THE USE OF A LIQUID PHASE BLOCKING ELISA KIT FOR DETECTION OF ANTIBODIES AGAINST FOOT-AND-MOUTH DISEASE VIRUS IN COLOMBIA
C. SANCHEZ MARTINEZ, M. QUINTERO
Laboratorio de Enfermedades Vesiculares,
ICA- CEISA, Avenida El Dorado no.42-42,
Santafé de Bogota, Colombia
Abstract
THE USE OF A LIQUID PHASE BLOCKING ELISA KIT FOR DETECTION OF ANTIBODIES AGAINST FOOT-AND MOUTH DISEASE VIRUS IN COLOMBIA.
The objective of this study was to undertake an interlaboratory comparison of a liquid pha blocking ELISA for detection of antibodies to FMD virus. For that purpo ra from 120 vaccinated, 120 infected and 120 FMD negative cattle were tested. All ra were tested in a screening assay at a dilution of 1/32. Positive ra were tested in a titration assay (1/10, 1/50, 1/250, 1/1250). For rotype
O1 Cruzeiro 108 ra from the FMD-free group were classified as negatives giving a specificity of 90%. For the same rotype the group of infected/ vaccinated cattle gave 114/115 positive results showing a nsitivity of 95% respectively 96%. For rotype A24 Cruzeiro from the FMD-free group 85 ra were classified as negatives giving a specificity 71%. For the same rotype the group of infected/ vaccinated cattle gave 90/99 positive results showing a nsitivity of 75% respectively 82%. The predictive value of the assay was good as results expected for the different rum categories were mainly confirmed in the test. Nevertheless a high number of plates were rejected due to “outside limits” and further adjustments are necessary to obtain more reliable results.
人口调查1. INTRODCUTION
In some regions of Colombia foot-and-mouth dia virus rotypes O1 Campos and A24 Cruzeiro exist endemically. At prent the country is involved in the hemispheric foot-and-mouth dia eradication plan. To achieve this objective it is necessary to u techniques with a higher nsitivity and specificity than the traditional diagnostic rological tests [1,2,3,4,5]. The u of a liquid pha blocking ELISA, LPBE is of great benefit in areas, where FMD prevention, control and eradication programs are carried out. The LPBE provides more reliable results becau it is very nsitive and specific. Other advantages are the fast delivery of results - usually within the same day - and the fact
that the technique is easy to perform and does not require special laboratory conditions
< cell culture or CO2 environment.
2. MATERIAL AND METHODS
The assay is bad on specific blocking of a defined amount of FMDV antigen by antibodies in the test sample during the liquid pha [6,7]. After the test rum is allowed to react with specific FMDV antigen, the test rum/antigen mixture is transferred to an ELISA plate coated with FMDV rotype specific trapping antibodies. The prence of antibodies to FMDV in the rum sample will result in the formation of immune complexes and conquently reduce the amount of free antigen trapped by the immobilized rabbit antira. In turn, less amount of guinea pig anti-FMDV detecting antibodies will react in the next incubation step. After the addition of enzyme labeled (horradish peroxida, HRP) anti-guinea pig immunoglobulin and substrate/chromogen solution a reduction of color development will be obrved when compared to control containing free antigens only. The bench protocol of the Joint FAO/IAEA Division was followed [8].
A total of 360 ra from cattle were tested from 3 different categories as shown below:
- 120 bovine ra from free areas of FMD (provided by CPFA)
鲢鱼怎么钓- 120 bovine ra from vaccinated cattle with trivalent vaccine (provided by CPFA)
- 120 bovine ra from FMD infected animals obtained from outbreaks which naturally occurred in different regions of Colombia.
3. RESULTS
For rotype O1 Cruzeiro 108 ra from the FMD-free group were classified as negatives giving a specificity of 90%. For the same rotype the group of infected/ vaccinated cattle gave 114/115 positive results showing a nsitivity of 95% respectively 96%. For rotype A24 Cruzeiro from the FMD-free group 85 ra were classified as negatives giving a specificity 71%. For the same rotype the group of infected/ vaccinated cattle gave 90/99 positive results showing a nsitivity of 75% respectively 82% (Table I).
TABLE I. RESULTS ACCORDING TO GROUP OF SERA AND SEROTYPE
Bovines Samples O Virus A Virus
P N R P N R Free 120 5 108 7 21 85 14 Infected 120 114 4 2 90 15 15 Vaccinated 120 115 0 5 99 10 11
P = positive
N = negative
R = retest
A high number of plates was classified “outside limits” becau the positive rum controls were out of the upper and lower limits, although the negative rum control and the antigen control were within limits.蘑菇面
阴阳师金鱼姬赞美女生的词4. CONCLUSIONS AND DISCUSSION
Although most of the plates were rejected due to “outside limits” the predictive value of the assay remained good as results expected for the different rum categories were confirmed in the test as shown in Table I.
Further adjustment for the upper and lower control limits is necessary to obtain reliable results. It could be obrved that the problem was more noticeable for the dia free ra, where a small percentage of the samples were positives. In the ca of ra from infected animals, the highest percentage was positive. A similar result was obrved in the group of ra from vaccinated animals.
In the group of ra from infected animals positive results were obtained for both rotypes. The reason for this is that the animals live in FMD endemic areas where additionally vaccination is carried out.
Once having standardized this technique it will be ud as a routine test all over the country to monitor the success of the vaccination programme, which is being applied systematically every six months.
ACKNOWLEDGEMENTS
We like to thank PANAFTOSA and the Joint FAO/IAEA Division for their support to carry out this project.
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天庭饱满面相
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[8] FMD ELISA KIT, Liquid pha blocking enzyme immunoassay for detection of antibodies
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