Microsoft word - legionala urinary antigen eia new.doc
An enzyme immunoassay (EIA) based system for the qualitative
detection of Legionella pneumophila serogroup 1 antigen
(L. pneumophila serogroup 1 antigen) in human urine as an adjunct
to culture for the presumptive diagnosis of past or current
The majority of Legionnaires' Disease is caused by Legionella pneumophila
serogroup 1 and is characterized as an acute febrile respiratory illness that
Legionella is common nosocomial infection
Legionella is present in water
Transmitted via aerosolized droplets
Affects immunocompromised or otherwise ill persons
Increasingly common in the community setting
Empiric therapy may not cover Legionella
Treatment of choice: azithromycin
A total of 294 patient urine specimens were evaluated using the Binax
Legionella Urinary Antigen EIA Test kit and a commercially available method.
The results of this correlation are as follows:
Sensitivity: 97.7% Specificity: 100 % Agreement: 99.0 %
Positive and negative controls (liquid)
All necessary reagents
851-000 NOW® Legionella Urinary Antigen EIA
2. Correlation of Results
A total of 294 patient urine specimens were evaluated using the Binax Legionella
Urinary Antigen EIA Test kit and a commercially available method.
The results of this correlation are as follows:
Legionella Urinary Antigen EIA
Sensitivity: 97.7% (0.951 to 1.00, 95% Confidence Interval)
I. PROPRIETARY NAME
Specificity: 100 %
Urinary Antigen EIA, 96 Wells
Agreement: 99.0 %
II. INTENDED USE
Statement of Cross-ReactivityOf the 164 negative specimens: 76 specimens were bacteremic (other than Legionella
sp.) pneumonia patients, 34 had urinary tract infections (UTI), 16 had other pulmonary conditions, 19
FOR IN VITRO DIAGNOSTIC USE
had Mycobacterial infections, and 2 were pneumonias caused by transtracheal aspirates (TTA).
This kit is an enzyme immunoassay (EIA) based system intended for IN VITRO
DIAGNOSTIC USE to qualitatively detect the presence of Legionella pneumophila
serogroup 1 antigen (L. pneu-mophila
serogroup 1 antigen) in human urine as an adjunct to culture for the presumptive diagnosis of past or current Legionnaires' Disease.
3. Binax Legionella
Urinary Antigen EIA Kit Precision
III. SUMMARY AND EXPLANATION OF TEST
Within Assay Precision
Legionnaires' Disease, named after the outbreak in 1976 at the American Legion convention in Philadelphia, is caused by Legionella pneumophila
and is characterized as an acute febrile res-
Within Assay Precision was determined on three urine samples containing different antigen concentrations. The samples were run in twenty replicates. Each sample was correctly identified as
piratory illness ranging in severity from mild illness to fatal pneumonia (1). Since that time, it has been recognized that the disease occurs in both epidemic and endemic form and that spo-
being positive or negative 100% of the time.
radic cases are not readily differentiated from other respiratory infections by clinical symptoms. It is estimated that about 25,000 cases of Legionella
infections occur annually (2). Known riskfactors include immunosuppression, cigarette smoking, alcohol consumption and concomitant pulmonary disease (2). The resulting mortality rate, which ranges up to 25% in untreated
Coefficient of Variation (%)
immunocompetent patients, can be lowered if the disease can be diagnosed rapidly and appropriate antimicrobial therapy is instituted early. Legionella pneumophila
is estimated to be respon-
sible for 80-85% of reported cases of Legionella
infections with the majority of cases being caused by L. pneumophila
serogroup 1 alone (3). Current methods for the laboratory detection of
pneumonia caused by Legionella pneumophila
require a respiratory specimen (e.g. expectorated sputum, bronchial washing, transtracheal aspirate, lung biopsy) or paired sera (acute and
convalescent) for accurate diagnosis. These techniques include Legionella
culture, direct fluorescent antibody (DFA), DNA probe, and indirect fluorescent antibody (IFA). All of these rely oneither obtaining an adequate respiratory specimen for sufficient sensitivity, or collecting sera at a two to six week interval. Unfortunately, one of the presenting signs of patients with Legionnaires'
Between Assay Precision
Disease is the relative lack of productive sputum. This necessitates in many patients the use of an invasive procedure to obtain a respiratory specimen. Diagnosis by serological techniques is
Between assay precision was determined on three urine samples containing different antigen concentrations. Each sample was analyzed on two lots of reagents in a total of 33 assays. The sam-
usually retrospective in nature, and even then, patient compliance in obtaining the necessary samples is poor.
ples were correctly identified as being positive or negative 100% of the time
It was shown as early as 1979 by Berdal (4), that a specific soluble antigen was present in the urine of patients with Legionnaires' Disease (5,6,7,8). The presence of specific antigen in urine
Coefficient of Variation (%)
makes this an ideal specimen for collection, transport and subsequent detection in early, as well as later, stages of the disease (9). The Binax Legionella
Urinary Antigen EIA Kit uses microtiter
0.047 0.013 27.7
ELISA methodology for the detection of soluble antigen in urine from patients with Legionella pneumophila
serogroup 1 infections.
