The yield of Limulus amoebocyte lysate (LAL), which is used for endotoxin detection and adjunct diagnosis of invasive fungal infection, was optimized with caffeine collection buffer

Abstract

Background: Limulus amoebocyte lysate (LAL) is a major component of the Fungal Infection Diagnosis Kit for the adjunct diagnosis of invasive fungal infections. During the previous bleeding season, PBS-collection buffer was found to prevent degranulation during the blood collection procedure. In addition, PBS-derived Limulus Amoebocyte Lysate (LAL) was used in both the chromogenic assay and the turbidimetric test. A similar phenomenon was observed with the caffeine collection buffer. This collection buffer is easy to prepare.

Methods: To further confirm these observations, we used caffeine collection buffer to collect blood from horseshoe crabs. Six crabs were bled per week, for a total of 60 crabs. Blood was collected from each crab via both caffeine collection buffer and 3% NaCl collection buffer. The cell pellets were then resuspended in LAL reagent water (LRW) or 5 mM CaCl₂. The final LAL activity was tested via chromogenic tests and turbidimetric assay methods.

Results: Caffeine collection buffer prevented degranulation for more than 1 h, and the yield of caffeine-derived LAL was much greater than that of the 3% NaCl solution. Notably, caffeine-derived LAL was found to work in both chromogenic tests and turbidimetric assays. The enzyme characteristics of the LAL were also determined.

Conclusion: Caffeine collection buffer prevents amoebocyte degranulation during blood collection and processing. The activity of caffeine LAL is much greater. Caffeine LAL works in both chromogenic tests and turbidimetric assays.

Keywords: Limulus amoebocyte lysate (LAL); caffeine buffer; endotoxin; exocytosis; fungal infection diagnosis.

Figure 1

Caffeine buffer inhibits exocytosis during the blood collection process. The blood of horseshoe crabs was collected via caffeine buffer, PBS buffer or 3% NaCl as described in the materials and methods section. (A) Comparison of the activity of LAL in PBS with that in 3% NaCl. (B) Comparison of the activity of LAL in PBS with that in caffeine. (C) After centrifugation, a large gel clot was observed in 3% NaCl solution-collected blood. (D) After being kept at 30 min at room temperature, most of the granules were released from 3% NaCl-collected amebocytes; however, almost all the granules were kept inside a cell in a caffeine collection buffer blood mixture under the microscope. (E) Comparison of the activity of caffeine LAL with that of 3% NaCl collected LAL. The results revealed that the caffeine-containing enzyme had greater activity (27.61 vs. 0.1578, p < 0.0001).

Figure 2

Comparison of LAL enzyme activity in the four different resuspension solutions. A total of 15 mL of blood was collected from one horse-shoe crab in 4 different corn tubes, each containing 15 mL of caffeine buffer. The blood buffer mixture was centrifuged at 3000 rpm at 4°C for 5 min. The cell pellet was washed twice with 3% NaCl. Then, LRW, 5 mM CaCl2, 5 mM MgCl2, and 5 mM NaCl were added to 4 different cell pellets at a ratio of 1 gram of cell pellet per 7 mL of resuspended solution. Afterward, the cell pellet was vortexed for 30 s, the mixture was decanted into 4 different glass flasks, and the flasks were shaken at 4°C overnight. The enzyme activity of the supernatant was tested via a chromogenic method. (A) Blood collection procedure. (B) The cells remained intact and not lysed after being shaken overnight, with no granules inside the cells. (C) Limulus amoebocyte lysate pathway. (D) Comparison of enzyme activity in 4 different resuspension solutions.

