Your Step-by-step Guide to

Interferon Preparation and

Detection (Part One)

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Introduction

In this paper, interferon-inducing agent was used to prepare human leukocyte

interferon as an example to introduce the preparation method of interferon.

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Experimental principle

Steroids are a class of highly active, multifunctional proteins produced by interferon-

inducing agents that act on biological cells. After it is produced and released from cells,
it acts on other cells of the same species to obtain anti-virus and anti-tumor immunity.

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Experimental principle

The so-called interferon inducer refers to a substance that can induce the
production of interferon by biological cells. In practice, two methods are
used to prepare interferon. The first is to use interferon inducer to induce
the production of interferon in certain biological cells, which can be used
after being extracted and purified and qualified. The cells used in this
method are mostly peripheral blood leukocytes. The second method is
carried out by genetic engineering, that is, the interferon gene is introduced
into Escherichia coli, and interferon is produced by culturing Escherichia coli.
At present, large-scale production of interferon mainly uses genetic
engineering methods.

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Major equipment

Cell culture equipment (such as culture flasks, porous culture plates, incubators,
microscopes, rotary incubators, etc.), water baths, etc.

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Experimental procedure

1. Preparation of inducing agent: using NDVF attenuated strain, stored in

chicken embryo allantoic fluid at -20 ° C.

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2. Preparation of induced cells: sterile human peripheral blood (using human
umbilical cord blood, or blood bank storage blood), placed in a sterile bottle
containing heparin, stored at 4 ° C for no more than 24h; induced cells
(white blood cells) are not extracted separately and replaced with whole
blood.

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3. Preparation of crude
interferon:

1) Add an inducer, add 1ml anticoagulant whole blood plus 0.2ml of

inducing agent (ie NDVF allantoic fluid, the blood coagulation titer is not less
than 1:640);
2) Warming adsorption, anticoagulant whole blood with antibiotic was
placed in a 37 ° C water bath for 1 h, shaking once every 15 min, so that
NDVF was adsorbed on white blood cells. Then centrifuge at 1000r/min for
20min, discard the supernatant, and leave a precipitate;

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3. Preparation of crude
interferon:

3) Incubation with nutrient solution, add Eagle nutrient solution to the

above precipitate according to the amount of anticoagulant whole blood 1
to 2 times, mix well, and rotate in a temperature of 35 ~ 36 °C for 18 ~ 20h;
4) Centrifugation and acid treatment; the above culture was centrifuged at
2000r/min for 30min, the supernatant was taken, the pH value was adjusted
to 2.0 with 6mol/L hydrochloric acid, and the NDV was inactivated in a
refrigerator at 4°C for 5 days;
5) Neutralize; after acidification for 5 days, adjust the pH to 7.2-7.4 with
6mol/L sodium hydroxide.

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To be continued…

Collected by Creative BioMart.

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