Evaluation of Coagglutination Test Kit for Red Sea Bream Iridovirus

Abstract

Coagulation test, in principle, is an immunodiagnostics technique, in which immunoglobuline G of antibody is bound to protein A from Staphylococcus aureus. The aim of study is to develop a rapid test kit for detecting iridovirus infection in fish. Method was summarized as follows: (1) vaccine of iridovirus was injected to rabbit four times with a dosage as 0.5 mL, 1 mL, 2mL, 3 mL each week. Serum was collected at the fifth week as a coagglutination test kit; (2) through the positif polymerase chain reaction (PCR) test, the kidney and spleen sample infected with iridovirus are homogenized by using the phosphate buffered saline (PBS) solution of pH 7.2 with ratio 1:2 (W/V); (3) the supernatant material is collected after centrifugation at 8,000 rpm for 15 min; (4) filtrate/supernatant from sample was dropped on a slide an added with coagglutination test kit with the same volume (1:1); (5) the agglutination observation is done after the 30, 60 and 90 min incubate at room temperature. The coagglutination test gave positive result in 25% of the test samples.

Rapid Diagnostic Test of Red Sea Bream Iridoviral Disease (RSIVD) in Grouper <i>Epinephelus</i> Sp. Based on Serological Co-Agglutination and Molecular Study

“…Antibodies have very specific reactivity properties against antigens that stimulate the formation of antibodies. The interaction of antigens with antibodies done outside the body is a very useful diagnostic tool for various diseases, and this approach is called "serology" [21]. This reaction can not always be observed either macroscopically or with the naked eye especially on viral antigens, so to be observed secondary reactions are needed.…”

“…Purification of immunoglobulins is done by precipitation of ammonium sulfate. The resulting precipitation solution is placed in a 15 mL conical tube, then it was centrifuged at a 6000 rpm for 30 min, the supernatant was removed by the pellet resuspended with physiological NaCl (0.85% NaCl) up to the initial volume, the anti-serum is then precipitated up to 3 times [21]. The dialysis process was carried out to remove the salt content contained in antiserum by means of an anti-serum suspension inserted into a 15 mL volume membrane of MWCO 6-8 kDa and was immersed in PBS pH 7.2 in a 300 mL beaker glass for 36 h with PBS replacement pH 7.2 new 12 h once.…”

“…The dialysis process was carried out to remove the salt content contained in antiserum by means of an anti-serum suspension inserted into a 15 mL volume membrane of MWCO 6-8 kDa and was immersed in PBS pH 7.2 in a 300 mL beaker glass for 36 h with PBS replacement pH 7.2 new 12 h once. The results of the dialysis process obtained immunoglobulin solution as the material of co-agglutination kits [21].…”

“…Staphylococcus aureus was harvested using ose and PBS pH 7.2, collected in a 15 mL conical tube and washing 3 times. The final wash pellet was added with 0.5% formalin in PBS pH 7.2 incubated overnight at room temperature [21]. The pellet was heated on a water bath with a temperature of 80 o C for 15 min and cooled rapidly, then washing 3 times with PBS pH 7.2, the final wash made 10% Staphylococcus aureus suspension in PBS pH 7.2 [21].…”

“…The 10% suspension of Staphylococcus aureus is added to the same volume as the immunoglobulin immunization result and is incubated overnight in room temperature. This mixture was then centrifuged at 3000 rpm for 10 min; the supernatant was discarded, it was resuspended back to initial volume with PBS pH 7.2 (obtained RSIV co-agglutination kit) [21].…”