Figure 3

Effect of ions on enzyme activity. (A) Effect of NaCl on enzyme activity. A total of 25 μL of LRW-collected LAL was incubated with 0, 50, 100, 150, or 200 mM NaCl in a 96-well plate for 5 min. Afterwards, 50 μL of the reaction mixture was added. The plate was placed in the plate reader, and any reactions were monitored for 1 h. The results showed that NaCl does not inhibit LAL activity. (B) Effect of CaCl₂ on enzyme activity. A total of 25 μL of LRW-collected LAL was incubated with 0, 50, 100, 150, or 200 mM CaCl₂ in a 96-well plate for 5 min. Afterwards, 50 μL of the reaction mixture was added. The plate was placed in the plate reader, and any reactions were monitored for 1 h. The results indicated that CaCl₂ inhibited LAL activity. (C) Effect of MgCl₂ on enzyme activity. Forty microliters of LRW-collected LAL were incubated with 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM MgCl₂ in a 96-well plate for 5 min. Then, 50 μL of the reaction mixture (0.14 M Tris, pH 7.4; 1.2 mM chromogenic peptide; and 5Eu/ml endotoxin) was added. The plate was placed in the plate reader, and the reactions were monitored for 1 h. The results showed that LAL has activity over a broad range of concentrations of MgCl₂. The optimum concentration of MgCl₂ was 40 mM.

Figure 4

Effects of temperature (A,C) and pH (B,D) on enzyme activity. (A,C) Effects of temperature on enzyme activity. Fifty microliters of the reaction mixture were incubated with 50 μL of LAL in 4 different 96-well plates. The plates were put into four plate readers, which were set at 25°C, 30°C, 37°C and 42°C. The biochemical reaction was monitored for 1 h. The results revealed that the optimum temperature for the LAL enzyme reaction was 37°C. (B,D) Effects of pH on enzyme activity. A total of 25 μL of six different pH buffers (200 mM pH 4.6 acetate buffer, 200 mM pH 5.6 acetate buffer, 200 mM pH 6.5 MES buffer, 200 mM pH 7.4 Tris-Cl buffer, 200 mM pH 8.2 Tris-Cl buffer, and 200 mM pH 8.4 Tris-Cl buffer) was incubated with 25 μL of LAL in 96-well plates for 5 min. Then, 50 μL of the reaction mixture (50 mM MgCl₂, 1.2 mM chromogenic peptide, and 5Eu/mL CSE endotoxin) was added. The plate was placed into the plate reader, and the biochemical reactions were monitored for 1 h. The results indicated that the optimal pH for the LAL enzyme reaction was 8.2.

Figure 5

CaCl₂ inhibited the effect of caffeine buffer on exocytosis. The crab blood was collected using pure caffeine collection buffer and caffeine collection buffer with an additional 50 mM CaCl₂. (A) The gel was formed in caffeine collection buffer with an additional 50 mM CaCl₂ blood buffer mixture after being incubated at room temperature for 1 h. (B) The results of the enzyme activity test revealed that increased enzyme activity was detected for the caffeine-containing enzyme. However, almost no enzyme activity was detected in the supernatant of the caffeine collection buffer supplemented with an additional 50 mM CaCl₂. (C) Crab blood was collected using pure caffeine collection buffer and caffeine collection buffer with an additional 50 mM MgCl₂. No gel formation was observed in either buffer mixture after being incubated at room temperature for 1 h. (D) The results of the enzyme activity test indicated that the enzyme activity in both the pure caffeine collection buffer and the caffeine collection buffer supplemented with 50 mM MgCl₂ was not significantly different.

Figure 6

LAL produced through caffeine buffer works in turbidimetric tests. A total of 120 μL of 1 M MgCl₂ was added to 3 mL of raw LAL in 50 mL glass flasks. One hundred microliters of 30% NaCl was added to one flask, and the same amount of LRW was added to the other. The mixture was shaken at room temperature for 10 min and transferred to 10 mL sterile glass vials for lyophilization at −50°C for 3 days. The tube-based reaction mixtures were composed of 100 μL of a solution containing 160 mM Tris-Cl (pH 7.4) and 0.0156, 0.031, 0.062, 0.125, 0.25, 0.5, 1, or 2 EU/ml endotoxin. The lyophilized LAL powder was dissolved in 2.5 mL of 0.2 M Tris-Cl, pH 7.4, at room temperature for 8 to 10 min. Then, 100 μL of dissolved LAL was added to the reaction mixture. The mixture was vortexed for 30 s before being placed in a 96-well PK Flex plate. The reaction was monitored at 660 nm for 180 min. (A) Caffeine LAL works in turbidimetric tests. NaCl seems to increase enzyme activity. (B) A gel formed at high concentrations of endotoxin ranging from 0.125 Eu/mL to 1 Eu/mL.