Patent Application
20090044643
1. Field of the Invention
The present invention relates generally to a method for accurately determining the cleaning effectiveness of various household cleaning solutions and implements. Specifically, this invention relates to a method for calculating the percentage of dust containing common household allergens, e.g., cat (Fel d1) and dog (Can F1), picked up from a surface using household cleaning products, and more specifically to a method for determining the percentage of allergens picked up from a surface using a dusting cloth and a furniture polish.
2. Discussion of the Related Art
In many household environments, a number of airborne particulates, e.g., allergens, dust, and/or other airborne matter, are present which can create respiratory problems for individuals living within the home, especially those with disease conditions such as asthma. Some airborne particulates can accumulate on various readily viewable surfaces within the home, which can be aesthetically displeasing.
To manage, control, or otherwise influence the accumulation of airborne particles, numerous known devices and procedures may be utilized. For example, dust may be removed with a rag or dish cloth. However, as shown in
With regard to the advertising of claims of allergen removal effectiveness, there is no adequate standardized objective tests to quantify the amount of allergens removed by a particular cleaning product. Therefore, a standardized, objective and repeatable test was developed.
According to the invention, a method for determining the percentage of allergens picked up from a surface with a dust cloth and a cleaning product is disclosed. The method is comprised of a series of procedures designed to account for variables and correct the test results based on these variables.
The three-pass/one-panel dusting method is a test method designed to determine the amount of dust pick-up from a finished wood panel. This dusting method includes the steps of applying a known amount of dust to a dust panel, picking up the dust with cleaning products, e.g., a cloth and furniture polish, extracting the dust and allergens into a buffer solution and assaying the solution to determine the concentration of allergens. Consequently, a number of variables must be accounted for including the concentration of allergens in a dust sample, the amount of dust and allergens lost to the air during dust application and the amount of allergens on a dusting cloth not extracted into a buffer solution. The disclosed test method determines these variables and accounts for them when calculating a corrected allergen pick up efficiency.
The inventive method is comprised of the following procedures:
Procedures #101 to #104 outline procedures for collecting four types of samples. The obtained samples are extracted and concentrated, if necessary, according to the procedures in Procedures #105 and #106. The process sample extracts are then assayed for Fel d1 and Can f1 allergens following Procedures #107 and #108.
These and other aspects and of the present invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying figures. It should be understood, however, that the following description, while indicating preferred embodiments of the present invention, is given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the present invention without departing from the spirit thereof, and the invention includes all such modifications. In describing the preferred embodiment of the invention, specific terminology will be resorted to for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that each specific term includes all technical equivalents, which operate in a similar manner to accomplish a similar purpose.
The figures help illustrate the best mode currently contemplated of practicing the present invention.
In the Figures:
In describing the preferred embodiment of the invention several aspects of which are illustrated in the drawings, specific terminology will be resorted to for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.
The present invention and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments described in detail in the following description.
A synthetic test dust material was used in the following inventive procedures to help ensure repeatable and reliable test results. The preferred test dust used is a combined mixture of fiber and particulate containing various substances intended to accurately reflect typical household soils. Such a dust is disclosed in U.S. Pat. No. 7,001,773 to Lepow et al., the disclosure of which is herein incorporated by reference. Other dust options are discussed in U.S. Pat. No. 6,777,064 to Brown and U.S. Pat. Pub. No. 2006/0051434 to Tsuchiya which are also incorporated by reference. The naturally occurring mean (and standard deviation) of allergen content in the combined dust used during this study was determined to be 90.4±3.0 μg/g-dust of Fel d1 allergens and 22.4±1.5 μg/g-dust of Can f1 allergens.
The cleaning product used for these tests was Pledge® Natural Beauty Lemon furniture polish and was applied directly to cotton dusting cloths according to the disclosed procedure. However, any type of cleaning products can be used for this test method including, but not limited to Pledge® Multi-Surface Clean & Dust™ Spray, other brands of furniture polish or dusting sprays, and disposable cleaning products such as Pledge® Grab-It® and Swiffer® dusters and the like.
The particular dusting cloths used were 100% cotton terry cloths obtained from Wal-Mart (RN #52469 white, twelve inches by twelve inches). The cloths were machine washed once with detergent (100 mL Sunlight Ultra Fragrance Free) and machine washed only with water two additional times to eliminate any residual detergent. The triple-washed cloths were then machine dried once, with any loose threads trimmed and stored in bags before used in the test. Prior to use in the procedures, the terry cloths were placed on a conditioning rack and conditioned for at least two hours at a constant temperature and relative humidity or until the cloth weight is stable.
For consistent standardized results, Procedures #101-#104 are preferably run in a controlled environment. These inventive test procedures were run in an environmentally-controlled simulated residential exposure room or test chamber, identified by reference number 10 in
For the test procedures involving the handling of potentially airborne dust, a half facepiece respirator (e.g., 3M 6000 series with 60921 cartridges for particulate and organic vapor) was worn by the testers. Disposable gloves (e.g., TNT blue nitrile gloves 92-675) were worn during the test steps involving handling of dust or buffer solution.
