Gotham Biotech has developed strong collaborative associations with Marc R. Couturier, PhD at ARUP Laboratories and Elitza S. Theel, Ph.D.at the Mayo Clinical Laboratories in an effort to evaluate a new ELISA for Detection of Blastomycesdermatitidis antigen in urine.
Blastomycesdermatitidis is the causative agent for blastomycosis, an endemic fungal infection prevalent in the Ohio and Mississippi River Valleys, Great Lakes region, and the Southeastern United States. It causes acute and chronic pneumonia, as well as disseminated extrapulmonary disease.
ABOUT THE KIT
This kit is for the quantitative determination of Blastomyces dermatitidis serum antigen in a human serum specimen. The Gotham Biotech Blastomyces dermatitidis Serum Antigen Detection Kit is a Research Use Only device for the determination of Blastomycosis in human patients as well as for monitoring of the efficacy of treatment.
SUMMARY AND EXPLANATION
Blastomycosis is a fungal infection in humans. The infection results in the release of antigen that may be detected in human serum. This assay employs antibodies specific to the Blastomyces dermatitidis antigen for the capture and detection of its presence in a human serum specimen. The detection method involves an enzyme/substrate system with the level of antigen in the serum proportional to the assay signal. The patient specimen result is compared to a standard curve of a series of assay calibrators to determine if the patient is positive or negative for Blastomycosis, and if positive, to determine the level of Blastomyces dermatitidis serum antigen in the specimen.
MEASUREMENT PRINCIPLE
The colorimetric reaction resulting from the detection of Blastomyces dermatitidis serum antigen in the specimen is measured on a spectrophotometer as absorbance at 450 nm, with a 650 nm filter blank.
REAGENTS
Allow all reagents to reach room temperature prior to use Assay Plate: 96 well microplate coated with Blastomyces dermatitidis specific antibody, and blocked. Store at 4- 8⁰ C. One plate included per kit 5X Wash Solution: A 5 fold concentrated wash solution employed in the wash steps of the assay. Store stock at room temperature.. Prepare working wash solution by adding 1 part 5X Wash Solution to 4 parts distilled water. 50 mL 5X stock included per kit. Working solution can be stored at 4- 8⁰ C for a period of 2 weeks.
Anti-Blastomyces dermatitidis-HRP Conjugate: This reagent, supplied as a 1000X stock is employed in the antigen detection step of the assay. Store stock at 4- 8⁰ C. The working stock is prepared by adding 1 part anti-Blastomyces dermatitidis-HRP conjugate stock to 999 parts Assay Conjugate Diluent. 20 μL 1000X stock is supplied with each kit. Prepare a fresh working stock for each assay.
Assay Conjugate Diluent: The assay conjugate diluent is used to dilute the 1000X anti-Blastomyces dermatitidis-
HRP conjugate. Store at 4- 8⁰ C. 30 mL of Assay Conjugate Diluent is included per kit
TMB Substrate: This reagent is the colorimetric substrate used in the development step of the assay. Store at 4- 8⁰ C. 30 ml of TMB substrate is included per kit.
Stop Solution: This reagent used to arrest the colorimetric reaction in the development step of the assay. Store at
4- 8⁰ C. 30 ml of TMB substrate is included per kit.
Materials and equipment required but not supplied:
- Pipette (p200) and pipette tips
- Multichannel pipette
- Reagent reservoirs
- Assay Plate Sealants
- Clinical Laboratory Grade Distilled Water
- Volumetric glassware or graduated cylinders
- Timer
- Liquid storage containers and test tubes (15 mL)
- Vortex
DETERMINATIONS PER KIT
It is recommended that the calibrators, controls and patient serum specimens be run in duplicate. The Blastomyces dermatitidis Serum Antigen Detection Kit contains microwells and reagents sufficient to test 40 patient serum specimens run in duplicate in one single run, or 24 patient specimens run in duplicate for three runs, accounting for the duplicate runs of each calibrator and control.
CALIBRATORS AND QUALITY CONTROLS
A set of assay serum calibrators will be included in the assay, at Blastomyces dermatitidis antigen levels of 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL . 6.25 ng/mL, 3.125 ng/mL, and 1.56 ng/mL and 0 ng/mL.
RESULTS
The results of the assay are reported in ng/mL Blastomyces dermatitidis serum antigen.
SPECIMEN COLLECTION
Human serum can be collected by any institutionally approved method
RECOMMENDED MEASURING RANGE AND DETECTION LIMIT
The assay has a quantitative dynamic range of 0-100 ng/mL Blastomyces dermatitidis serum antigen, and a lower
limit of quantitation of 5 ng/mL Blastomyces dermatitidis serum antigen. Specimens quantitating greater than 100 ng/mL will have to be diluted and re-assayed.
ASSAY PROCEDURE
- Add 100 μL/well of undiluted Calibrators, Serum Samples and Controls in duplicate to designated assay wells
- Incubate samples for 1 hour at room temperature
- Decant plate and Wash 4 times with 300 uL/well Wash Buffer (Prepare from 5X Stock)
- Tap and blot plate dry after the last wash
Incubate at 100 μL/well with a 1:1000 - Dilution of anti-Blastomyces-HRP 1000X Stock, diluted in Conjugate Buffer, for 30 minutes at room temperature
- Decant plate and Wash 4 times with 300uL/well Wash Buffer (prepare from 5X Stock)
- Tap and blot plate dry after the last wash
- Add 100 μl/well of TMB substrate and incubate for 5 minutes at room temperature
- Add 100 uL of Stop Solution to each well of the assay plate and read the plate absorbance at 450nm (with 650 nm filter if available)
CALCULATIONS
The concentration of each of the assay calibrators is plotted against the absorbance mean at 450 nm for each of the assay calibrators, using a 4 parameter logit-log curve fitting algorithm. Patient specimen values can subsequently be calculated from the equation of the standard curve.
RESULT INTERPRETATION AND REPORTING
- If the calculated value is less than 5 ng/mL report as “Below Limit of Quantification”.
- If the results are between 5 ng/mL and 100 ng/mL, report as “Detected, XX ng/mL”.
- If the results are above 100 ng/mL, report as “Detected, Above Limit of Quantification”