Indirect Enzyme Linked Immunosorbent Assay Sebagai Metode untuk Melacak Bruselosis pada Sapi Perah (INDIRECT ENZYME IMMUNOSORBENT ASSAY (I-ELISA) AS METHOD FOR DETECT BRUCELLOSIS IN DAIRY COW)

Rinaldi Ghurafa,Denny Widaya Lukman,Hadri Latif

doi:10.19087/jveteriner.2019.20.1.30

Abstract

Brucellosis has become a zoonotic disease that received attention in efforts to prevent and eradicate strategic infectious animal diseases in Indonesia. Brucellosis can be detected early by the rose bengal test (RBT), followed by complement fixation test (CFT) and by enzyme linked immunosorbent assay (ELISA). The aims of this research was to study the indirect enzyme linked immunosorbent assay test (I-ELISA) as an alternative test for detecting brucellosis in dairy cattle. The method was used by conducting tests of RBT, CFT, I-ELISA and commercial I-ELISA to test brucellosis. The test results were calculated sensitivity and specificity, as well as analyzed by calculating the kappa value. The method was used by conducting tests of RBT, CFT, I-ELISA and commercial I-ELISA to test brucellosis. The test results were calculated for sensitivity and specificity, as well as analyzed by calculating the Kappa statistical value. The results of the sensitivity and specificity calculation showed that the indirect enzyme linked immunosorbent assay (I-ELISA) test developed a higher sensitivity (100%) compared to RBT test (93.75%) and commercial I-ELISA (93.75%). The developed I-ELISA specificity (74.68%) was still lower than RBT (89.87%), but higher than commercial I-ELISA (70.52%). The calculation of the statistical value of kappa RBT with CFT showed the kappa value 0.7120 which meaned it had a good agreement, commercial I-ELISA with CFT showed kappa value 0.6165 which meaned it had good suitability, whereas I-ELISA developed with CFT showed kappa value 0.4984 which meaned having a moderate agreement.In conclusion, the indirect enzyme linked immunosorbent assay (I-ELISA) which had been developed had low specificity, but the sensitivity was the highest compared to the commercial I-ELISA test and RBT, so this test was appropriate to be used as a screening test, especially in dairy cows movement into brucellosis-free areas or regions.

Highlights

Brucellosis has become a zoonotic disease that received attention in efforts to prevent and eradicate strategic infectious animal diseases in Indonesia
The method was used by conducting tests of rose bengal test (RBT), complement fixation test (CFT), I-enzyme linked immunosorbent assay (ELISA) and commercial indirect enzyme linked immunosorbent assay (I-ELISA) to test brucellosis
The test results were calculated for sensitivity and specificity, as well as analyzed by calculating the Kappa statistical value

Summary

Indirect Enzyme Linked Immunosorbent

Fernandez-Lago dan Diaz (1986); Alton et al. (1988); Adji et al (2015); dan Mujiatun et al. Hasil titrasi ini selanjutnya dijadikan dasar dalam melakukan pengenceran antigen, serum, dan konjugat pada uji I-ELISA. Pengujian I-ELISA dilakukan menggunakan antigen LPS dari B. abortus strain S99 yang dilarutkan dalam phosphat buffer saline (PBS) dengan pH 7,2. Antigen 100 μL yang telah dilarutkan dalam PBS pH 7,.2 dimasukkan ke dalam setiap sumur microplate (Maxisorp, Nunc, Denmark), kemudian microplate ditutup dengan plastik adesif dan diinkubasi selama 24 jam pada suhu 4°C. 100 μL ke dalam setiap sumur microplate dan selanjutnya diinkubasikan selama satu jam pada suhu ruang di atas microplate shaker. PBST 0,05% sebanyak tiga kali dan selanjutnya konjugat protein dilarutkan dalam PBSTC 0,2%. Konjugat terlarut kemudian dimasukkan sebanyak 100 μL ke dalam sumur microplate, selanjutnya diinkubasi selama 1 jam pada suhu ruang di atas microplate shaker. Microplate dicuci kembali dengan PBST 0,05% sebanyak tiga kali dan selanjutnya ditambahkan substrat. Nilai 3 0,81 kesesuaian sangat baik, 0,60-0,80 kesesuaian baik, 0,41-0,60 kesesuaian sedang, 0,21-0,40 kesesuaian kurang, 0,00-0,20 kesesuaian sedikit sekali, dan 0 tidak ada kesesuaian (Thrusfield 2005)

HASIL DAN PEMBAHASAN Hasil Pengujian Sampel

Uji Laboratorium

Findings

Nilai statistik Kappa