At the beginning of each test day, a NIST-certified thermohygrometer (i.e., dual temperature/humidity gauge) and the humidifier were turned on. A preferred thermohygrometer is Model 4185 made by the Control Company of Friendswood, Tex. The environmental conditions within the test chamber were adjusted until they were within the desired range. The jar 46 containing test dust was rotated upside down ten times to break up any dust stratification and thoroughly mix the dust.
The computer 42, balances 26, 33, 34, and anti-static unit 36 were also turned on. After the computer and balances were operational, the spreadsheet software application used for data recording, e.g., Microsoft Excel, was opened. The analytical balance communication software application, e.g., Mettler Toledo BalanceLink, was also opened. In order to verify that the analytical balance 32 was communicating properly with the computer/database software application, the balance 32 was made to send a stabilized weight reading to the spreadsheet application. Finally, the balances were calibrated with NIST-certified standard calibration weights (including 20 mg, 100 mg, 1 g, 10 g, 100 g, 200 g, 300 g, 1 kg, 5 kg).
Once a dusting methodology was finalized, a definitive study was conducted using the inventive method for product claim support. The study was completed over a two week period. A total of ten control dust samples (obtained via Procedure #101), ten deposition efficiency (DE) tests using the wash method (Procedure #102), and ten dusting tests (Procedure #104) were conducted. Fifteen samples were collected during each study week. Five samples were each collected on Monday, Tuesday and Wednesday. The samples were extracted (Procedure #105) and centrifuged (Procedure #106) on the same day. The samples to be tested for Can f1 were further concentrated with a Centricon device (Procedure #106). The samples were stored in a refrigerator at 4° C. until the ELISA assay (Procedures #107 and #108). Each sample was replicate assayed on Thursday and Friday. The data obtained was then applied to support a cleaning product claim regarding allergen pick-up effectiveness from a surface. At the end of each test day, the test chamber was cleaned and the test materials are returned to storage. At the end of the test study week, the test chamber was deep cleaned.
The test method described in further detail below, sets forth the best mode of determining the percent allergen pick-up of a cleaning product.
The concentration of allergens in a test material, i.e., synthetic household dust in this example, can be determined by extracting a known mass of the dust applied to a dusting cloth with a known volume of extraction buffer. The buffer is then extracted and assayed to determine the allergen concentration in the dust. The following procedure outlines the steps to obtain and prepare control dust samples to evaluate the allergen concentration in the synthetic household dust.
A sample jar is prepared by adding 150 mL of extraction buffer to the jar and place a label on it. The label includes a database file name and a unique sample ID to uniquely identify each sample. Before applying any dust, the dust supply jar is rolled at least fifteen times within a roller box to mix up the particulate and fibers.
The weight of a 3″×3″ aluminum foil piece is tared. Using a clean spatula, approximately forty-five milligrams of test dust is measured out onto the foil. Forty-five milligrams of dust mimics the amount that deposits on a dusting panel when applying seventy-five milligrams of dust with a sieve application method such as that disclosed in Procedure #104. After the balance reading has stabilized, the dust weight is recorded.
The dust and foil is carefully placed into the sample jar. The dust is not permitted to fall freely into the buffer but rather the dust is slowly immersed into the buffer solution to minimize any dust suspension loss. The preferred extraction buffer solution is not a standard ELISA wash buffer (as used for the Fel d1 and Can f1 assays in Procedures #107 and 108). Instead, the ELISA wash buffer is reformulated with a higher concentration, i.e., 3.5%, of Tween® 20 non-ionic surfactant. The reformulated buffer solution is necessary because the silicone content of the cleaning product prevents water-based and other organic solvent buffer solutions from effectively extracting allergens. However, standard ELISA wash buffer may still be used for plain dust samples with no cleaning products containing silicone on them.
These steps are repeated as necessary to collect the desired number of samples.
The test dust ID and the mass of dust used in each run is reported. After the sample extraction procedure (Procedure #105) and ELISA assays (Procedures #107 & 108), the allergen concentration in the control dust samples is reported.
where,
Callergen-in-dust is allergen concentration in surrogate household dust (μg/g-dust),
Callergen-buffer is concentration of allergen in extraction buffer (μg/mL),
Vbuffer is volume of extraction buffer used to extract household dust (mL),
Mdust-CT is mass of dust used in this sample (g).
Table 1 presents allergen concentrations in the synthetic test dust (Callergen-in-dust) for the ten control dust samples. The mean allergen concentrations in the synthetic dust were 90.4±3.0 μg/g-dust for Fel d1 allergens and 22.4±1.5 μg/g-dust for Can f1 allergens. The standard deviation was low, indicating that allergens were distributed uniformly with the bulk dust.
