Development of immunochromatographic Lateral flow test for rapid detection of Clostridium perfringens α, β and ε toxins in clinical samples

Abstract

In this study a lateral flow immunochromatographic kits have been developed for rapid detection of Clostridium perfringens toxins types, alpha (α), beta (β) and epsilon (ε) in clinical samples. Standard bacterial strains of C. perfringens bacterium types A, B and D were obtained from the Anaerobic vaccine Department in Veterinary Serum and Vaccine Research Institute, Abbasia, Egypt. Molecular identification of C. perfringens strains and their toxin genes were done. The C. perfringens α, β and ε toxins were prepared, purified and inactivated with 0.2% formalin according the manual of that department. Polyclonal antibodies specific to C. perfringens types α, β and ε toxoids have been prepared in rabbits and guinea pigs. Purification of toxins-specific IgG from Rabbit and Guinea pigs polyclonal antibodies was done using Caprylic acid. The purified toxin-specific polyclonal IgG prepared in rabbits was conjugated and labeled with gold chloride nanoparticles, (colloidal gold (CG), then The prepared toxin-specific rabbit and guinea pigs antibodies plus goat anti-rabbit antibodies were utilized in development of C. perfringens toxins lateral flow immunochromatographic test (LFT) that have been evaluated for detection of C. perfringens α, β and ε toxins in clinical samples. The sensitivity, specificity and accuracy of the developed LFT in detection of C. perfringens toxins were determined using a commercial ELISA kits as standard test. The Least amount of the C. perfringens α toxins that can be detected using the prepared LFT was 2ug/ml. It was 250ng/ml for the β toxin and 60ng/ml for the ε toxin. The developed kits gave positive results when tested with the corresponding standard C. perfringens α, β and ε toxins. No cross reactivity was recorded. The Validity testing of the developed LFT for detection of different C. perfringens toxins was determined through examination of one hundred fecal samples from diseased chickens collected from a boiler poultry farm suspected suffering clostridium infection. The chickens showed undigested food, diarrhea and low conversion rate. The collected samples were tested by the developed LFT kits and by three types of commercially available ELISA kits specified for detection of alpha, beta and epslon C. perfringens toxins (Bio-X Diagnostic, Belgium, Europe. cat. no. for Alpha toxin BIO K 289/2, for Beta Toxin BIO- X K267/2 for Epsilon toxin BIO-X K 277/2). The result by both kits were compared and divided into four groups; true positive (T+), false positive (F+), false negative (F-) and true negative (T-) for each type of toxins. The T+, F+, F- and T- for Alpha toxin were 30, 3, 7 and 60, respectively. In case of the Beta toxin were 23, 1, 7 and 69, respectively, while for Epsilon toxin were 8, 1, 4 and 87, respectively, The validity test for LFT that depends on the sensitivity, specificity and accuracy determination was calculated and it was 81%, 95.2% and 90%, respectively, for Alpha toxin, 76.6%, 98.5% and 72%, respectively, for Beta toxin and 66.6%, 98.8% and 95%, respectively, for Epsilon toxin.