(1→3)-β-d-glucan as a measure of active mold

Abstract

Electrokinetic devices and methods are described with the purpose of collecting assayable agents from a dielectric fluid medium. Electrokinetic flow may be induced by the use of plasma generation at high voltage electrodes and consequent transport of charged particles in an electric voltage gradient. Actively growing mold releases the carbohydrate cell wall component (1.fwdarw.3)-β-D-Glucan into the air. The invention recognizes that the airborne fraction is that which affects respiratory health and selectively tests for a free form which is soluble in aqueous medium. The sample to be analysed is preferably collected by the electrokinetic propulsion method described, but any air sampling method such as filtration, impactor or impingement may be applicable.

Claims

1. A method for analyzing aerosol particles, comprising capturing aerosol particles using an air sampling device, suspending said captured aerosol particles from the air sampling device in a extraction fluid, performing centrifugation of said suspension, directly adding with no other processing all or part of supernatant from said centrifugation to a reaction mixture for analysis of said supernatant for (1.fwdarw.3)-β-D-glucan.

2. A method according to claim 1 wherein the air sampling device is based on electrokinetic propulsion.

3. A method according to claim 1 where the sampling device is based on electrostatic precipitation.

4. A method according to claim 1 comprising determining (1.fwdarw.3)-β-D-glucan level by a limulus-amebocyte based assay.

5. A method according to claim 1 comprising determining (1.fwdarw.3)-β-D-glucan level by an immunoassay.

6. A method according claim 1 wherein the sampling device is based on filtration.

7. A method according claim 1 wherein the sampling device is based on impingement.

8. A method according claim 1 wherein the sampling device is based on an impactor.

9. A method for analyzing aerosol particles, comprising: capturing aerosol particles on an electrode of an electrokinetic propulsion device from a volume of air propelled electrokinetically through said device, said electrode being removably attached to a carrier mounting; removing said electrode from said carrier mounting and placing said electrode in an extraction vessel; adding a predetermined volume of extraction fluid to said extraction vessel; agitating said electrode in said neutral extraction fluid for a predetermined time; and directly adding with no other processing all or part of said neutral extraction fluid to a reaction mixture for analysis of said aerosol particles for (1.fwdarw.3)-β-D-glucan.

10. A method according to claim 9 comprising determining (1.fwdarw.3)-β-D-glucan level by a limulus-amebocyte based assay.

11. A method according to claim 9 comprising determining (1.fwdarw.3)-β-D-glucan level by an immunoassay.

Description

BRIEF DESCRIPTION OF THE INVENTION

(1) FIG. 1 represents a generic electrokinetic flow device.

(2) FIGS. 2A-2D represent an assembled carrier with removable electrodes.

(3) FIG. 3 is a graphical representation of the statistical analysis of a comparison of (1.fwdarw.3)-β-D-glucan compared with presence or absence of mold allergen in those same airborne samples.

(4) FIG. 4 is a graphical representation of the statistical analysis of the comparison of (1.fwdarw.3)-β-D-glucan determination on a set of airborne fractions treated or not treated with NaOH.

(5) FIG. 5 is a graphical representation of the distribution of (1->3)-β-D-glucan in 76 homes throughout the United States.

DETAILED DESCRIPTION OF THE INVENTION

(6) Air is sampled by a sampling device which maybe as described in detail in prior patents issued to the Applicant, namely U.S. Pat. Nos. 8,038,944, 9,216,421, 9,360,402, 9,481,904 and 9,618,431. The specification of each of these patents is incorporated by reference herein. Particular attention is drawn to U.S. Pat. No. 9,360,402 for the analysis of bio-specific material using a cartridge with removeable electrodes. This device provides the convenience of extraction of the bio-specific agents by immersing the detached electrodes directly in buffer extraction medium and shaking in a centrifuge tube with a vortex mixer, as is described in detail below.

Example 1

(7) FIG. 1 represents a basic ion propulsion device with a housing, 1, with high voltage wire seen in cross-section, 2, and symbolic representation of voltage contours, grounded plate electrodes 3. Resulting ion flow is represented by arrow 4. Resulting net air in-flow is represented by arrow 5 and outflow by arrows 6. Advantageously, the electrodes 3 are removable and may be mounted to a carrier, which is removable from the housing 1, as shown in U.S. Pat. No. 9,360,402.

Example 2

(8) FIGS. 2A and 2B illustrates a removable carrier assembly 21 that could be used with the housing of FIG. 1 as an alternative to fixed electrodes. The carrier assembly 21 is described in detail in U.S. Pat. No. 9,360,402. The carrier assembly 21 includes a one-piece plastic carrier 23, a latch 25 and capture electrodes 27. The carrier 23 is removeable from the housing 1. Moreover, the capture electrodes 27 are removably secured to the carrier 23. The latch 25 supports the electrodes 27, see FIGS. 2C and 2D. The latch 25 is adapted to secure the capture electrodes 27 to the carrier 23. For testing, the carrier 23 can be removed from the housing 1. The latch 25 and electrodes 27 can then be removed from the carrier 23 to facilitate testing.

Example 3

(9) Samples were run in a variety of mold positive and mold negative home environments. The presence of airborne mold allergens were determined by multiplex immunoassays using MARIA kits from Indoor Biotechnologies and the MagPix instrument supplied by BioRad Inc. Samplers were routinely run for 5 days and the allergens and (1.fwdarw.3)-β-D-glucan containing material extracted from the detached electrodes as described in detail in Example 4, and the supernatants tested by MARIA and by Glucatell assay kit (Associates of Cape Cod Inc., East Falmouth, Mass.) following manufacturers protocol for kinetic rate assay.

