Method for assaying luteoloside and special enzyme-linked immunosorbent assay kit thereof
An enzyme-linked immunosorbent reagent, luteolin technology, applied in the biological field, can solve the problems of uneven quality of honeysuckle medicinal materials, affecting the development and application of honeysuckle, and achieve a highly specific effect
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Embodiment 1
[0045] Example 1, the acquisition of hybridoma cell lines and the preparation of monoclonal antibodies against luteolin
[0046] 1. Synthesis of luteolin-BSA immunogen and luteolin-OVA coating agent
[0047] 1. Weigh luteolin-7-O-β-D-glucuronide standard substance (purchased from China Institute for Food and Drug Control, product catalog number is 111968, chemical structural formula is as follows: figure 1 Shown) 4.40 mg, dissolved in 1 mL of anhydrous N,N-dimethylformamide (purchased from Sigma, catalog number D4551), and magnetically stirred at room temperature to obtain luteolin-7-O-β- D-glucuronide standard solution.
[0048] 2. Add 2.13 mg of N-hydroxysuccinimide (purchased from Sigma, catalog number 56480) to the luteolin-7-O-β-D-glucuronide standard solution in step 1 to avoid After light stirring for 30 min, 3.73 mg of dicyclohexylcarbodiimide (purchased from Sigma, catalog number D3128) was added, stirred at 4°C for 4 h, centrifuged (3000 g, 5 min), and the supernat...
Embodiment 2
[0082] Example 2. Determination of cross-reactivity of monoclonal antibodies by indirect competitive ELISA
[0083] About 30 kinds of flavonoids in honeysuckle medicinal materials have been isolated, among which luteolin, luteolin, rutin and quercetin are relatively high in content; in addition, flavonoids with similar structures include apigenin and puerarin , naringin, hyperoside, and isoorientin, etc., so in this embodiment, the monoclonal antibody against luteolin obtained in step 5 of Example 1 and luteolin, rutin, and luteolin, rutin, and Cross-reactivity of quercetin, apigenin, puerarin, naringin, hyperoside, and isoorientin. Specific steps are as follows:
[0084] 1. Coating: Take a 96-well ELISA plate and coat it with luteolin-OVA solution, 100μL / well; the concentration of luteolin-OVA solution is 1000ng / mL; coat at 37°C for 3 hours, wash with washing solution Wash 4 times.
[0085] 2. Add samples: Add 50 μL of the following compound solutions to each well: luteoli...
Embodiment 3
[0094] Embodiment 3, the sensitivity detection of monoclonal antibody
[0095] The standard curve was established by indirect competition ELISA experiment, and the detection sensitivity was determined to be 42.26ng / mL, and the linear detection range was 9.07-258.07ng / mL. The specific method is as follows:
[0096] 1. Coating: Take a 96-well ELISA plate and coat it with luteolin-OVA solution, 100 μL / well; the concentration of luteolin-OVA solution is 1000 ng / mL; coat at 37°C for 3 hours, and wash with washing solution 4 times.
[0097] 2. Add standard solution and sample solution: add 50 μL luteolin standard solution and sample solution to be tested in each well, the concentrations of luteolin standard solution are: 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL mL, 25ng / mL, 12.5ng / mL and 6.25ng / mL; the control well is 50μL of sample diluent.
[0098] 3. Add antibody: Add 50 μL of the diluent of the anti-luteolin monoclonal antibody solution (diluted to 1000 ng / mL with the sample dil...
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