Recipient Organization
VECTOR TEST SYSTEMS
3537 OLD CONEJO RD STE 101
NEWBURY PARK,CA 913206555
Performing Department
(N/A)
Non Technical Summary
African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs, which is responsible for serious economic and production losses. Prevention and early detection can play a key role in the control strategy for ASF. VecTOR Test Systems, Inc. will work on developing rapid immuno-chromatographic assays for detection of the ASF viral antigens as well as the tests for detection of ASF-specific IgG and IgM in infected animals. In Phase I, feasibility of the concept will be confirmed by developing prototype assay(s) that utilizes the most promising reagents available currently. The technology is based on the simple and reliable immuno-chromatographic detection method by which sensitive test-strip based assays provide results in less than 30 minutes. The proposed assays will be easy to perform and results easy to interpret with minimal training. The assay will be made available in a kit with components required to perform the assay. The assay kit will be stable at ambient storage conditions.
Animal Health Component
20%
Research Effort Categories
Basic
10%
Applied
20%
Developmental
70%
Goals / Objectives
The objective of this project is to develop a hand-held, field-deployable assay capable of detecting ASFV-specific IgM and IgG and identifying African Swine Fever (ASF) virus antigen in veterinary samples (blood, serum, nasal or body fluids, and other biological materials).Technical Objectives for the project:1. Identify and apply reagents for detection of ASF-specific IgM and IgG antibodies in porcine samples. Utilize and compare the use of various proteins that are available at KSU (Kansas State University, laboratory of Dr Juergen Richt) as recombinant antigens. ASFV p30/p32 and p54 will be prioritized based on our current knowledge (studies ongoing at KSU and elsewhere globally).Evaluate various preparations of the recombinant ASF proteins (different purification methods); different expression systems (e.g., E.coli vs. Baculovirus expression systems) and determine ease of scale-up.2.Identify and apply reagents for detection of ASFV antigen in porcine samples:Utilize existing monoclonal antibody reagents available (against structural ASFV proteins); compare these reagents with ASF antigen tests (pen-side/ELISA) at the BRI, KSU.Utilize monoclonal antibody reagents developed by Meridian Life Science. MAbs to ASFV p30 (Cat # CO1881M and Cat CO1882M) will be evaluated. Compare and identify MAb reagents that can be utilized for ASF antigen product development3. Develop easy to implement method for sample testing including sample prep and processing.Initially, specimens to be tested will be serum samples positive for ASF virus or ASFV-specific antibodies. Future testing will also include swine oral fluid (SOF) samples. Phase I studies will include development of sample dilution buffer for lateral-flow assay, and use of capillary tube for blood/serum..Evaluate above tests with KSU archived samples in order to determine proof of concept.Deliver prototype assays for evaluation to Kansas State UniversityIn summary, in order to meet Phase I objectives the following criteria are desired capabilities of the final product: (i) Rapid and accurate detection of ASFV-specific IgM and IgG antibodies and ASFV-specific antigen/s. (ii) The assay should be specific and exhibit similar sensitivity and specificity as current gold-standard antigen or serological assays. (iii) The assay must be rapid (<30 min), make use of a one- or two-step format, and have an acceptable shelf life (stable at 35oC for 18 months to 2 years). (iv) The assay must be user-friendly (i.e., easy to operate), inexpensive, and portable. (v) The assay should be based on heat-stable reagents, without the need for special storage requirements. (vi) The visually interpreted test developed in Phase I should be modifiable with minimal adjustments to work with a semi-quantitative reader to be developed in Phase II.