0.187 0.036 19.5
IV. PRINCIPLES OF PROCEDURE
The microtiter wells contained in the kit are coated with polyclonal rabbit antibody specific for L. pneumophila
serogroup 1 antigen. Patient urine is added to the rnicrotiter well and any L.
serogroup 1 antigen present is captured by the antibody coated microtiter well (solid phase). Anti-Legionella
HRP conjugate added at the same time as the patient specimen binds
1. Frazer, D.W., T.R. Tsai, W. Orenstin, WE. Parkin, H.J. Beecham, R.G Sharrar, J. Harris, G.F. Mallison, S.M. Martin, J.E. McDade, C.C. Shepard, P.S. Bracham, and the Field Investigation Team.
to L. pneumophila
serogroup 1 antigen. After an incubation, the wells are decanted and washed to remove any unbound antigen and conjugate. A color developer is added to the wells and
1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297: 1189-1197.
incubated. The assay reaction is stopped using stop solution and the resultant absorbances of color are read on a microplate reader. Samples with absorbances greater than or equal to 3 times
2. Kohler, R.B. 1984. Antigen detection for the rapid diagnosis of Mycoplasma and Legionella pneumonia. Diagn. Microbiol. Infect. Dis. 4:475-595.
the negative control are considered positive.
3. Reingold, A.L., B.M. Thomason, B.J. Brake, L. Thacker, H.W. Wilkinson, and J.N. Kuritsky. 1984. Legionella pneumonia in the United States: the distribution of serogroups and species caus-
ing human illness. J. Infect. Dis. 149:819.
V. MATERIALS PROVIDED
4. Berdal, B.P., C.E. Farshy, and J.C. Feeley. 1979. Detection of Legionella pneumophila
antigen in urine by enzyme-linked immunospecific assay. J. Clin. Microbiol. 9:575-578. 5. Tilton, R.C. 1979. Legionnaires' disease antigen detected by enzyme-linked immunosorbent assay. Ann. Intern. Med. 90:697-698.
Microtiter Wells - Each kit contains 96 Microtiter Wells, coated with purified rabbit anti-Legionella pneumophila
serogroup 1 IgG. Ready for use. Store at 2-8°C.
6. Kohler, R.B., S.E. Zimmerman, E. Wilson, S.D. Allen, P.H. Edelstein, L.J. Wheat, and A. White. 1981. Rapid radioimmunoassay diagnosis of Legionnaires' Disease. Ann. Intern. Med. 94:601-605.
Wash Concentrate - One (1) vial with 40mL of 0.1 M Phosphate Buffered Saline, detergent and preservative. Dilute the contents to 400 mL with distilled or deionized water for use. Store at 2-
7. Bibb, W.F., P.M. Arnow, L. Thacker, and R.M. McKinney. 1984. Detection of soluble Legionella pneumophila
antigens in serum and urine specimens by enzyme-linked immunosorbent assay
with monoclonal and polyclonal antibodies. J. Clin. Microbiol. 20:478-482.
Positive Control Urine - One (1) vial with 2 mL of human urine containing Legionella pneumophila
serogroup 1 antigen and preservative. Ready for use. Store frozen at -10 to -20˚C in aliquots
8. Tang, P. W., and S. Toma. 1986. Broad-spectrum enzyme-linked immunosorbent assay for detection of Legionella soluble antigens. J. Clin. Microbiol. 24:556-558.
or at 2-8°C as supplied.
9. Kohler, R.B., W.C. Winn, Jr., and L.J. Wheat. 1984. Onset and duration of urinary antigen excretion in Legionnaires' disease. J. Clin. Microbiol. 20:605-607.
Negative Control Urine - One (1) vial with 2 mL of normal human urine and preservative. Ready for use. Store frozen at -10 to -20°C in aliquots or at 2-8°C as supplied.
XVIII. ADDITIONAL BIBLIOGRAPHY
HRP Conjugate - One (1) vial with 15 mL of purified rabbit anti-Legionella pneumophila
serogroup 1 IgG conjugated to horseradish peroxidase (HRP) in a Tris buffer with a protein stabiliz-
Klaucke, D.N., R.L. Vogt, D. LaRue, L.E. Witherell, L.A. Orciari, K.C. Spitalny, R. Pelletier, W.B. Cherry and L.F. Novick. 1984. Legionnaires' Disease: the epidemiology of two outbreaks
er. Contains less than 0.02% thimerosal as a preservative. Ready for use. Store at 2-8°C.
in Burlington Vermont. Am. J. Epidemiol. 119:382-391.
