PubMed · 480121
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A Puech. [The limulus test: introduction to the round table].. https://pubmed.ncbi.nlm.nih.gov/480121/
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The gel formation by endotoxin with limulus amoebocyte lysate (LAL) solution requires the presence of free calcium ions. The chelating agents and radiopharmaceuticals prepared from the chelating agents always reduce the available free calcium levels present in the kits used for the testing of apyrogenicity and thus inhibit gel formation of LAL with E. Coli endotoxin (ET). This inhibition of gel formation could be reversed by the addition of excess free calcium ions or the excessive dilution of radiopharmaceuticals and chelating agents. The tests of positive control (0.2 ml ET units and LAL), inhibition control (0.1 ml sample, 0.1 ml ET and LAL), and negative control (0.1 ml sterile saline and LAL) were carried out with the fresh preparation (0.1 ml) of samples (triplicate), tropolone, ACD anticoagulant, and 99mTc-labeled radiopharmaceuticals. In the Ca-supplemented tests, 0.1 ml of 60 mM sterile calcium chloride solution was added to the test solutions and incubated for 60 min at 37 degrees centigrade. The results of gel formation (+ve and -ve) and normalization of inhibition control tests with Ca-supplement indicate that commercial LAL kits need extra calcium ions for the correct testing of the apyrogenicity of chelate-containing radiopharmaceuticals and chelating agents.
Inhibition of the Limulus Amoebocyte Lysate (LAL) assay for the detection of endotoxin is often a problem when testing parenteral solutions with actual concentrations of chemical substances. Seven samples of sterile, pyrogen-free solutions widely used in Norwegian hospitals, with inhibiting effect on the LAL test, were spiked with endotoxin, and ultrafiltered with different volumes of diluent. For six of the seven samples the inhibition was reduced or eliminated.
A simple micromethod of Limulus test was studied. Briefly, this method is as follows. A 20 microliter aliquot of Pregel lysate solution and the same amount of sample are dropped on the same place on the silicone coated slide glasg and mixed well. Then it is incubated at 37 degrees C for 30 minutes in a moisture chamber. After the incubation, the vibration of the droplet surface is observed. When the vibration is no longer observed, it is judged as positive reaction, and when the vibration is still observed as before the incubation, it is judged as negative reaction. Compared with the conventional tube method, this method has advantages as below: 1) the judgement of the result is relatively objective, 2) shorter incubation period is enough for the reaction to take place and 3) smaller amount of Limulus lysate is enough for each sample. Besides these merits, the detection limit of this method is not inferior to that of the tube method.