When dust is applied onto a test panel, not all of the dust and allergens are deposited onto the panel. Instead, a measurable amount of dust and allergens are lost to the air or to the sides of the containment box. The percentage of allergen deposited on the testing panel compared to the total amount applied must be determined in order to calculate the corrected allergen pick-up efficiency. This “allergen deposition efficiency” factor (DE) is defined as the percentage of allergens on the panel as compared to the total allergens in the approximately seventy-five milligrams of dust applied.
It was initially assumed that the deposition efficiency of allergens was the same as the applied dust. Therefore, the percentage mass of dust deposited on the panel was compared with total dust mass to estimate the deposition efficiency. However, this procedure was not as successful due to the difficulties associated with accurately measuring the small mass increase on the panel and significant run-to-run and operator-to-operator variation. A direct allergen measurement technique that used a variety of cloth wipes, wet and dry, to collect deposited dust and allergens from the panel was then attempted. The wipes were extracted and ELISA assayed to assess allergen deposition efficiency. This technique also had problems due to the failure of cloth wipes to effective pick-up all of the deposited allergens.
Finally, a wash method was designed and proven to produce a reproducible allergen deposition efficiency determination. The procedure includes applying a known amount of dust to a dust containment unit or box 50 (see
After the test dust was applied, each plate was sequentially washed in three buffer containers with a known volume of buffer to ensure maximum recovery. The wash buffer was extracted and ELISA assayed to determine the actual mass of deposited allergens. From this value, the allergen deposition efficiency can be calculated.
The weight of one empty extraction jar is measured and recorded. A sample label is placed on the jar. The label includes a database file name and a unique sample ID. The label information is sufficiently unique to identify each sample.
The deposition plate is prepared by first placing a piece of clean aluminum foil on the designated dust application area. Next, the twelve deposition plates are laid down in two rows, six plates each, on the foil. The plates are overlapped by at least two millimeters to prevent any dust loss in gaps. The overall area of the deposition plates is slightly larger than the dust application area.
The dust containment box 50 is cleaned with a piece of wet KleanWipes® paper and let air dry. Before using the test dust, the dust jar 46 is rolled at least fifteen times within the roller box 48, along the 17″ side, to mix the particulate and fibers. An aluminum weighing boat is tared and, using a clean spatula, approximately seventy-five milligrams of dust is measured out into the weighing boat. After the balance reading has stabilized, the dust weight is recorded.
The dust containment box 50 is then placed on top of the deposition plates. The 100 mesh sieve is checked to ensure there is no contamination dirt on it. If necessary, the sieve is tapped to clean any contamination. The sieve is not solvent- or water-cleaned under normal use to ensure a constant layer of dust on the sieve, necessary for reproducible dust application. The dust is sieved five to seven inches above the plates within the containment box 50. Caution is taken to prevent dust piles, i.e., “hot spots.” Fingers are kept on the edge of the sieve during dust application and the dust layer on the plates is not disturbed. After applying the dust, the dust is allowed to settle for sixty seconds. The containment box 50 is slowly removed away from the dust application area to avoid any further suspension loss.
Next, three wash pans and a plastic scraper are placed in sequential order beside the deposition area and 50 mL of ELISA wash buffer is added to each pan. Each wash pan is tilted about 30° to one side so that the washing buffer accumulates in one side to facilitate plate washing. The top most deposition plate is carefully collected and slowly transferred to the first wash pan in a horizontal orientation to minimize dust loss. Once the plate is on top of the first wash pan, one side of the plate is slowly tilted into the wash buffer. The scraper is used to slowly brings wash buffer over the plate and wet the dust layer. The wet dust is then scraped into the wash buffer. The plate is washed two or three times until most of the dust is washed down into the first wash pan. Before moving to the second wash pan, any remaining dust attached to the back of the deposition plate is scraped down, including any dust in the immersed area. The washing/rinsing procedure is repeated for the deposition plate in the second and third wash pans. The washing steps are then repeated in a sequential order, from top to bottom, for the remaining eleven plates.
After washing the dust from all twelve deposition plates, the first wash pan is rinsed and the wash buffer poured into a sample jar. The second wash pan is rinsed and the wash buffer contained therein is used to rinse the first wash pan. This second wash buffer portion is then poured into the sample jar. The third wash pan is then rinsed with the wash buffer used to rinse the second and first wash pans sequentially. After consecutive rinses, the third wash buffer portion is poured into the sample jar. 50 mL of clean buffer solution is used to rinse the third, second, and first wash pans sequentially and then poured into the sample jar. The final weight of the sample jar is measured and the actual amount of wash buffer used is calculated. The sample jar is stored in a safe place before extraction.
After completing the procedure, the stainless steel plates, wash pans, and scrapers are rinsed with tap water to remove any residual wash buffer. The plates are rinsed again with DI water to remove any residual tap water minerals. The cleaned materials are wiped dry or allowed to air dry before being used again. This method may be repeated to collect additional samples as needed.