Color Developer - One (1) vial with 25mL of chromogenic substrate solution containing tetrarnethylbenzidine (TMB) and hydrogen peroxide. Ready for use. Store at 2-8°C.
Sathapatayavongs, B., R.B. Kohler, L.J .Wheat, A. White, W. C. Winn Jr., JC. Girod, and P.H. Edelstein. 1982. Rapid diagnosis of Legionnaires' Disease by urinary antigen detection. Am.
Stop Solution - One (1) vial with 10 mL of 1N H2S04. Store at 2-8°C.
Kohler, R.B., M.L.V. French, L.J. Wheat, P.L. Meenhorst, W.C. Winn, Jr., and P.H. Edelstein. 1985. Cross-reactive urinary antigens among patients infected with Legionella pneumophila
serogroups 1 and 4 and the Leiden 1 strain. J. of Inf. Diseases. 152:1007-1011.
Kohler, R.B., L.J Wheat. 1982. Rapid diagnosis of pneumonia due to Legionella pneumophila serogroup 1. J. of Inf. Diseases. 146:144.
Sathapatayavongs, B., R.B. Kohler, L.J. Wheat, A. White and W.C. Winn, Jr. 1983. Rapid diagnosis of Legionnaires' Disease by latex agglutination. Am. Rev. Respir. Dis. 127:559.
HANDLE URINE SPECIMENS AND POSITIVE AND NEGATIVE CONTROL URINES AS IF CAPABLE OF TRANSMITTING AN INFECTIOUS AGENT.
XIX. TECHNICAL ASSISTANCE
Control urines supplied in the kit have been autoclaved for a minimum of 15 minutes at 121°C, filtered through 0.2µm membrane filter and contain 0.8% boric acid as preservative. Althoughprocessed by these accepted methods of microorganism inactivation, it should not be assumed that this will provide full inactivation.
For further information contact Technical Services at 1-800-323-3199 or 1-207- 772-3988.
Do not pipet by mouth. Do not eat, smoke or drink in areas in which specimens or kit reagents are handled. Wear disposable gloves throughout the test procedure. Dispose of gloves in bio-
hazard waste. Thoroughly wash hands afterwards. Avoid contact with skin or mucous membranes.
851-000 Binax Legionella
Urinary Antigen EIA 96 Test Kit
Stop Solution contains sulfuric acid. Contact with skin or eyes may result in severe burns. Wear eye protection and impermeable gloves. If exposure occurs, wash with large quantities of water.
Color Developer contains N,N-Dimethylformamide as part of a mixture. May be harmful if swallowed, inhaled or absorbed through the skin.
217 Read Street Portland, Maine 04103 USA
HRP Conjugate contains an ingredient listed under California Chapter 3, Safe Drinking Water and Toxic Enforcement Act of 1986, Section 12000, Chemicals Known to Cause Cancer or
TEL: 207-772-3988 or 800-323-3199
Reproductive Toxicity. The listed ingredient is thimerosal, known to the State of California to be a reproductive toxicant.
Do not use kit components beyond expiration date. The date is printed on individual component labels and on the kit box.
Do not substitute reagents from other kit lots. Reagents are kit specific. Each kit lot has been optimized to detect Legionella pneumophila
serogroup 1 antigen directly from clinical specimens.
Dilution and alteration of reagents may result in loss of sensitivity.
Avoid microbial contamination of reagents. Microbial contamination may interfere with the sensitivity of the assay.
XIII. QUALITY CONTROL
Do not interchange vial or bottle caps and stoppers; this will lead to cross contamination of reagents.
The Positive and Negative Control Urine must be included with each run of patient specimens in order to calculate patient test results and to monitor test performance. These control samplesare ready to use as supplied, and must be tested in the same manner as patient specimens. Additionally, known positive and negative urine specimens may be run as controls.
Incubation times or temperatures other than those specified may give erroneous results.
The average absorbance of the Negative Control Urine should be less than or equal to 0.100. The average absorbance of the Positive Control Urine must be greater than or equal to three (3)
Color developer is light sensitive. Store in amber bottle. If using reagent boats, dispense only a quantity appropriate for run size, approximately 2mL for each microtiter strip.
times that of the Negative Control Urine.
Store diluted Wash Solution in a clean container to avoid contamination. Do not discard unused diluted Wash Solution until all Microtiter Wells have been utilized or passed expiration date.
Results of each replicate of the duplicate wells from patient samples, Positive Control Urine and Negative Control Urine must correspond (both score as either positive or negative in the assay).
