Preparation And Characterization Of Monoclonal Antibody Against Palytoxin

OBJECTIVES: Palytoxin is one of the most highly toxic marine substances as known. It can bind to Na+/K+ pumps in the plasma membrane of neurons and muscle cells, and cells death are also induced by the effects of PTX. People will be intoxicated because of ingestion of the seafood polluted by PTX. Their symptoms were mainly featured by severe muscle pain, low back pain, and discharge of black urine, even death. To our knowledge, however, there's no medicine produced in cure of intoxication by PTX. Our lives are threatened by it.During these years, with the concept of the food safety and health going more and more deeply, to establish a useful assay to detect the PTX become more and more important in the world. Some countries had used HPLC,FETAX and other assays to detect and quantitate PTX in past years.In our study, we used chemistry methods to prepare the artificial antigen of PTX, and immunized BALB/c mice by Freund's adjuvant immunization method. After that, the antiserum was induced in mice. The hybridoma cell lines that secreted anti-PTX McAb with high affinity were established by hybridoma technique. The method could be applied to detect the Palytoxin in seafood and has great potential to be used in toxicological researches in future. METHODS: This article is composed of three parts:1. Preparation of anti-PTX antiserum. PTX was coupled with the carrier protein KLH and BSA to provide PTX the immunogenicity. The PTX-PDP-KLH conjugates were used to immunize several 6-8 weeks old female BALB/c mice. The antiserum was attained by retrobulbar venous plexus blood law. The potency test refers to the indirect ELISA. The control group we could choice the healthy BALB/c mice.2. Preparation of the anti-PTX McAb hybridoma cell lines. By fusing murine NS-1 cells and spleen cells from mice immunized which obtained a hybridoma cell line steadily secreting monoclonal antibodies against Palytoxin. Furthermore, by indirect ELISA, we screen clones which could secrete specific monoclonal antibodies.3. Identification and production of the anti-PTX McAb. After karyotype and specificity analysis, the positive fusion cells injected into the healthy BALB/c mice by intraperitoneal injection. About 14 days later, ascites can be collected,and to be the McAb (ascitic fluid) after purification.RESULTS: 1. By Ultraviolet-visible Spectrophotometer, the PTX-PDP-KLH conjugates had the peak value in 267 nm, and the peak value of the PTX-PDP-BSA conjugates was in 273 nm. We used the PTX-PDP-KLH conjugates to immunize the 6-8 weeks old female BALB/c mice. The potency test which refers to the indirect ELISA shows that the difference between antiserum of immunized mice and healthy mice are obvious (OD > 7, 1: 2500).2. The hybridoma fusing ratio was 21.43 %, and cloning ratio was about 9.1%. The monoclonal antibodies from A6 & B8 showed highly significant reactivity by indirect ELISA.3. By karyotype analysis, we know that the hybridoma cell chromosome number is about 94 to 104. No cross-reactivity among BSA,KLH in specificity analysis. After immunization, mice will induce 4-5ml ascites. SDS-PAGE indicates the anti-PTX McAb have 2 protein bands: one is in 25 kD, the other is in 50 kD. CONCLUSIONS: 1. We have successfully made the PTX couple the carrier protein KLH,BSA by the chemistry methods, and overcome the influence that PTX's molecular weight is too small to induce neutralization. The PTX-PDP-KLH conjugates have immunogenicity to induced antiserum which neutralize PTX in BALB/c mice.2. It is feasible to obtain hybridoma cell lines which could secrete monoclonal antibodies A6 & B8 that neutralize the PTX by hybridoma technology. The McAb secrete from this cell lines can bind to PTX specifically. It means that we can use this type of McAb to identify and detect PTX in service use.