Project Methods
ASF-specific IgM/IgG antibody detection assay: For the ASF-specific IgM/IgG antibody detection assay, a specific combination of reagents is expected to be utilized for initial evaluations. For detection of IgM and IgG antibodies in porcine serum, reagents that detect porcine IgM and reagents that detect IgG will be immobilized on the membrane. The gold label will be adsorbed with a variety of protein formulations, initially p30/p32 and/or p54 ASFV proteins, for comparative analyses; the assay will use alternative ASFV-specific proteins (see Table below) if p30/32 and/or p54 are not suitable as target antigens in our system; in addition, various purification levels of the p30/32 and p54 proteins will be utilized; and/or the different expression systems (E.coli vs. baculovirus) utilized for production of ASFV proteins.Table : African Swine Fever Virus proteins available at KSU (Sunwoo SY, et al. 2019)ProteinStructure and functionp54Integral membrane protein; Highly antigenic; virus morphogenesis attachmentP30/p32Highly antigenic, early infection, virus attachmentpp62 (p15, p35)Polyprotein (processed to p15, p35), assembly and maturation of virion's corep15Expressed late in infection, resides at the core shell structure proteinp35Expressed late in infection, resides at the core shell structure proteinp72Virus surface protein promoting neutralizing antibodiesCD2vVirulence, hemadsorption, pathogenesis and virus disseminationp17Transmembrane protein of viral internal envelope; Virus assemblyp22Envelope protein; early structural proteinAssembly of the assays: We plan to use currently established, commercially available materials for use in lateral and other immuno-chromatographic assays.Preparations of colloidal gold labeled antigens (for IgM/IgG antibody tests) or gold labeled antibodies (for antigen detection tests) that are used for conjugate pad preparation will have the following sub-tasks:Preparation of colloidal gold or other proprietary particulate label.Perform adsorption reaction. Initially this will be carried out at different pH conditions, or different concentrations of antigen or antibodies used in order to find the amount of protein/antibody required to stabilize the gold solution, to evaluate the degree of adsorption, and to optimize the colloidal stability and retention activity; this approach is generally used for this format of assays.Preparation of test assays will consist of the following sub-tasks:Preparation of membrane component. In this task, suitable concentrations of antigen or antibody will be printed in a specific region on the membrane using methods currently in use in the industry.Preparation of conjugate pads.Specific treatments to materials such as membrane, conjugate pads or the wick.Lamination of different components of the strip.Cutting laminated material into strips by using standard strip cutting equipment.Assembly of strips into plastic cassette and in kit package format.The prepared test strips will be evaluated and specific manufacturing conditions standardized. Tests containing various reagent combinations present either on gold or immobilized on membrane will be prepared and evaluated. The best reagent pairs will be selected and panels or individual test strips prepared.ASF virus Antigen Detection Assay:For the ASF antigen detection test, the immuno-chromatographic prototype assay will be a dual antibody sandwich immunoassay where two ASF-specific antibodies are utilized for detection of the respective ASF antigen. Monoclonal Antibody reagents from commercial sources (e.g. Meridian BioSciences) as well as those developed at KSU will be evaluated for developing the antigen assay. At KSU, two monoclonal antibodies have been made for p54, and a series of different hybridomas for p32. Each monoclonal was screened for specificity to recombinant antigen by indirect ELISA. In addition,five monoclonal antibodies for p32 (2B8, 2B9, 1D8, 5C1, 3H1) have been shown to be reactive to ASFV genotype I and II isolates.Evaluations:Materials for evluations will include recombinant proteins produced at KSU (Dr Richt's laboratory) for detection of the ASFV and stored archived samples for Phase I evaluations.Evaluation of prototype assays: A scoring system is applied to "read" the visual intensity of the signal. For example, the strength of the signal is +++ (strong signal) to + (weak signal) and +/- for instances where the reader is uncertain. The developed assay's performance can be compared to a gold standard assay, e.g. ELISAs for antigen and antibodies, qPCR, IFA, virus titers or other methods in practice.In Phase I, reasonable amount of information will be sought from the assay evaluations performed. For the antigen assay, a critical criteria would be the amount of recombinant antigen (nanograms) that can be detected and if such concentrations are available in the to be assayed porcine samples. Attempts will be made to quantify or obtain information on actual samples collected and archived, and attempts will be also made to evaluate the antigen detection capacity with prototypes prepared in Phase I. A realistic estimation is necessary for improvements that can be made in Phase II using new methodology which can provide amplification of signals.Evaluation of the detection capacity of ASF-specific IgM/IgG antibody assay prototype will also be sought. Here, the detection of ASF -specific antibodies at defined time points after infection is critical. Commercially available ELISA kits detect ASF-specific antibodies in blood of experimentally infected pigs around day 10 p.i. Comparative analyses will be performed. Once the required feasibility for ASFV antigen and/or IgM/IgG antibody assays is confirmed, preliminary plans will be made for possible field trial sites. Actual trials would be performed in Phase II. Typically, trials are performed at three or more field sites. The locations of these laboratory or sites should be pertinent to ASF disease incidence/prevalence and the locations where the tests will be used. The purpose is to show that the test's results are reproducible at different sites and that its performance characteristics are confirmed independent of the manufacturer. Studies of the assay's specificity, sensitivity, interfering substances, and cross-reactivity will be performed, as are comparisons to established reference methods.