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Development And Application Of Enzyme-linked Immuno Sorbent Assay Kits For The Detection Of Haemocytes In Scallop Chlamys Farreri

Posted on:2012-04-20Degree:DoctorType:Dissertation
Country:ChinaCandidate:T T LinFull Text:PDF
GTID:1113330338965632Subject:Aquaculture
Abstract/Summary:PDF Full Text Request
Bivalves frequently exposed to environmental stress and invasive or pathogenic microbes, and their survival depends to a large extent on the circulating cells collectively known as haemocytes. Haemocyte count and intracellular enzyme activity can be directly or indirectly used to evaluate the physiological state of a host or to estimate its immunity and potential defence capability against disease agents. In this dissertation, variations in haemocytes of scallop Chlamys farreri after viral and polysaccharidal stimulations were investigated. Then, C. farreri haemocytes were classified and separated. Subsequently, monoclonal antibodies (MAbs) against different types of C. farreri haemocytes were produced using the separated haemocytes as antigens. Afterwards, enzyme-linked immunosorbent assay (ELISA) kits for detection scallop haemocytes were prepared by employing these MAbs as primary antibodies. Finally, variations in scallop haemocytes in natural or stressed conditions were detected by ELISA kits. The main aim of this dissertation was to provide promising tool and theoretical basis for the assessment of health conditions of cultured scallops. This dissertation included the following 5 parts:1 Variation in haemocytes of C. farreri after a viral infectionDifferent concentrations (50, 5-1, 5-2, 5-3, 5-4, 5-5, 5-6 and 5-7) of acute viral necrobiotic virus (AVNV) were used to infect scallops C. farreri at 25°C. After infection, survival rates in each concentrations were counted, after that, among them, three concentrations of 5-3, 5-4 and 5-5 were picked out as the desired infection concentrations. Subsequently, AVNV concentrations of 5-3 and 5-5 were used to infect scallops C. farreri at 25°C, and total haemocyte count (THC) and activities of acid phosphatase (ACP), alkaline phosphatase (ALP), superoxide dismutase (SOD), myeloperoxidase (MPO), phenoloxidase (PO) and peroxidase (POD) in haemocytes were measured every day till 15 days, respectively. The results showed that THC in viral groups was significantly lower than that in control group, while activities of ACP, SOD, MPO, PO were significantly higher than control group in the first 8 or 9 days, and went back to control group gradually after the 10th day. Moreover, these five immune parameters in group of 5-3 varied more than that in group of 5-5. Differently, POD activity was reduced sometimes or induced other times, and showed no regular trends, while ALP activity wasn't detected. The results clearly illustrated that THC and intracellular enzyme activities varied greatly after a viral infection, validating that haemocytes play a crucial role in the process of C. farreri against AVNV invasion.2 Variation in haemocytes of C. farreri after a polysaccharidal stimulationScallops C. farreri were treated with 0.5, 1.0 or 2.0 mg/ml ofβ-glucan suspensions at 15°C (the optimum temperature for C. farreri) or 25°C (the temperature at which the disease rate is elevated), then THC, and the activities of PO, ACP, SOD, POD and ALP were measured in haemocytes every 12 h for seven days. The results showed that at 15°C, compared to the control, THC and PO in 0.5, 1.0 and 2.0 mg/ml, ACP, SOD and POD in 1.0 and 2.0 mg/ml, MPO and ALP respectively in 1.0 and 2.0 mg/mlβ-glucan were significantly increased. While at 25°C, THC and SOD in 1.0 mg/ml, PO in 0.5 and 1.0 mg/ml, ACP in 1.0 and 2.0 mg/mlβ-glucan were significantly induced, however, no significant induction of POD or MPO activity was observed, surprisingly, ALP activity was undetectable at 25°C throughout the entire experimental period. Moreover, the extent and duration of inducement as well as the time of peak emergence for these seven immune parameters appeared higher, longer and earlier at 15°C than 25°C in allβ-glucan treatments. These results clearly illustrated that THC and intracellular enzyme activities varied greatly, validating that haemocytes also play a crucial role in the process of C. farreri againstβ-glucan and high water temperature stresses, and high temperature may weaken or delay immune reactions to exogenous stress, thereby increasing the probability of being infected. 