1. Introduction Aim of study
IMMUNOANALYTICAL SYSTEMS FOR HEPATITIS A VIRUS DETERMINATION L. Michelia,e, A. Attarc,De Stefanoa, D. Doniab, M. Diviziab, A. Aminec, G. Palleschia,e, P. A. Salazar Carballod, D. Mosconea,e a. Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma “Tor Vergata” – Via della Ricerca Scientifica Rome, Italy b. Dipartimento di Medicina sperimentale e Chirurgia, Università di Roma “Tor Vergata”, Via Montpellier, 1 – Rome, Italy c. Faculty of Sciences and Techniques, University Hassan II of Casablanca, BP 146, Mohammedia 20650, Morocco d. Laboratorio de Neuroquimica y Neuroimagen, Facultad de Medicina, Universidad de La Laguna, Campus de Ofra s/n Tenerife, España e. Consorzio Interuniversitario Biostrutture e Biosistemi “INBB”, Viale Medaglie d’Oro 305, Rome, Italy
Introduction
Hepatitis A virus (HAV) causes an acute hepatitis associated with a significant morbidity and occasional mortality. Outbreaks of waterborne diseases are certainly underestimated, due to the lack of adequate programs for the epidemiological surveillance. Current legislation for water, shellfish (EC 2073/2005 EC B53/2004) and plant (EC 2073/2005) does not provide for any limitation to the presence of HAV and other enteric viruses in the irrigation and housing waters. In addition, there is no official method for the detection of these viruses. The presence of virus is alleged by rise of symptoms in consumer and more specifically through the search for anti-HAV IgM and/or anti-HAV IgG antibodies in blood [1]. Immunoanalytical approaches are reported as methods to determine HAV in drinking water before its use, thus avoiding the infectious disease.
Aim of study
The idea of this study is to provide an "alarm" system o for possible contamination of HAV of food and environment using a sensitive and fast screening system. For the achievement of this objective, two electrochemical, competitive and sandwich, Enzyme Linked Immuno-MagneticElectrochemical assays (ELIME) have been developed. These systems are based on the use of new polydopamine-modified magnetic nanobeads (MNPs – pDA) as solid support for the immunochemical chain, and an array of 8 screen printed electrodes as a sensing platform. The electrochemical technique used for the final detection was DPV. This rapid and low-cost analysis method involves the use of a portable instrument, able to perform measurements directly in the field. All operative parameters were studied, such as blocking reagents, coating and labeling reagent concentration, etc.
Methods and materials
Magnetic NanoParticles
(MNPs-pDA)[2]
Competitive and Sandwich formats
developed on MNPs - pDA
DVP PARAMETERS
Potential Range – 600 mV
Pulse Width ms
Pulse Amplitude mV
Scan Speed mV/s
Pulse Repetition ms
MNPs – pDA
Antigen (HAV)
Monoclonal antibody
Secondary polyclonal antibibody labelled with AP (alkaline phosphatase)
Primary monoclonal antibibody labelled with AP
(alkaline phosphatase)
Array of 8 screen printed electrodes
Main results
Calibrations curves
Study of blocking step for both formats
Shaking 45min
DPV detection
MNPs-pDA-HAV (40 ng/mL) + BSA 3%
MAb (20 μg/mL) in PBS
PAb-AP 1: v/v in PBS
1-NPP ( 5 mg/mL) in DEA
Shaking 45min
DPV detection
MNPs-pDA (25 mg/mL) + MAb (20 ng/mL)
HAV (0 – 10-2 IU/mL) in PBS
MAb-AP 1:1.000 v/v in PBS
1-NPP (5 mg/mL) in DEA
Competitive format
Sandwich format
PVA = polyvinyl alcohol; BSA = bovine serum albumin
Cross-reactivity study
for chosen monoclonal antibody against HAV
Antigen
%CR
HAV
100%
Cox* 1%
LOD
8∙10-7 IU/ml
Working range
2∙ ∙10-2 IU/ml
RSD% (n=3) 3%
LOD
1∙10-9 IU/ml
Working range
5∙ ∙10-2 IU/ml
RSD% (n=3) 7%
Conclusion and future work
The commercial monoclonal antibody against HAV used in both ELIME assays is selective for HAV (low cross-reactivity for Coxsackie B4 (Cox), entorovirus present in sewage and water).
Results showed that sandwich formats is more sensitive of competitive one in terms of LOD and working range.
Preliminary results showed no matrix effect on both ELIME formats.
Next steps will be the comparison of the proposed ELIME systems with the qRT-PCR analysis, technique routinely applied by the controllers in the evaluation of contaminated matrices by enteric viruses, using spiked and real samples of water and sewage.
The proposed ELIME systems, coupled to a portable instrumentation, can be used for HAV determination for analysis of sewage, tap and well water directly in “situ”.
[1]Hollinger FB and Ticehurst JR (1996), in Fields virology. 3rd Edition. Philadelphia: Lippincott-Raven, ; [2] M. Martín,   P. Salazar,   et al.  (2014), J. Mater. Chem. B, 2,