The development of enzyme-linked immunosorbent assay (elisa) for diagnostics of leptospirosis in animals
Journal Title: Бюлетень "Ветеринарна біотехнологія" - Year 2019, Vol 34, Issue
Abstract
Introduction. Leptospirosis is the most wide spread zoonosis worldwide, which is present in all continents except Antarctica and evidence for the carriage of Leptospira has been found in virtually all mammalian species examined. The wide spectrum of symptoms confuses the clinical diagnosis and makes it undependable. The laboratory diagnosis of leptospirosis, a prerequisite for treatment, is usually achieved either by isolation of the causative organisms or by serological evidence indicating recent infection. The microscopic agglutination test (MAT) is the reference test for diagnosis and detects antibodies at serovar levels. MAT has many advantages, but there are significant deficiencies. The maintenance of stock cultures and use of live organisms creates a risk of laboratory-acquired infection. Therefore, several methods have been developed for use in diagnosis of leptospirosis as an alternative to MAT, of which IgM ELISA is the most promising and detects genus-specific antibodies. The goal of the work was to develop a modification of ELISA for the diagnosis of leptospirosis in animals that would correspond to the following requirements: 1) ability to detect antibodies against serogroups of Leptospira, that are recommended for the diagnosis of this zoonoses in Ukraine (to have high sensitivity and specificity); 2) minimize the risk of laboratory-acquired infection at preparation of reagents for ELISA and its conducting. Materials and methods of research. During the validation, we have conducted several technological stages. The first of them was performed by selection and determination the optimal concentration and titers of ELISA main components (antigen, enzyme conjugate, enzyme substrate and blocking substance) and the second was carried out a statistical analysis of the results after testing ELISA on the reference panel of sera blood samples. To perform this research, ELISA was carried out with field samples of blood sera from sick and healthy for Leptospirosis dogs, pigs and cattle and seven reference sera (OIE), that were obtained from the Royal Tropical Institute, Amsterdam. Results of research and discussion. As the antigen was used recombinant protein LipL 32, which is a modified compound one of the major outer membrane lipoprotein of pathogenic Leptospira species. To determine the optimal dose of sensitizing antigen, conducted its sorption within 0.5–4 μl volume. At the same time we have tested substances that eliminate the not specific interaction of antibodies with antigen – edible gelatin and skimmed milk powder (respectively, 0.5% and 5% solutions). Analysis of titration showed, that the optimum amount of LipL 32 in both cases was 1 μl volume. However, was registered high background reaction in all investigated wells of microplate when dilution of blood sera samples by phosphate buffer with 0.5% gelatin solution. When a 5% solution of skimmed milk was used, the indicators of optical density in negative controls were much smaller, and correlation coefficients of positive and negative sera were more. By analyzing the results of conjugate titration, the highest correlation coefficients of optical density between positive and negative samples of blood sera were registered in its titer 1/500 and dilution of sera 1/2.5 (40 μl volume). A comparison enzyme substrates showed, that the indicators of optical density in positive samples of blood sera by MAT were significantly higher by using TMB solution, than indicators by using OFD chromogen, respectively, 0.836±0.089 o.d. against 0.601±0.066 o.d. The difference of optical values in both cases was significant (р˂0.05). At the same time, the optical indicators of negative blood sera samples by MAT significantly were not different in both cases (0.098±0.002 o.d. against 0.085±0.0017 o.d.). The last technological stage was carried out a statistical analysis of the results after testing ELISA on the reference panel of sera blood samples. Established that the sensitivity of the developed ELISA is less than its specificity (89.8% against 96.7%), but overall efficiency of this method is high and equals 93.6%.
Authors and Affiliations
A. Pyskun, V. Ukhovskyi, G. Aliekseieva, O. Pyskun
Keywords
Related Articles
- EP ID EP556393
- DOI 10.31073/vet_biotech34-12
- Views 91
- Downloads 0
Micromycetes species identification and study of their ability to produce fusariotoxins
Introduction. Among many toxic environmental factors, more attention is attracted to the metabolites of mold fungi - micotoxin. More than 45 of only trichothecene mycotoxins have been isolated and studied. T-2 toxin is o...
Liposomal emulsion of hermatranol influence on the lipid peroxidation process in cows body and on their postnatal period course
Introduction. Experiments on animals indicate that Germanium is rapidly adsorbed in any method of administration, does not accumulate in certain organs and rapidly excreted with the urine. It should be noted that the eff...
Molecular genetic characterization of street rabies virus isolates selected in Ukraine
Introduction. Studies of molecular genetic characteristics of different isolates of rabies virus, and in particular their nucleotide sequences allows to determine phylogenetic relationships within phylogroup and genotype...
Obtaining cultural antigen for diagnostics equine infectious anemia in the DPR test
Introduction. One of the most important challenge in horse breeding is equine infectious anemia (EIA) that constantly registers in many countries with the developed breeding horses. Diagnostics of EІA is a basic chain in...
Methodical approaches to the radial hemolysis reaction use for equine infectious anemia diagnostics
Introduction. Of Equine Infectious Anemia (EIA) is one of the most important issue that constantly registers in many countries with the developed horse breeding. Diagnostics of EІA is a basic element of the preventive me...