Endotoxins cause major contamination in biologically active substances, as well as pyrogenic reactions in host organisms. Lipopolysaccharides (LPS), the components of Gram-negative bacterial cell walls, are bacterial endotoxins. LPS is heat stable and independent on the bacterial cell. Endotoxin level detection and endotoxin removal are critical quality control procedures for various products, especially for drugs.
Endotoxin Level Detection Methods
1. The Limulus Amebocyte Lysate (LAL) Test
This commercial test is the most common way to detect endotoxin. LAL is an enzyme produced by the blood cells of the horseshoe crab (Limulous), which serves as a primitive immune system to bind and inactivate endotoxin from invading bacteria. A clot can be formed based on endotoxin inactivation by LAL, protecting the horseshoe crab from infection. LAL reagent can be added to the products to be tested, assaying for clot formation. The gel clot assay with the LAL test is semi-quantitative, which sensitivity can be reached to 0.06 EU/ml. The gel clot method is considered as the most accurate and sensitive way to test endotoxin content with fewer false results, however, this method is time-consuming since there is no available automated systems for it.
2. The Chromogenic Method
A serine protease (coagulase) can be activated by the endotoxin. This activation process is exploited to develop the USP chromogenic method for endotoxin detection, leading to clotting cascade. There are two available methods to quantify the endotoxin concentration in the sample. One is the kinetic method based on the particular absorbance (405 nm) of the sample, the other one is the endpoint chromogenic method based on the measurement of p-nitroaniline. Both methods require a standard curve for endotoxin quantification. The chromogenic method is very user-friendly and time-saving.
Endotoxin Removal Methods
Methods to remove endotoxins are based on the molecular weight or the chemical properties of the endotoxin, or both. There are four different basic methods to efficiently remove endotoxin from aqueous solutions:
1. Adsorption by activated charcoal and asbestos
Endotoxins can be adsorbed by activated charcoal and asbestos. The adsorption procedure is used to effectively eliminate endotoxin before using ultrafiltration. However, this method is inconsistent, accompanied by high losses of the target products, and highly dependent on the endotoxin concentration in the solution.
Ultrafiltration is a highly efficient way to remove endotoxin from the contaminated solutions. To apply this method, you need to know the physical size difference between the endotoxin and the target protein.
3. Ion exchange chromatography
This method provides following advantages:
a. high capacity;
b. relatively mild conditions;
c. large sample volumes can be processed.
Using anion exchangers can successfully remove endotoxins since endotoxins are negatively charged.
4. Affinity chromatograph
Chemical adsorption of endotoxins is widely used in both practical clinical treatment and industrial purification. Polymyxin B sulfate can be used to neutralize the biological activity of endotoxin because it binds to endotoxins with high affinity.
It is with great importance to determine the endotoxin level accurately and effectively in various products since endotoxins cause pyrogenic or immunoresponsive reactions. Our resourceful technical support platform helps you in endotoxin level detection and endotoxin removal. Please see the related services for more information and feel free to Contact Us.