The mass of dust used in each run and type of containment box 50 used is recorded as is the mass of wash buffer in each sample. After the sample extraction (Procedure 105) and ELISA assay (Procedures 107 & 108), the allergen deposition efficiency is determined and recorded.
where,
DE is allergen deposition efficiency (%),
Callergen-WS is allergen concentration in wash sample extract (μg/mL),
VWS is volume of wash buffer (mL),
Mdust-WS is mass of dust used in this wash sample (g), and
Callergen-in-dust is allergen concentration in synthetic dust (μg/g-dust).
Table 2 presents the allergen deposition efficiency (DE) for the ten wash samples. The mean deposition efficiency for Fel d1 was 84.2±7.1% and 71.7±10.1% for Can f1.
The allergens assayed on a dusting cloth sample are typically not fully extracted into the buffer solution due to the interaction between the cleaning product, e.g., Pledged and the dusting cloth. This so-called “extraction efficiency” (EX) must be determined with simulated dusting samples in order to correct for the allergen pick-up efficiency. The simulated samples were processed with the same extraction procedure and ELISA methods to mimic the extraction efficiency for an actual dusting sample with cleaning product.
The simulated dusting samples were prepared by applying approximately forty-five milligrams of dust on a piece of 7″×4″ dusting cloth with a cleaning product, e.g., Pledge® previously applied. Forty-five milligrams of dust mimics the percentage (approximately 60%) of dust that typically deposits on the dusting area in Procedure #104. The dust was well mixed with the cleaning product to simulate the dust-cleaning product mixture on a typical dusting cloth.
A piece of 25″ aluminum foil is placed on a table away from the testing materials to avoid contamination. The target mass of cleaning product applied to each terry cloth is 1.5±0.1 g.
Next, a sample jar is prepared by adding 150 mL of modified extraction buffer to the jar. A label having a unique sample ID is placed on the jar. Before using any dust, the dust jar 46 is rolled fifteen times within the roller box 48, along the 17″ side, to homogenize the particulate and fibers. Next, one 3″×3″ aluminum foil piece is placed on the balance and tared. Using a clean spatula, approximately forty-five milligrams of dust is measured out onto the foil. As previously discussed, forty-five milligrams of dust is approximately the average amount of dust that deposits onto a dusting panel when applying seventy-five milligrams of dust with the sieve application method discussed in Procedure #104. After the balance reading has stabilized, the weight of the dust is recorded.
Next, a 6″×9″ aluminum foil piece is placed on the tabletop balance and the balance is tared. One 4″×7″ terry cloth is placed onto the foil and the initial weight of the cloth is recorded. Another clean 6″×9″ aluminum foil piece is then placed in the central area of the cleaning product application station and the terry cloth is moved to this foil. The 7″ side of the cloth is aligned horizontally to the cleaning product applicator.
The can of cleaning product spray is vigorously shaken for at least thirty seconds to ensure a homogeneous mixture. The nozzle of the cleaning product container is kept approximately eight inches above the cloth. The cleaning product is applied to the terry cloth with a three-pass method. During the three-pass application, the cleaning product trigger nozzle is continuously pressed to deliver a consistent spray volume. If the application speed is controlled at approximately one second per pass, approximately 1.5 g of cleaning product will be deposited on the cloth, though practice may be required to achieve consistent results. The three-pass application preferably is started outside the cloth at the left-hand-side. After the cleaning product can is emitting a stable spray, the cleaning product container is moved left-to-right across the cloth until the spray stream passes the cloth. The application direction is reversed to complete the second pass right-to-left and reversed yet again to complete the third pass left-to-right.
The final weight of the cloth is measured and recorded immediately after the three-pass application of cleaning product. From this weight, the mass of the applied cleaning product is calculated. The target range for cleaning product mass on the cloth is 1.5±0.1 g. If the mass of the cleaning product is less than 1.4 g, a fine-tune spray application of cleaning product is necessary. If the mass of the cleaning product is over 1.6 g, the cloth is discarded and the three-pass application redone with a new cloth. To ensure a consistent application rate, a can of cleaning product should be decommissioned when its mass has decreased by half, e.g., about 230 g for a standard container of Pledge®.
After the application of the cleaning product, the dust is carefully placed on the cloth while taking caution to prevent suspension dust loss. The dust is spread across the cloth and pressed into the cloth with a force similar to that of panel dusting. All of the dust particles ideally are in contact with the cleaning product in order to accurately mimic actual dusting operations. Next, the cloth is folded and the reverse side is used to wipe any residual dust on the 3″×3″ foil. After checking the sample ID, the cloth sample is carefully placed into the sample jar. Using tweezers, the cloth is opened and “washed,” i.e., agitated, several times inside the jar to extract most of the dust particles. The lid is then closed and the sample jar is stored in a safe place before extracting dust following the steps of Procedure #105—Sample Extraction. This extraction procedure is repeated as necessary to collect additional samples.
