinical, molecular and demographic features of CJD patients