I don't know if you often need to purchase consumables. If you often purchase laboratory consumables, you may find that various manufacturers may label their products as enzyme free, pyrogen free and endotoxin free, especially for consumables involving cell biology and analytical biology, such as common 15ml and 50ml centrifuge tubes, cell culture bottles, cell culture dishes, cell culture plates, cryopreservation tubes PCR board and other products. Products with these descriptions may also be more expensive than ordinary similar products. So what are the meanings of these statements and how do they affect our experiments? How should we make choices when making purchases? Here's a brief introduction.
Definition
1.Pyrogen
Pyrogen refers to the pyrogenic substance that can cause abnormal rise of body temperature after being injected into the body. In a broad sense, pyrogen includes bacterial pyrogen, endogenous macromolecular pyrogen, endogenous low molecular pyrogen and chemical pyrogen. The "pyrogen" here mainly refers to bacterial pyrogen, which is the metabolite of some microorganisms, bacterial corpses and endotoxin.
2.Enzyme
Enzymes were learned in junior high school. As a biological catalyst, enzymes can efficiently catalyze various biochemical reactions in our organisms. However, when most products say no enzyme, actually more accurately, they do not mean all enzymes, but nucleases. Many manufacturers also write DNase free and RNase free. Nuclease can decompose nucleic acid molecules. It can specifically degrade DNA or RNA, and it can also act on both DNA and RNA molecules with low specificity.
3.Endotoxin
Endotoxin is a general term for toxic substances existing in gram-negative bacteria. It is the cell wall component of a variety of gram-negative bacteria. It is the toxin released after the cell is cracked, and the component is lipopolysaccharide. It is generally believed that endotoxin is a pyrogen. It is generally acceptable during drug production: if there is no endotoxin, there is no pyrogen. Therefore, during production, only endotoxin test may be performed without pyrogen test.
Influence
Pyrogen and endotoxin can stimulate the body and cause fever. In pharmaceutical, clinical and other fields, for the sake of human safety, there are usually requirements for the amount of pyrogen and endotoxin. In the Chinese Pharmacopoeia, specific methods have been given for the detection of pyrogen and endotoxin. When it comes to molecular laboratories, nuclease may degrade target molecular fragments because it can decompose DNA. For example, in PCR experiments, if nuclease is contaminated, there may be no CT value, resulting in false positive results. Therefore, consumables such as sucker and PCR are usually nuclease free.
Detection of pyrogen and endotoxin
The following is a brief introduction to the pyrogen and endotoxin tests in the pharmacopoeia.
1.Pyrogen testing
The rabbit method for pyrogen detection is described in the General Rule of Chinese Pharmacopoeia 1142. Use healthy and qualified rabbits, inject a certain amount of test substance into rabbits intravenously, and observe the temperature rise of rabbits within the specified time. The test utensils in contact with the test article shall be sterile and pyrogen free. The pyrogen is usually removed by dry heat sterilization (250 ℃, more than 30 minutes), or other appropriate methods. If the temperature of the tested rabbit rises to a certain standard within a certain period of time, it is considered that the pyrogen test of the test article does not meet the requirements.
2.Endotoxin test
Before endotoxin test, the limulus test was used internationally. Specific methods include gel method and photometric method. Limulus reagent can agglutinate with bacterial endotoxin to produce gel or turbidity change, and endotoxin content can be determined by measuring this change. Tachypleus amebocyte lysate is a kind of reagent extracted from the blood of marine arthropod Tachypleus amebocyte lysate. Therefore, Tachypleus amebocyte lysate has been caught and killed in large quantities worldwide, and has also become a national second-class protected animal in China. At present, FDA has approved the recombinant C-factor method to replace the TAL reagent. This law has also been mentioned in the guiding principles of the Chinese Pharmacopoeia.
How to select
In fact, it is not difficult to choose after understanding these concepts. It is mainly based on your own needs. Generally, consumables without enzymes and pyrogens are common, and products without endotoxin are relatively few. If the experiment itself has no high requirements for these, ordinary consumables can be used. For example, when centrifuging cells or related components, it is better to use a centrifuge tube without enzymes and pyrogens, while ordinary centrifuge tubes can actually be used for ordinary liquid centrifugation.
In addition, this is also a problem that many users will have. When purchasing centrifuge tubes from some businesses, they will ask whether the centrifuge tubes can be reused. In fact, this problem will be discussed according to the situation, because the centrifuge tubes have been polluted to a certain extent after being used once, and even pressure steam sterilization can not completely eliminate the pollution, Therefore, if you use it again, it depends on the user's own needs for the experiment.
In the final analysis, all products are for experimenters and experiments. We can't influence our experiments because of the products, but we need to choose the appropriate products according to our experiments.
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