3 Production of MAbs against different types of C. farreri haemocytesScallop C. farreri haemocytes were separated by Percoll discontinuous density gradient (10, 20, 30, 40 and 50%) centrifugation, and the haemocytes at each resulting interface were collected and identified using flow cytometry and Giemsa staining, finding 30-40% interface of hyalinocytes, and 40-50% of granulocytes. Subsequently, separated hyalinocytes and granulocytes were respectively used as antigens to immunise mice. After cell fusion, screen using an indirect immunofluorescence assay (IIFA) and clone, the resulting MAbs were characterised using a flow cytometric immunofluorescence assay (FCIFA) and western blotting assay (WBA), respectively. The results showed that more than fifty hybridomas secreting positive antibodies were obtained, however, most of the antibodies showed specificities for both hyalinocytes and granulocytes, such as MAb 1F7, 4D5, 5C6, 4G11, 6A6, 2H11 and 4E2, among them one MAb designated as 6H7 presented a specificity only for granulocytes, and no MAbs showed a specificity only for hyalinocytes. MAb 1F7 had a positive rate of 82±2.4%, among them, positive hyalinocytes (PH) of 34±2.2%, positive granulocytes of 48±3.1%, it had abundant epitopes, reacted with many haemocyte proteins. MAb 4D5 was 76±2.3%, PH of 37±2.4%, PG of 39±1.1%, it reacted with three haemocyte proteins of 16.7, 87 and 96 kDa. MAb 5C6 was 70±2.6%, PH of 32±1.9%, PG of 38±2.4%, it reacted with 16.7 kDa haemocyte protein. While MAb 6H7 was 62±2.5%, PG of 54±3.0%, PH of 8±2.3%, it reacted with 155 kDa haemocyte protein.4 Preparation of ELISA kits for detection scallop haemocytesSpecific MAb suitable for primary antibody of ELISA kit for detection C. farreri haemocytes was firstly screened out using IIFA, FCIFA and WBA. Then the optimal using dilution was found out using ELISA chessboard titration by respective gradient dilution for antigen and screened MAb. Afterward, scallops C. farreri were stimulated by high water temperature (25°C) and 5-4AVNV, respectively, and haemocyte variation in challenged scallops was detected by ELISA. Subsequently, an evaluation criteria was completed by finding a critical range between normal physical and environmental stress from the ELISA data comparison of normal temperature and high temperature group, a critical range between environmental stress and pathogenic infection from the ELISA data comparison of non-infected and infected groups. Finally, ELISA kit was assembled and prepared. The results showed that MAb 1F7 with a high positive rate, abundant epitopes, against both hyalinocytes and granulocytes, was suitable for primary antibody of ELISA kit for detection C. farreri haemocytes. The optimal using dilution of antigen and antibody was that antigen diluted two times, and antibody diluted four times. The critical range between normal physical and environmental stress was 0.57±0.025, between environmental stress and pathogenic infection was 0.47±0.019. This ELISA kit contained ELISA plate, blocking solution, MAb 1F7, alkaline phosphatase labeled goat anti-mouse antibody (AP-GMA), colour developmental solution, standard samples and evaluation criteria, etc., with a sensitivity of 200 ng/ml.Granulocyte antigenic cross-reactivity of other two scallop species Patinopecten yessoensis and Argoecten irradians was carried out using MAb 6H7 against C. farreri granulocytes, illustrating MAb 6H7 also specifically reacted with the granulocytes of P. yessoensis and A. irradians, not with hyalinocytes, therefore, MAb 6H7 was chosen as primary antibody of ELISA kit for detection scallop granulocytes. After that, the optimal using dilution was found out using ELISA chessboard titration by respective gradient dilution for three antigens and MAb 6H7. Finally, ELISA kit was assembled and prepared. The results showed that not diluted antigen and antibody had a best reaction. This ELISA kit contained ELISA plate, blocking solution, MAb 6H7, AP-GMA, colour developmental solution and standard samples of three scallop species, etc., with a sensitivity of 5μg/ml.5 Application of ELISA kits for detection scallop haemocytesScallops C. farreri were collected monthly from Shandong area during the period from March 2009 to January 2010. After measurement of shell height, a relationship between haemocyte count and growth was investigated using ELISA kit. Scallops C. farreri were infected with 5-4 AVNV, and granulocyte count was measured using ELISA kit every day for 9 days. Scallops A. irradians were divided into two groups: one group fed every day, the other one not fed, afterward, granulocyte count in two group was measured using ELISA kit every week for 5 weeks. The results showed that in April, May and June, C. farreri grew fast, correspondingly, in this period, C. farreri had a large number of haemocytes, while in August, September and October, C. farreri grew slowly, with a small number of haemocytes. After AVNV infection, C. farreri granulocytes count significantly increased on day 1, then decreased on days 2, 3 and 4, thereafter, rebounded and approached to a second peak on day 6, finally went down gradually to the control level on day 8. After starvation stress, A. irradians granulocyte count significantly decreased in first 3 weeks, thereafter, decreased slightly, basically kept at a stable level, while in fed group, granulocyte count was maintained at the same level within 5 weeks. Conclusively, the results of these applications showed that ELISA kits developed in this dissertation had a promising prospect.
Keywords/Search Tags:Scallop Chlamys farreri, Haemocytes, Enzyme-linked immunosorbent assay kit, Monoclonal antibody
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