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Dear Reader,
Part II: Enhancement
Dr. K. Nagarajan and Dr. P. K. Chitnis
I hope that you would have found our attempt to address the issues
related to Inhibition encountered during LAL testing useful. In Introduction
continuation of the same, I wish to bring to you the next part of the Limulus Amebocyte Lysate (LAL) test is the assay of choice to
detect endotoxin because of its sensitivity and specificity
newsletter devoted to the complex issue of Enhancement.
towards bacterial endotoxins {Lipopolysaccharide (LPS) from
the gram negative bacterial cell wall}1,2. However, a few
The phenomenon of enhancement is probably the most complex and polymeric forms of glucose namely glucans have been shown to
intriguing topic for anyone analyzing bacterial endotoxins by the activate the LAL cascade of enzymes when they are present in
Limulus Amebocyte Lysate test. The alternative pathway that sufficient quantities in the sample solution3. The glucans
include -(1,3)-D-glucans, which are found in the cell wall of
works through the Factor G is the cause for the phenomenon of
fungi, yeasts and alage4,5 and LAL-reactive material (LAL-RM)
enhancement. Factor G is a 'biosensor' which interacts with presumably -(1,4)-D-glucan derived from a hollow-fiber
'glucans' or 'LAL Reactive Material' and in turn activates the hemodialyzer with a saponified cellulose acetate or Cuprophan
'enzymatic cascade' to form a gel, which is the root cause for a lot membrane6. It is very interesting to note that not all of the
of debate and discussion. polymeric forms of glucose react with the LAL. A better
understanding of this phenomenon of non-reactivity of some
types of glucans to LAL relates back to the principles of the LAL
The topic, therefore, is exhaustive and has many significant test itself.
implications viz. causing the 'false positives', thus making it very
important to delve on the relevant aspects at length. The clotting mechanism of the blood of the horseshoe crab is
designed to prevent the spread of bacterial/fungal
contamination throughout the circulatory system of the
We have made an attempt to provide an insight with a view to
horseshoe crab7. When the LPS of Gram-negative bacteria/-
design a strategy to overcome this interference caused by the (1,3)-D-glucans come in contact with the blood cells of the
enhancement and enable successful LAL testing. horseshoe crab an enzymatic cascade is triggered (Figure.1).
The reaction pathway consists of two biosensors, Factor C and
You are always welcome to send your suggestions and comments to Factor G, two proenzymes (Factor B and proclotting enzyme)
and a clottable protein (coagulogen). This enzymatic cascade
our email id : india.customercare@crl.com
forms the defense mechanism of the horseshoe crab aimed at
detecting bacterial endotoxins with the help of Factor C and
Sincerely, glucans with the help of Factor G. The two pathways merge at
the activation step of the proclotting enzyme (Figure 1)7.
Dr. P. K. Chitnis
Director
-(1,3) Glucans8 Depending upon the concentration and size of the glucans,
they might interfere in the LAL testing by causing the formation
(1,3) Glucans are those polymers of D-glucose linked of a gel clot in the absence of endotoxin. This gelation in
glycosidically with a -(1,3) linkage (Figure 2). absence of endotoxin is contrary to the expected result and is
therefore referred to as a False Positive.
Figure 2. (a) Glucose molecule showing carbon numbering notation and - orientation 4. Chloroform extraction of LAL increased sensitivity of the reagent to
(b) Showing orientation and location of different -glucan linkage endotoxin, lipid A and LAL-RM/Glucans.
Preliminary Testing
Results
d) The use of Tris-GB buffer will be the best solution for the BET for
Sandostatin and CM-cellulose related product.
e) Best recommended method for the BET of Sandostatin and CMC Figure 6. Glucan Cartridge Setup
related product is as below:
1) Sample: 10-fold dilution with Tris-GB buffer
Conclusion
It would be most appropriate, therefore, to surmise that
2) Method: KTA with the microplate method
Enhancement is more of a Glucan Interference to LAL testing
than anything else.
Rapid method to Detect of Glucan using Portable Test
System (PTS) Further to this, considering the fact that the coagulation system
of the Horseshoe crab is activated by two pathways viz. by
Charles River Laboratories offer a new test to help ensure that
endotoxin (LPS) and by (1,3)--D-Glucans, a differentiation
the products are as free of glucans. The Endosafe-PTS glucan
between the two can be achieved based on their reactivity
assay is a rapid test designed to detect and quantify (1,3)- -D-
towards LAL reagent. Thus, glucan interference of LAL test can
Glucans in a sample.
be managed in two ways:
This test employs the Limulus Enzyme Cascade to detect (1,3)- 1. The first approach combines the use of glucan sensitive and glucan
-D-Glucan (Figure.5.) in the sample. The Endosafe-PTS blocked LAL to differentiate endotoxin and -glucan and to identify sources
cartridge and its interface with the reader have been designed of glucan contamination with the aim of eliminating them from the product
and,
on the principles of the Kinetic Chromogenic (KCA) method to
measure the color intensity which is directly related to the (1,3)- 2. The second approach uses glucan-blocked reagents for release of testing
of certain products, such as medical devices and carboxymethylated
-D-Glucan concentration in a sample. polysaccharides, where the objective is to test for endotoxin specifically.
15. (a) J. F. Cooper, M. E. Weary and F. T. Jordan, J. Parenter. Sci. Technol. 51, 2-
6 (1997) (b) J. F. Cooper, Journal of parenteral Science and Technology, Nr.
44:1 Page No: 13 (1990).
16. H. S. Kumar, LAL Newsletter (Salesworth India Pvt. Ltd.), 6, 1-6 (1998).
Contact Us
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Tel: 080-25588175 - 177. Fax: 080-41659281. Email: india.customercare@crl.com