The test dust lot ID and the mass of dust used in each run is recorded. The cleaning product ID and mass of cleaning product on each sample is also recorded. After the sample extraction (Procedure #105) and ELISA assays (Procedures #107 and #108), the allergen extraction efficiency (EX), calculated with the following formula, is recorded.
where,
EX is extraction efficiency (%),
Callergen-EX is the allergen concentration in EX sample extract (μg/mL),
VEX is volume of extraction buffer (mL),
Mdust-EX is the mass of dust used in the EX sample (g), and
Callergen-in-dust is allergen concentration in synthetic household dust (μg/g-dust).
Tables 3 and 4 present the extraction efficiency (EX) data for Fel d1 and Can f1, respectively. The extraction efficiency for Fel d1 allergens was 90.5±4.4% and 86.5±10.9% for Can f1 allergens.
The allergen pick-up efficiency (PU) is defined as the percentages of allergens picked up by a piece of cloth treated with a cleaning product. A number of factors, or variables, must be determined through the steps in Procedures #101-#103 to provide a corrected, i.e., more accurate, allergen pick-up efficiency.
A piece of 25″ aluminum foil is placed on an application table. The application table is located away from the testing materials to avoid contamination. The target mass of a cleaning product on a terry cloth is 1.5±0.1 g. A sample jar is prepared by adding 150 mL of modified extraction buffer to a sample jar and a sample label is placed on the jar. The label includes a database program file name and a unique sample ID. The label information is sufficiently unique so as to easily identify each sample.
The dusting panel 22 is aligned within the designated area on a support table 23. The left side 25 of the panel 22 extends by about one inch outside the table 23 so that any push-off dust (i.e., dust “pushed” off the panel 22 but not retained on the cloth) will fall on a piece of push-off collection foil located on the push off collection cart 24. Mineral spirits are applied to the panel 22 in an “s” shaped pattern and then the panel 22 is wiped with a clean shop towel to remove the mineral spirits. The cloth is reversed and the panel 22 is cleaned a second time. The same cloth is used to clean the panel edges. Special caution is taken to clean the push-off edge, i.e., left side 25, as dust may become trapped there. The mineral spirit cleaning procedure is repeated one time.
When the panel 22 is dry, the panel 22 is cleaned with a deionized water-saturated KleanWipes® paper to eliminate any static electricity that built-up during the mineral spirits cleaning. A dry KleanWipes® paper is used to dry the panel 22. The timer is started and the panel 22 is air dried for at least 8 minutes. If the panel 22 is not used within thirty minutes, the panel cleaning procedures are redone to ensure consistent surface conditions. During the panel 22 drying period, the dust containment box 50 is cleaned with a wet KleanWipes® paper and air dried. While the panel 22 is drying, it is beneficial to practice consistently applying a 1.4 kg dusting force with a piece of practice cloth on the Ohaus tabletop balance 34.
During the eight minute panel drying period, a terry cloth 60 is placed onto a 9″×6″ aluminum foil piece 62 as shown in
The push-off collection foil 52 is prepared by cutting a piece of 30″ aluminum foil. This is done at the beginning of a study day so that foil can also be conditioned with the chamber atmosphere. The foil is folded and placed in a 16 oz. plastic cup. The initial weight of the foil and cup is measured with the Mettler analytical balance. After the balance reading has stabilized, the weight is recorded in the spreadsheet.
The synthetic household dust is prepared by first rolling the dust jar 46 fifteen times within the roller box 48 as explained above. The weight of an aluminum weighing dish is tared on the Mettler analytical balance 32. Using a clean spatula, approximately 75 mg of dust is measured out onto the weighing dish. When the balance reading has stabilized, the dust weight is recorded.
After the test panel 22 has dried, the dust containment box 50 is placed onto the panel 22 with the left side 54 of the box 50 aligned with the push-off edge 25. As shown in
The 100 mesh sieve must not have any visible dirt on it. If necessary, the sieve is tapped to remove any contaminants. The sieve is not solvent or water cleaned under normal use to ensure a constant layer of dust on the sieve for reproducible dust applications. Next, the dust is sieved from a height of five to seven inches above the panel 22.
The cleaning product is applied to the terry cloth 60 with the three-pass method described in Procedure #103. The panel 22 is dusted immediately after applying the cleaning product to ensure that the cleaning product is not dry. The folded terry cloth 60 is run across the panel 22 with the cleaning product side down. The three-pass dusting procedure, as depicted below, is performed with approximately 1.4 kg of dusting force:
After completing the three-pass dusting procedure, the cloth is held dust side up to prevent any falling dust loss and moved carefully to a cutting board. The cloth is laid flat with the dusting side up and the cloth is unfolded carefully. The 4″×7″ dusting area is cut with the roller cutter, folded, and carefully put into a sample jar after checking the sample ID. The tweezers is used to agitate the cloth several times to facilitate dust extraction. The sample jar is stored in a safe place before performing the steps of Procedure #105—Sample Extraction. The dust application and dusting procedures are repeated as necessary to obtain the desired number of samples.
