PSMD4 protein ELISA detection kit as well as detection method and application thereof
A detection kit, PSMD4 technology, applied in the fields of immunology and biology, can solve problems such as simplicity
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Embodiment 1
[0074] Preparation of detection ELISA plate: Coating buffer 1×PBS, pH: 7.4 Dilute the antibody (rabbit anti-human PSMD4 polyclonal antibody) to 2.0ug / ml, add 100 μl to each well of the ELISA plate, Seal the plate with blocking solution containing 3% bovine serum albumin + PBS solution and incubate overnight at 4°C for later use.
[0075] Detection of serum samples and recombinant proteins: serum samples were diluted 1:1 with sample diluent 1×PBS (pH:7.4), added at a volume of 100 μl / well, and incubated at room temperature for 2 hours; PSMD4 recombinant protein was diluted with sample diluent In different concentration gradients, add samples at a volume of 100 μl / well, and incubate at room temperature for 2 hours; shake off the liquid in each well, and add ELISA plate washing solution (1×PBS solution containing 0.05% Tween-20), each well 300 μl, washed 3-5 times, and dried; Dilute the PSMD4 detection antibody to 0.5 μg / ml, 100 μl per well, incubate at room temperature for 1 hou...
Embodiment 2
[0076] Example 2: Optimization
[0077] 1. Optimization of coating antibody concentration:
[0078] Select different coating antibodies rabbit anti-human PSMD4 polyclonal antibody (PTG company and Abnova company), choose different concentrations (7.5μg / ml, 5μg / ml, 2.0μg / ml, 1μg / ml, 0.5μg / ml, 0.25 μg / ml) coated ELISA plate, detected according to the operating steps in Example 1, adding known PSMD4 protein standards of different concentrations. According to the obtained OD value, select the blank group with the smallest OD value and the closest linear relationship between the OD value and the standard protein concentration as the best antibody (PTG company). The coating antibody concentration is 2.0 μg / ml.
[0079] 2. Optimization of blocking solution:
[0080] Choose different blocking solutions, containing 1% bovine serum albumin + PBS solution, containing 2% bovine serum albumin + PBS solution, containing 3% bovine serum albumin + PBS solution, containing 4% bovine serum al...
Embodiment 3
[0087] Embodiment 3: Specificity and sensitivity evaluation of the ELISA kit of PSMD4 protein
[0088] 1. Specificity test
[0089] The ELISA method established in Example 1 was used to detect the serum samples of 100 liver cancer patients, 50 normal people and 20 patients with liver cirrhosis (the healthy people were selected from the physical examination samples of our hospital staff; The pathological diagnosis was primary liver cancer; the liver cirrhosis sample was from the Department of Infectious Diseases of Changhai Hospital, and the clinical diagnosis was liver cirrhosis), PSMD4 recombinant protein was used as a positive control, and a standard curve was drawn according to the obtained standard OD value and the corresponding concentration to calculate the Serological levels of PSMD4 protein in .
[0090] The result is as figure 2 As shown, the serum level of PSMD4 protein in liver cancer group (Median388.2pg / mL, IQR348.3pg / ml) was significantly higher than that of l...
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