Gotham Biotech

ELISA Blastomyces dermatitidis Antigen Urine Test

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Contact us at info@gothambiotech.com for pricing

Description:
To find out more about and to purchase our products, contact us at info@gothambiotech.com.

Gotham Biotech conducts research for immunodiagnostics with utility in the fields of human, veterinary as well as environmental testing. One of our specialties is the non-invasive detection of human pathogens with extensive experience in human fungal pneumonia pathogens.

Additionally, Gotham Biotech has developed strong collaborative associations with ARUP Laboratories as well as the Mayo Clinic for the purpose of evaluating a new ELISA for Detection of Blastomyces dermatitidis antigen in urine (Blasto Urine Test).
BLASTOMYCES DERMATITIDIS URINARY ANTIGEN DETECTION KIT
(Version 001, Revision 003)

INTENDED USE

This kit is for the quantitative determination of Blastomyces dermatitidis urinary antigen in a human urine specimen. The Gotham Biotechnology Blastomyces dermatitidis Urinary 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 can be detected in human urine. This assay employs antibodies specific to the Blastomyces dermatitidis urinary antigen for the capture and detection of its presence in a human urine specimen. The detection method involves an enzyme/substrate system with the level of antigen in the urine 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 urinary antigen in the specimen.

MEASUREMENT PRINCIPLE

The colorimetric reaction resulting from the detection of Blastomyces dermatitidis urinary 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
10X Wash Solution: A 10 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 10X Wash Solution to 9 parts distilled water. 50 mL 10X stock included per kit. Working solution can be stored at 4- 8⁰ C or at room temperature 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 1000 parts Assay Conjugate Diluent. 30 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 urine specimens be run in duplicate. The Blastomyces dermatitidis Urinary Antigen Detection Kit contains microwells and reagents sufficient to test 40 patient urine 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 calibrators will be included in the assay, at Blastomyces dermatitidis antigen levels of 1.25 ng/mL, 2.5 ng/mL, 5 ng/mL, 10 ng/mL and 20 ng/mL. The negative control is at 0 ng/mL Blastomyces dermatitidis antigen and the positive control is at 4-6 ng/mL Blastomyces dermatitidis antigen.

RESULTS

The results of the assay are reported in ng/mL Blastomyces dermatitidis urinary antigen.

SPECIMEN COLLECTION

Human urine can be collected by any institutionally approved method

RECOMMENDED MEASURING RANGE AND DETECTION LIMIT

The assay has a quantitative dynamic range of 0- 20 ng/mL Blastomyces dermatitidis urinary antigen, and a lower limit of quantitation of 1.25 ng/mL Blastomyces dermatitidis urinary antigen. Specimens quantitating greater than 20 ng/mL will have to be diluted and re-assayed.

ASSAY PROCEDURE

Add 100 µL/well of undiluted Calibrators, Urine 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 10X 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 10X Stock)

Tap and blot plate dry after the last wash

Add 100 µl/well of TMB substrate and incubate for 30 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 650nm 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 five 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 1.25 ng/mL report as “Below Limit of Quantification”.

If the results are between 1.25 ng/mL and 20 ng/mL, report as “Detected, XX ng/mL”.

If the results are above 20 ng/mL, report as “Detected, Above Limit of Quantification”.