Evaluation of Direct Rapid Immunohistochemical test (DRIT) of Canis lupus familiaris hippocampal touch impression smears using a monospecific polyclonal antibody for rabies virus detection

@#<p style="text-align: justify;"><strong>BACKGROUND:</strong> Rabies is an important zoonotic disease that needs to be eradicated worldwide. It is still prevalent in the Philippines, thus development of a relatively affordable but still accurate and rapid post-mortem detection test for the virus is desired, especially in regional laboratories.<br /><strong>METHODS:</strong>The study evaluated the Direct Rapid Immunohistochemical Testing (DRIT) of hippocampal touch impressions of suspected rabid Canis lupus familiaris using monospecific N protein polyclonal antibody developed by the Research Institute for Tropical Medicine (RITM). One hundred sixty (160) acetone-fixed hippocampal touch impressions were subjected DRIT.<br /><strong>RESULTS:</strong> One hundred thirteen (70.6%) out of 160 samples tested positive for rabies viral antigen (RVA) and 47 (29.4%) out of 160 samples tested negative for RVA. No false positive and false negative results were obtained. The results agree with the gold standard, dFAT.<br /><strong>CONCLUSION:</strong> DRIT was able to detect low to high concentrations of RVA in the hippocampal touch impressions based on the grading distribution. DRIT had 100% sensitivity, specificity and over-all accuracy using monospecific polyclonal antibodies, which suggests its use as a more affordable alternative to the gold standard dFAT.</p>

Preparation of Monospecific Polyclonal Antibodies Against Glechoma hederacea Agglutinin / 生物化学与生物物理进展

Glechoma hederocea agglutinin (Gleheda) is a novel glycosylated lectin isolated from the leaves of G. hederacea. Like other glycosylated proteins, the detection of Gleheda by immunological methods is often hampered by the cross-reactivity of the polyclonal antibodies with unrelated glycoproteins. Hence a protocol to purify monospecific polyclonal antibodies from a crude antiserum raised against Gleheda was developed. After selective ammonium sulfate precipitation and successive affinity chromatography on columns of Sepharose 4B with immobilized Gleheda and Robinia pseudoacacia agglutinin (RPA), respectively, ion-exchange chromatography on a column of Q Fast Flow was used for further purification. The specificity of the antibody fractions from each step was tested by double immunodiffusion assay and analyzed by Western blot. Results revealed that affinity chromatography of the immunoglobulin fraction on the immobilized Gleheda antigen yielded an antibody preparation that still cross-reacted with many proteins in leaf extracts. Depletion of nonspecific cross-reacting antibodies directed against the glycan part of the glycoprotein by affinity chromatography on immobilized RPA removed most but not all nonspecifically reacting antibodies. Only upon further purification by ion exchange chromatography an IgG fraction of monospecific antibodies that reacted exclusively with Gleheda could be obtained and accordingly was suitable for immunodetection studies. This antibody purification procedure promises simplicity and efficiency. In addition, this method does not require expensive facilities.