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      Development and clinical evaluation of a rapid diagnostic kit for feline leukemia virus infection


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          Feline leukemia virus (FeLV) causes a range of neoplastic and degenerative diseases in cats. To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 × 10 9 and 0.86 × 10 9, respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 × 10 4 IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats.

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          Most cited references37

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          The Bradford method for protein quantitation.

          N Krüger (1993)
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            Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay.

            A simple, general procedure is described for the determination of the dissociation constant (KD) of antigen-antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of KD found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, or fluorescence transfer). Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody-antigen complexes can be determined even with crude preparations of monoclonal antibody. The sensitivity of the ELISA used permits the detection of very small concentrations of antibody and the determination of KD values as small as 10(-9) M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required.
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              Validation of serological assays for diagnosis of infectious diseases.

              Assay validation is a series of the following interrelated processes: an experimental process: reagents and protocols are optimised by experimentation to detect the analyte with accuracy and precision, and to ensure repeatability and reproducibility in the assay. a relative process: its diagnostic sensitivity and diagnostic specificity are calculated relative to test results obtained from reference animal populations of known infection/exposure status. a conditional process: classification of animals in the target population as infected or uninfected is conditional upon how well the reference animal population used to validate the assay represents the population to which the assay will be applied (accurate predictions of the infection status of animals from test results and predictive values of positive and negative test results are conditional upon the estimated prevalence of disease/infection in the target population) an incremental process: confidence in the validity of an assay increases over time when use confirms that it is robust as demonstrated by accurate and precise results (the assay may also achieve increasing levels of validity as it is upgraded and extended by adding reference populations of known infection status) a continuous process: the assay remains valid only insofar as the assay continues to provide accurate and precise results as proved through statistical verification. Therefore, validation of diagnostic assays for infectious diseases does not end with a time-limited series of experiments based on a few reference samples. Rather, it is a process that also requires constant vigilance and maintenance, along with reassessment of its performance characteristics for each population of animals to which it is applied. It is certain that the current movement to develop and implement accreditation criteria for veterinary diagnostic laboratories may be of little worth unless there is some assurance that the assays conducted in such laboratories are properly validated. Fully accredited laboratories may generate highly reproducible test results, but the results may still misclassify animals as to their infection status due to an improper assay validation process. Therefore, assay validation is foundational to the core product of veterinary diagnostic laboratories--test results and their interpretation.

                Author and article information

                J Vet Sci
                J. Vet. Sci
                Journal of Veterinary Science
                The Korean Society of Veterinary Science
                March 2014
                19 March 2014
                : 15
                : 1
                : 91-97
                [1 ]Department of Clinical Pathology Laboratory Science, Daejeon Health Science College, Daejeon 300-711, Korea.
                [2 ]Department of Biochemistry and CBITRC, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea.
                [3 ]Water Analysis and Research Center, K-water, Daejeon 306-711, Korea.
                [4 ]Bacteriology and Parasitology Division, Animal and Plant Quarantine Agency, Anyang 430-824, Korea.
                [5 ]Seoul Metropolitan Government Research Institute of Public Health and Environment, Gwacheon 427-070, Korea.
                [6 ]Department of Public Health, Graduate School of Public Health & Social Welfare, Dankook University, Cheonan 330-714, Korea.
                Author notes
                Corresponding author: Tel: +82-41-550-1939, Fax: +82-41-550-1998, pp99pp@ 123456dreamwiz.com

                The first two authors contributed equally to this work.

                © 2014 The Korean Society of Veterinary Science.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                : 08 March 2013
                : 23 May 2013
                : 09 July 2013
                Funded by: Daejeon Health Sciences College (Korea)
                Award ID: 2012013
                Original Article

                Veterinary medicine
                anti-felv p27 monoclonal antibody,feline leukemia virus,polymerase chain reaction,rapid diagnostic test,recombinant felv p27


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