The test dust lot ID, mass of dust used, type of containment box 50 used, cleaning product ID and mass of cleaning product used in each dusting procedure is recorded. After completing the sample extraction steps (Procedure #105) and ELISA assays (Procedure #107 and #108), the allergen pick up percentage before correction is calculated and recorded using the following formula.
where,
PU is the allergen pick up efficiency (%),
Callergen-PU is the allergen concentration in pick up sample extract (μ/mL),
VPU is the volume of extraction buffer (mL),
Mdust-PU is the mass of dust used in this pick up experiment (g), and Callergen-in-dust is the allergen concentration in synthetic household dust (μg/g-dust).
However, the allergen pick up efficiency must also be corrected for both the allergen deposition efficiency (DE—calculated in Procedure #102) and cleaning product-cloth sample extraction efficiency (EX—calculated in Procedure #103) using the following formula.
where,
PUcorrected is corrected allergen pick-up efficiency (%),
PU is allergen pick up efficiency (%, before correction),
EX is allergen extraction efficiency from pick-up sample (%), and
DE is allergen deposition efficiency (%).
Table 5 presents corrected allergen pick-up (PUcorrected) for ten dusting samples obtained with terry cloths and an aerosol Pledge® cleaning product. The mean allergen pick-up efficiency for Fel d1 allergens was 93.0±9.0% and 94.6±13.7% for Can f1 allergens.
The samples collected from Procedures #101 to #104 are extracted with the following sample extraction procedure.
The sample jars are placed in an extraction box with spacers placed between the jars to prevent collisions. The extraction box is securely affixed onto a horizontal shaker with appropriate elastic straps. The shaker is turned on, adjusted to a shaking frequency of 250 RPM, and run for two hours.
Four tubes of extracted buffer are centrifuged for each sample. Two of tubes are for the Can f1 Centricon sample concentration procedure (Procedure #106), one tube is for the Fel d1 assay (Procedure #107), and one is saved as a backup.
The centrifuge tubes are labeled with the appropriate sample ID numbers. The Finnpipettes® are set at 4.5 mL volume. Three pipettes of extraction buffer are transferred into each of the four centrifuge tubes. The sample jar is shaken by hand immediately before pipetting to ensure representative sampling. If the sample contains a terry cloth, a tweezers is used to mix the sample buffer before pipetting. Next, the centrifuge tubes are placed in tube adapters and the assembly is placed into the centrifuge. The weight of opposite swing buckets are balanced using dummy tubes if necessary. The samples are centrifuged at 4000 RPM for twenty minutes and then carefully placed the centrifuged tubes on a tube rack. Special caution is taken as the silicon and water layers may be mixed by even a slight agitation.
The Finnpipette® is set at 4.0 mL volume. The pipette tip is carefully inserted into the centrifuged tube and a water sample is collected for a Centricon device. This step is repeated for another centrifuged tube to collect a total of 8 mL in the Centricon device. The final weight of the Centricon is measured and the mass of the extraction buffer is calculated. Centricon samples are processed following Procedure #106.
The pipette tip is carefully inserted into the third centrifuged tube to collect a 2 mL water sample into a 2 mL sample vial. The 2 mL sample vials are stored in a refrigerator at 4±2° C. before the Fel d1 ELISA assay (Procedure #107). The sample jars and the fourth centrifuged tubes as backup are stored in a freezer at −20±5° C.
The timing of each step is recorded in the spreadsheet as is the mass of extraction buffer in each Centricon device.
Preliminary ELISA assays indicated that 150 mL of extraction buffer was an appropriate volume for Fel d1 allergen extraction. The concentration of Fel d1 is such that it can be directly ELISA assayed (Procedure 107) after the centrifuge step (Procedure 105). However, the concentration of Can f1 was near or below the detection limit (1 ng/ml) of a standard ELISA assay. It was concluded that the extraction buffer must be concentrated prior to the ELISA assay for Can f1 (Procedure 108). Experimental results indicated that a Centricon Plus-20 concentration device would concentrate Can f1 to 20-30 ng/ml, which is in the linear range of the calibration curve. Experiments were conducted to develop the following definitive concentration procedure.
The Centricon devices are placed into centrifuge adapters and the Centricon samples obtained in Procedure #105 are centrifuged at 4000 g (4325 RPM for the Eppendorf 5804 Centrifuge) for twenty minutes. After the samples have been centrifuged, the Centricon devices are carefully placed on a tube rack.
The initial weight of a 200 μL pipette tip and labeled 20 mL vial is measured with an analytical balance. This pipette tip is used to collect unfiltered retentate in to the vial. The pipette tip is placed in the vial and the vial cap is closed to avoid any evaporation loss.