(10) FIG. 3 shows a statistical analysis of comparison of presence of mold allergens determined by the MARIA™ multiplex immunoassay method for the mold allergens Alt a 1 and Asp f 1) and (1.fwdarw.3)-β-D-glucan. The box plots show the 90 percentile ranges and the full range of values. The results summarized in FIG. 3 show a significant relationship between the absence of mold allergen and the absence of (1.fwdarw.3)-β-D-glucan in the airborne samples. A higher mean is also observed in the mold allergen positive group (7.19) compared to the negative group (2.42). Data from FIG. 3 shows a possible significant relationship between airborne mold allergen and the presence of (1.fwdarw.3)-β-D-glucan.

Example 4

(11) Effect of NaOH extraction on measured (1.fwdarw.3)-β-D-glucan.

(12) TABLE-US-00001 TABLE 1 Sample NaOH Treated (fg/L) NaOH Untreated (fg/L) 1 9.49 5.86 2 21.60 15.28 3 4.75 7.02 4 3.36 3.96 5 4.27 2.05 6 3.83 4.10

(13) The Inspirotec air sampling device, described above, was run for 5 consecutive days in each test environment. Following testing, stainless steel electrode strips were removed from cartridges, located in the device, and transferred to 15 ml centrifuge tubes. One ml of PBS with 0.02% TWEEN® 20 was added to the tubes and vortexed intermittently over 10 minutes. Samples were removed from the centrifuge tubes and transferred to 2 mL screw-cap tubes, then centrifuged at 15,000 g for 30 minutes. The supernatants were removed and placed in new 2 ml screw-cap tubes. In another 2 mL screw-cap tube, 80 μl of these samples were brought to 0.05N NaOH by addition of 20 uL of 2.5N NaOH. Samples were shaken for 2.5 hrs at room temperature o an orbital shaker, neutralized by addition of 100 μL of 2M Tris-HCL (1M Tris-HCL final). (1.fwdarw.3)-β-D-glucan levels were determined using the Glucatell assay kit (Associates of Cape Cod Inc., East Falmouth, Mass.) following manufacturer's standard protocol for kinetic rate assay.

(14) The results are also shown graphically in FIG. 3 as analyzed statistically with the JMP package, JMP® Pro 13.0.0 (SAS Institute Inc. Cary, N.C.).

(15) The slope of the best fit straight line is 1.41. This shows that when samples are collected and analyzed in this manner, 41% of the (1.fwdarw.3)-β-D-glucan is in an insoluble fraction. The current invention focuses attention on the soluble fraction, as determined by the supernatant from centrifugation and no extraction.

Example 5

(16) Distribution of (1.fwdarw.3)-β-D-glucan levels in homes throughout the U.S.

(17) Air samples were collected from 76 homes across the United States, using the Inspirotec device. In each home, the device was run for a period of 1 to 5 days. After completion of running period, stainless steel electrode strips were removed from cartridges, located in the device, and transferred to 15 mL centrifuge tubes. One mL of 1×PBS with 0.02% Tween® 20 was added to the tubes and vortexed intermittently over a period of 10 minutes. Samples were removed from 15 mL centrifuge tubes and transferred to 2 mL screw-cap tubes. They were then centrifuged at 15,000 g for 30 minutes. The supernatants were removed and placed in new 2 mL screw-cap tubes. (1.fwdarw.3)-β-D-Glucan concentration was measured using the Glucatell assay kit (Associates of Cape Cod Inc., East Falmouth, Mass.) following manufacturers protocol for kinetic rate assay.

(18) The Results are shown graphically in FIG. 4 as analyzed statistically with the JMP package, JMP® Pro 13.0.0 (SAS Institute Inc. Cary, N.C.). Values below the mean values of zero time field controls for the assay were assigned a value of field control/2 and number of occurrences of log.sub.10 of the values were plotted in bins as shown on the x-axis.

(19) (1.fwdarw.3)-β-D-Glucan was detected in 62% of homes.

(20) It is apparent from the foregoing that measurement of (1.fwdarw.3)-β-D-Glucan in the soluble fraction of samples collected from airborne material is a representation of the fraction of free (1.fwdarw.3)-β-D-Glucan in the air that is material released from actively growing molds and will penetrate most deeply into the respiratory system with impact on respiratory health. This fraction may also parallel the release of allergens from molds so that it is also be a surrogate assay for airborne allergen exposure. Previous art ignored the soluble fraction and focused on material extractable from larger complexes. It will be obvious to those skilled in the art that the soluble fraction may be prepared by the preferred method of centrifugation as described here, or by other well known methods such as filtration or settling.

(21) Thus, there is described herein a method for analyzing aerosol particles, wherein said aerosol particles are captured by an air sampling device, extracted from the sampling medium and soluble fraction analyzed for (1.fwdarw.3)-β-D-glucan. The air sampling device may be based on electrokinetic propulsion or on electrostatic precipitation. (1.fwdarw.3)-β-D-glucan may be determined by a limulus-amebocyte based assay or by an immunoassay. The sampling device may be based on filtration, on impingement, or on an impactor.

(22) There is also described a method for analyzing aerosol particles, wherein said aerosol particles are deposited on an electrode of an electrokinetic propulsion device from a volume of air propelled electrokinetically through said device, said electrode being removably attached to a carrier mounting; said electrode is removed from said carrier mounting and placed in an extraction vessel; a predetermined volume of extraction fluid is added to said extraction vessel; said electrode is agitated in said extraction fluid for a predetermined time; and all or part of said extraction fluid is added to a reaction mixture for analysis of said aerosol particles for (1.fwdarw.3)-β-D-glucan.