Test paper strip for semi-quantitative detecting Heterosigma akashiwo, its preparation and using method
A technology for the semi-quantitative detection of I. red tide algae, which is applied in the field of semi-quantitative immunochromatographic detection and analysis test strips and its preparation and detection of I. red tide algae. It can solve the problems of long time and achieve evaluation and management promotion Effects of oceanographic research and environmental research
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Embodiment approach 1
[0045] Embodiment 1: Preparation of immunogen
[0046] The vigorously growing I. akashiwo algae cell culture solution was fixed overnight with formaldehyde at a final concentration of 2%, centrifuged at 10,000 r / min for 10 min, and the algal cell pellet was collected. The algal cell aggregates were washed once with distilled water and twice with PBS, and the algal cells were collected by centrifugation each time, and the centrifugation conditions were 10000r / min, 10min. The collected algae cells were divided into 1.5ml Eppendorf tubes, the residual water was shaken off, and stored at -20°C for later use. Take it out just before use, and resuspend it evenly with 5ml of sterile PBS. The standard solution of algae cells was subjected to ultrasonication and alternate heating and cooling (-75°C, 15min: 100°C, 5min, three cycles) to obtain a broken algae cell standard solution with known cell density.
Embodiment approach 2
[0047] Embodiment 2: Determination of antibody titer.
[0048] Use the indirect ELISA method to determine the titer of the antibody, the main steps are as follows:
[0049] (1) Coating of seaweed or crude seaweed extract (10 5 -10 6 cells / m1), 100 μl / well, overnight at 4°C in a humid chamber;
[0050] (2) Wash the plate, wash 3 times with 0.5% PBST, wash 2 times with distilled water, and dry the remaining liquid;
[0051] (3) For blocking, dilute with 1% BSA coating buffer solution, 120 μl / well, at 37°C for 3 hours or overnight at 4°C;
[0052] (4) Wash the plate, wash 3 times with 0.5% PBST, wash 2 times with distilled water, and dry the remaining liquid;
[0053] (5) Add the polyclonal antibody gradient dilution solution of seaweed, 100 μl / well, 37°C, 1.5h:
[0054] (6) Wash the plate, wash 6 times with 0.5% PBST, rinse with distilled water 2 times, and dry the remaining liquid:
[0055] (7) Add the dilute solution of the enzyme-labeled secondary antibody, 100 μl / well,...
Embodiment approach 3
[0061] Embodiment 3: Determination of antibody specificity.
[0062] The specificity of the antibody was determined by indirect ELISA method, and the specific steps were as follows:
[0063] (1) Class A seaweed or seaweed crude extract coating (10 5 -10 6 cells / ml), 100 μl / well, overnight at 4°C in a humid chamber;
[0064] (2) Wash the plate, wash 3 times with 0.5% PBST, wash 2 times with distilled water, and dry the remaining liquid;
[0065] (3) For blocking, dilute with 1% BSA coating buffer solution, 120 μl / well, at 37°C for 3 hours or overnight at 4°C;
[0066] (4) Wash the plate, wash 3 times with 0.5% PBST, wash 2 times with distilled water, and dry the remaining liquid;
[0067] (5) Add polyclonal antibody dilution solution of type b seaweed, 100 μl / well, 37°C, 1.5h;
[0068] (6) Wash the plate, wash 6 times with 0.5% PBST, rinse with distilled water 2 times, and dry the remaining liquid;
[0069] (7) Add the dilute solution of the enzyme-labeled secondary antib...
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