The Centricon is attached with a labeled retentate collection cup and the assembly is reverse spun at 1000 g (1250 RPM for the Eppendorf 5804 Centrifuge) for one minute. The recovered retentate is transferred with the same pipette tip to the mL vial. The final weight of the 20 mL vial with the pipette tip is measured and the total recovery of retentate is calculated by subtracting the initial weight of the empty vial and tip. The reverse-spin recovered retentate is viscous due to the higher concentration of Tween® 20. The pipette tip is used to mix any recovered retentate and the retentate is pipetted into a labeled 2 mL vial for Can f1 ELISA assay (Can f1 ELISA Procedure 108).
The Finnpipette is set at 100 μL volume and the weight of a 0.5 mL labeled centrifuge vial is tared with the analytical balance. Next, 100 μL of retentate is pipetted into the vial, the vial cap closed and the vial final weight recorded. the retentate density and recovered sample volume is calculated and the retentate is returned to the 2 mL vial. The 100 μL retentate is not discarded due to the minimal volume of recovered retentate. The 2 mL vial is stored in a refrigerator at 4±2° C. until the Can f1 ELISA assay of Procedure #108.
The recovered retentate mass is recorded and the retentate density and volume of recovered retentate is calculated.
where,
D is retentate density (g/mL),
M is mass of 100 μL retentate (g),
Samples are ELISA assayed according to the following steps to determine the amount of Fel d1 allergens contained therein.
1. Coat Nunc™ 96-well Microplates with Antibody Anti-Fel d1.
A 1/1000 dilution of a Fel d1 antibody, preferably MA-6F9 (available from Indoor Biotechnologies, Inc. of Charlottesville, Va.), is prepared using 50 mM carbonate-bicarbonate buffer. The total required volume (V) of coating buffer needed for a batch of assay is calculated by determining the number of microplates (n) and using the following equation:
V=10×n+1=______mL.
Each well is coated with 100 μL of diluted MA-6F9 solution. A visual inspection is performed to ensure that each well has the same volume of solution. Next, the microplates are wrapped with plastic wrap, e.g., Saran Wrap, and incubated overnight at 4° C. or for at least 8 hours. The incubation start time is then recorded. The microplates are washed five times with 250 μL of PBST buffer and the washing time is recorded.
2. Block Microplates with a 1% BSA PBS-T Buffer Solution.
The total required volume of 1% BSA PBST solution is prepared in a pipette basin. Each well is blocked with 100 μL of 1% BSA PBST solution and visually checked to see if each has a similar volume of solution. The microplate is then incubated at room temperature for 30 minutes. After incubation, the microplates are again washed five times with 250 μL of PBST buffer and the washing time is recorded.
The microplate layout is determined as shown in Table 6.
The standard concentration and sample dilution is determined as shown in Table 7.
The volume of BSA PBST buffer in each well is determined as shown in Table 8.
The sample and standard volume is determined as shown in Table 9.
The total required volume (V) of 1% BSA PBST solution is prepared in a pipette basin. The determined volume of BSA-PBST is added into each wells as is the determined volume of sample. Serial 2× dilutions are performed four times from D1/H1 sequentially to D4/H4 as shown in Table 7. Serial 2× dilutions are also performed four times from C5/D5 & F5/H5 sequentially to C8/D8 & F8/H8 and from C9/G9 sequentially to C12/G12.
20 uL of Fel d1 standard (also from Indoor Biotechnologies Inc.) is added in wells A1, B1, and C1. Serial 2× dilutions are performed from A1/B1 sequentially to A10/B10. Serial 2× dilutions are performed from C1 sequentially to C4, and then from E5 sequentially to E8, and then from H9 to H10.
The microplates are then incubated at room temperature for two hours with the start time recorded. The microplates are washed five times with the 250 μL PBST buffer and the washing time is recorded.
4. Add Biotinylated anti-Fel d1 Monoclonal Antibodies.
The total required volume of 1/1000 dilution of an anti-Fel d1 monoclonal antibody, preferably BI-3E4 (also from Indoor Biotechnologies Inc), with 1% BSA-PBST buffer is prepared in a pipette basin. 100 uL of diluted BI-3E4 solution is pipetted into each well and incubated at room temperature for one hour. The start time is recorded. The microplates are then washed five times with a 250 uL PBS-T buffer and the washing time is recorded.
The total required volume of 1/1000 dilution reconstituted Streptavidin-Peroxidase with BSA-PBST is prepared. 100 uL of diluted Streptavidin-Peroxidase is pipetted into each well. The microplates are incubated at room temperature for 30 minutes. The start time is recorded. The microplates are then washed five times with a 250 uL PBS-T buffer and the washing time is recorded.
The total required volume of 1/1000 dilution of 30% H2O2 with 1 mM ABTS-70 mM citrate phosphate buffer is prepared. 100 uL of diluted H2O2 solution is pipetted into each well. At least one minute passes prior to developing the next plates to allow sufficient time to read a plate before the next plate finishes color development. Each microplate is read at 405 nm repeatedly until the optical density of wells A1 and B1 reaches 2.0-2.4. The reading time is recorded and the ELISA procedure is recorded with ELISA reports.
