Limulus amebocyte lysate assay

The MAP1981c gene was amplified from genomic DNA from MAP ATCC 19698 and the following primers: forward primer; the Nde I site is underlined, 5′-CGCCATATGAAAGCCGATGTAGCACAGCAG-3′ and reverse primer; the Hind III site is underlined, 5′-CCCAAGCTTCTGACCGGACCCCTTGACC-3′. The PCR-amplified full-length open reading frame (ORF) of MAP1981c gene was inserted into the plasmid pGEMT Easy Vector and sequences of MAP1981c gene in plasmid were confirmed. Next, the MAP1981c gene was cloned into the pET-22b (+) vector (Novagen, Madison, WI, USA) and then the clone was transformed into E. coli BL21 cells. The recombinant MAP1981c was expressed after induction with 1 mM isopropyl-β-D-thio-galactoside (IPTG) at 37°C for 6 h and then lysed and sonicated in lysis buffer supplemented with 20 mM Tris-HCl (pH 8.0), O,5 M Nacl, 20 mM imidazole, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The recombinant proteins were purified by using an immobilized nickel-chelate (Ni-NTA) column (Invitrongen, Carlsbad, CA, USA) in accordance with the manufacturer's instruction. Endotoxin contamination was removed from the recombinant MAP1981c by incubation with polymyxin B (PMB)-agarose (Sigma) for 8 h at 4°C. Also, Residual LPS in the MAP1981c preparation was measured by an Limulus Amebocyte Lysate (LAL) assay (Lonza, Basel Switzerland) and was < 7 pg/mL (< 0.1 UE/mL) in MAP1981c preparations.

IL-6, IP10 and sCD14 levels in the plasma samples from HIV infected patients and healthy controls were determined via specific ELISAs. For IL-6 determination, the Quantikine HS ELISA kit (R&D Systems) was used following the manufacturer’s protocol. The kit is designed to measure human IL-6 in serum, plasma and urine and has a sensitivity of 0.016–0.110 pg/mL. IP10 and sCD14 levels were determined using the Quantikine ELISA human IP10 and CD14 Immunoassay (R & D Systems) following the manufacturer’s protocol. The sensitivity of IP10 detection using the kit is 0.41–4.46 pg/mL and the minimum detectable dose (MDD) of human CD14 is less than 125 pg/mL. Plasma LPS levels were determined using the endpoint chromogenic Limulus Amebocyte Lysate (LAL) assay (Lonza) using the manufacturer’s recommendations. The kit shows linear absorbance at 405–410 nm in the concentration range of 0.1–1.0EU/mL endotoxin.

Cockroach species were identified and counted according to their morphological features [13 (link)]. Feces on traps were picked, weighed (fresh weight), and extracted for allergen and endotoxin analysis following the method of Schram et al. [14 (link)]. Endotoxins were extracted with 0.05% Tween-20 in pyrogen-free water, and allergens were extracted with PBS-0.045% Tween-20. Endotoxins and allergens were measured using the kinetic chromogenic Limulus Amebocyte Lysate (LAL) assay (Lonza, Allendale, NJ, USA) and Bla g 1 and Bla g 2 monoclonal antibody-based sandwich ELISA assays (Indoor Biotechnologies, Inc., Charlottesville, VA, USA), respectively. According to the manufacturer, each unit of Bla g 1 measurement is equivalent to about 100 ng of Bla g 1 protein.

Dried Ocs (100 g, Sworm Co., Seoul, Korea) was ground for 1 min using a manual grinder; the dried powder was mixed with 600 mL demineralized water containing 0.16% ascorbic acid, and this was subsequently incubated at 4 °C for 1 h. Following incubation, samples were ground again for 1 min, and the obtained solutions were centrifuged for 30 min at 70,000 rpm. Collected supernatants were filtered through Whatman No. 4 filter paper (Florham Park, NJ, USA) to remove the debris. Filtered solutions were dried in a vacuum freeze drier (VD-800F; Taitec, Saitama-ken, Japan), and freeze-dried powders (percentage yield of Ocs-P: 3.81%) were resuspended in phosphate-buffered saline (PBS, Biowest, Nuaille, France) and filtered through a 0.45 μm filter (Corning, NY, USA). Finally, we confirmed the absence of endotoxin or LPS contamination of purified Ocs-P using the Limulus Amebocyte Lysate (LAL) assay (Lonza, Basel Switzerland), following the manufacturer’s instructions.