Method for detecting adsorption completeness of recombinant novel coronavirus vaccine

A coronavirus and detection method technology, applied in the field of recombinant novel coronavirus vaccine adsorption completeness detection, to achieve the effect of accurate and reliable experimental results

Active Publication Date: 2020-10-02
天津中逸安健生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, since the existing vaccines for this disease are all in experiments, there are few reports on how to verify and detect the completeness of adsorption. The detection of completeness of adsorption is of reference value for further research on vaccine efficacy and quality control

Method used

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  • Method for detecting adsorption completeness of recombinant novel coronavirus vaccine
  • Method for detecting adsorption completeness of recombinant novel coronavirus vaccine
  • Method for detecting adsorption completeness of recombinant novel coronavirus vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Solution preparation:

[0049] Coating solution: weigh 2.93 g NaHCO 3 , 1.59 g Na 2 CO 3 Dissolve in 800 ml, adjust the pH to 9.5-9.6 with 1M NaOH, and dilute to 1000 ml.

[0050] Blocking solution: Weigh 2.0 g of bovine serum albumin, dissolve in 100 ml of PBS solution, mix well and set aside.

[0051] Stop solution: Measure 42 ml of concentrated sulfuric acid, slowly add to 400 ml of water, stir while adding, and dilute to 750 ml after cooling.

[0052] Sample Diluent / Wash: PBS with 0.05% Tween-20.

[0053] Test product / reference product treatment solution: Measure 1.25 ml of 20% diethanolamine and 0.20 ml of 10% Triton X-100, add 8.55 ml of PBS, mix well and set aside.

[0054] Reference and test product handling:

[0055] Desorption of the test product: Precisely measure 0.2 ml of the same batch of test product 1, test product 2 and test product 3 and place them in three 1.5ml EP tubes, add 0.2 ml of test product / reference product respectively for treatment ...

Embodiment 2

[0061] A method for detecting the completeness of recombinant novel coronavirus vaccine adsorption, comprising the following steps:

[0062] Coating: After diluting the RBD antibody with a concentration of 2.1 mg / ml to 5 μg / ml, pre-coat it into a 96-well microtiter plate with a coating volume of 100 μl / well, and place it at 6°C for 15 hours.

[0063] Sealing: Take the coated plate out from 2-8°C, wash the plate 4 times, each washing volume is 300 μl / well, pat dry on absorbent paper, add blocking solution, 300 μl / well, cover the plate membrane , and incubate at 37±1°C for 90 minutes.

[0064] Adding samples: Take out the closed ELISA plate, add 300 μl / well of washing solution, shake gently for about 30 seconds, discard the washing solution, pat dry on absorbent paper, and repeat washing 4 times; dilute the solution prepared in Example 1 well The working reference substance, the quality control substance, the desorption diluent of the test product, and the undesorbed supernatan...

Embodiment 3

[0070] data processing:

[0071] 1) Standard curve drawing: take the concentration value of the reference product as the abscissa (ng / ml), and the corresponding average OD value after deducting the negative control absorbance (OD) value as the ordinate, perform four-parameter fitting to generate the S curve and If the four-parameter equation, standard curve and quality control data meet the criteria for system applicability, the fitting is passed;

[0072] Fitting curve R 2 If it is less than 0.990, the experiment should be repeated.

[0073] The resulting standard curve equation is as follows:

[0074] y = (A - D) / [1 + (x / C) B ] + D type c;

[0075] In the formula, A= 2.00742; B = -0.82100; C= 314.08530; D= -0.09311.

[0076] R 2 It is 0.99981, which meets the requirements.

[0077] 2) Calculation of the recovery rate of quality control products: Substitute the absorbance values ​​of QCH and QCL after subtracting the OD value of the negative control into the standard...

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Abstract

The invention discloses a method for detecting the adsorption completeness of a recombinant novel coronavirus vaccine, and belongs to the technical field of biology. The method comprises the followingsteps: pre-coating a detection plate with an RBD antibody to obtain a coated plate; closing the coating plate, and incubating to obtain a closed elisa plate; carrying out gradient dilution on a reference substance; desorbing a test sample, and preparing a desorbed diluent and a non-desorbed supernatant; respectively adding the reference substance, the test sample desorption diluent and the test sample non-desorbed supernatant with various concentrations into plate holes, and incubating by taking the sample diluent as a negative control; adding an enzyme labeled secondary antibody, and incubating; adding a substrate color developing solution, and developing in a dark place; and terminating color development, reading, and counting and reading an absorbance value. The method can be used foradsorption completeness detection and related research work of the recombinant novel coronavirus vaccine semi-finished product, so that the experimental result is accurate and reliable.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for detecting the adsorption completeness of a recombinant novel coronavirus vaccine. Background technique [0002] Novel coronavirus pneumonia is mainly manifested by fever, dry cough, fatigue, etc., and a small number of patients are accompanied by upper respiratory tract and digestive tract symptoms such as nasal congestion, runny nose, and diarrhea. Severe cases often develop dyspnea after one week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, difficult-to-correct metabolic acidosis, coagulation dysfunction, and multiple organ failure. It is worth noting that severe and critically ill patients may have moderate to low fever or even no obvious fever during the course of the disease. Mild patients only showed low-grade fever, mild fatigue, etc., without pneumonia. Most patients have a good prognosis, and a few pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/535
CPCG01N33/535G01N33/6854G01N2333/165
Inventor 魏文进高辉褚晓明
Owner 天津中逸安健生物科技有限公司
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