Concentrated samples are ELISA assayed according to the following steps to determine the amount of Can f1 allergens contained therein.
1. Coat Nunc™ 96-well Microplates with Antibody Anti-Can f1.
A 1/1000 dilution of a Can f1 antibody, preferably MA-6E9 (Indoor Biotechnologies Inc.), is prepared using 50 mM carbonate-bicarbonate buffer. The total required volume (V) of coating buffer needed for a batch of assay is calculated by determining the number of microplates (n) and using the following equation:
V=10×n+1=______mL.
Each well is coated with 100 μL of diluted MA-6E9 solution. A visual inspection is performed to ensure that each well has the same volume of solution. Next, the microplates are wrapped with plastic wrap, e.g., Saran Wrap, and incubated overnight at 4° C. or for at least 8 hours. The incubation start time is then recorded. The microplates are washed five times with 250 μL of PBST buffer and the washing time is recorded.
2. Block Microplates with a 1% BSA PBS-T Buffer Solution.
The total required volume (V) of 1% BSA PBST solution is prepared in a pipette basin. Each well is blocked with 100 μL of 1% BSA PBST solution and visually checked to see if each has a similar volume of solution. The microplate is then incubated at room temperature for 30 minutes. The microplates are again washed five times with 250 μL of PBST buffer and the washing time is recorded.
The microplate layout is determined. The preferred layout is shown in Table 6 of Procedure #107.
The standard concentration and sample dilution is determined as shown in Table 10.
The volume of BSA PBST buffer in each well is determined as shown in Table 11.
The sample and standard volume is determined as shown in Table 12.
The total required volume (V) of 1% BSA PBST solution is prepared in a pipette basin. The determined volume of BSA-PBST is added into each wells as is the determined volume of sample. Serial 2× dilutions are performed four times from D1/H1 sequentially to D4/H4 as shown in Table 7. Serial 2× dilutions are also performed four times from C5/D5 & F5/H5 sequentially to C8/D8 & F8/H8 and from C9/G9 sequentially to C12/G12.
20 uL of Fel d1 standard (Indoor Biotechnologies Inc.) is added in wells A1, B1, and C1. Serial 2× dilutions are performed from A1/B1 sequentially to A10/B10. Serial 2× dilutions are performed from C1 sequentially to C4, and then from E5 sequentially to E8, and then from H9 to H10.
The samples are then incubated at room temperature for two hours with the start time recorded. The microplates are washed five times with the 250 μL PBST buffer and the washing time is recorded.
4. Add Rabbit anti-Can f1 Antibody.
The total required volume (V) of 1/1000 dilution of an anti-Can f1 monoclonal antibody, preferably PA-CF1 (Indoor Biotechnologies Inc.), with 1% BSA-PBST buffer is prepared in a pipette basin. 100 μL of diluted PA-CF1 solution is pipetted into each well and incubated at room temperature for one hour. The start time is recorded. The microplates are then washed five times with a 250 uL PBS-T buffer and the washing time is recorded.
5. Add Peroxidase Conjugated Goat anti Rabbit IgG
The total required volume (V) of 1/1000 dilution reconstituted Goat anti Rabbit IgG with BSA-PBST is prepared. 100 uL of diluted Goat anti Rabbit IgG is pipetted into each well. The microplates are incubated at room temperature for one hour. The start time is recorded. The microplates are then washed five times with a 250 uL PBS-T buffer and the washing time is recorded.
The total required volume (V) of 1/1000 dilution of 30% H2O2 with 1 mM ABTS-70 mM citrate phosphate buffer is prepared. 100 uL of diluted H2O2 solution is pipetted into each well. At least one minute passes prior to developing the next plates to allow sufficient time to read a plate before the next plate finishes color development. Each microplate is read at 405 nm repeatedly until the optical density of wells A1 and B1 reaches 2.0-2.4. The reading time is recorded and the ELISA procedure is recorded with ELISA reports.
Using the methodology described above, a ten replicate definitive study was conducted for an aerosol Pledge furniture polish cleaning product. This product was able to pick-up 93.0±9.0% of Fel d1 allergens and 94.6±13.7% of Can f1 allergens. This data may be used to support a product claim regarding allergen pick-up for this product such as is shown in
The novel test method, with or without modifications, generates reproducible and reliable results to provide product claims for other cleaning products as well. This information may be communicated to consumers with the knowledge that the data is obtained from a reliable, repeatable and accurate test.
Although the best mode contemplated by the inventors of carrying out the present invention is disclosed above, practice of the present invention is not limited thereto. It will be manifest that various additions, modifications, and rearrangements of the features of the present invention may be made without deviating from the spirit and scope of the underlying inventive concept.
It is intended that the appended claims cover all such additions, modifications and rearrangements. Expedient embodiments of the present invention are differentiated by the appended claims.