The wash step is critical. Insufficient washing can cause erroneous results. Use of an automated washing apparatus may require additional washes. If the negative control absorbance is over
Otherwise, the test result is not valid and should be repeated.
0.100 absorbance units, increase the number of washes until the negative control absorbance is in the acceptable range (< 0.100 absorbance units).
Do not report patient test results if controls do not meet above criteria for acceptability*.
Cross contamination of patient samples could cause false results.
* Quality Control References:
Control urines, samples, and reagent solutions must be pipetted directly to the bottom of each well.
1. Federal Register, Vol. 57, No.40, February 28, 1992 (CLIA '88 Final Rule)
VII. PREPARATION OF REAGENTS
2. "Draft FDA Guidance to Manufacturers of In Vitro Analytical Test Systems for Preparation of Premarket Submissions Implementing CLIA", December 17, 1992.
All reagents must equilibrate to room temperature before use.
3. NCCLS Document C24-A, "Internal Quality Control Testing: Principles and Definitions", 1991.
Wash Solution - Dilute the entire volume (40 mL) of the Wash Concentrate to 400 mL with distilled or deionized water. Mix well. Do not discard unused diluted Wash Solution until the kit lot
XIV. CALCULATION OF RESULTS
expires or is used up. NOTE: Upon storage at 2-8°C, wash concentrate may form a crystalline precipitate. Before use, warm to room temperature and be sure all of the crystals have dissolved.
The absorbance of the well A1 (substrate blank) should be subtracted from the absorbance of each well, to obtain a corrected absorbance for use in the calculation of patient results.
Positive and Negative Control Urines - Control Urines which have been stored frozen must be thawed and mixed sufficiently before use.
Calculation of Mean Absorbance
All other reagents are supplied ready to use.
Determine the mean absorbance of the duplicate Positive Control Urine, Negative Control Urine and patient urine by:
VIII. STORAGE INSTRUCTIONS
MEAN Absorbance = Absorbance (1) + Absorbance (2)
Positive and Negative Control urines may be stored at -10 to -20°C if stored in aliquots or at 2-8°C as supplied. All other kit components should be stored at 2-8°C.
Calculation of RATIO to Negative
Color developer is light sensitive and must be stored in an amber bottle at 2-8°C.
Diluted Wash Solution may be stored at 2-30°C until the expiration of the specific kit lot in which it was a reagent.
Determine the ratio of Positive Control Urine absorbance to Negative Control Urine absorbance and of patient urine absorbance to Negative Control Urine absorbance in the following manner:
IX. INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS
RATIO = MEAN Positive Control Urine or Patient Urine Absorbance
Changes in the physical appearance of the reagents supplied may indicate instability or deterioration of these materials. Do not use reagents which are visibly turbid.
MEAN Negative Control Absorbance
Reproducible decrease in Positive Control Urine ratio results below 3.0 may indicate instability of reagents or coated microtiter wells
A typical positive result might be 0.300 Absorbance = 7.5 RATIO
X. SPECIMEN COLLECTION AND PROCESSING
Interpretation of Results
Urine specimens should be collected in standard sterile containers, stored at room temperature or refrigerated (2-8°C), and assayed within 24 hours of collection. Alternatively, specimens maybe stored at 2-8°C for up to 14 days or at -10 to -20°C for longer periods before testing. Long term storage conditions have not been determined. Boric acid may be used as a preservative.
Urine samples that have a RATIO value greater than or equal to 3 are to be considered positive for the presence of Legionella pneumophila
serogroup 1 antigen. Positive specimens may be
Other preservatives have not been evaluated.
retested to confirm results.
Whenever possible, urine specimens should be shipped in leakproof containers at 2-8°C or frozen.
Urine samples that have a RATIO value less than 3 are considered negative. A negative test result does not rule out the possibility of Legionella
infection due to other serogroups or species of
Urine specimens stored at -20 to 8°C which contain excess urates, phosphates or other dissolved salts may be found with salt crystals after storage. It is acceptable to warm these specimens to
37˚C in order to facilitate dissolving these crystals before assay.
Urine specimens exhibiting large particulates should be filtered before running in the assay, as this may indicate contamination of the specimen.
XI. MATERIALS REQUIRED BUT NOT PROVIDED
≥ 3.0 Presumptive positive for the presence of L. pneumophila
serogroup 1 antigen in urine, suggesting current or past infection.
Pipettors and/or pipets that can accurately and precisely deliver 50, 100, 200, 250 µL volumes.
Presumptive negative for L. pneumophila
serogroup 1 antigen in urine, suggesting no recent or current infection. Legionnaires' disease cannot be ruled out since other serogroupsand species may also cause disease.
Microtiter plate reader at 450 nm.
Graduated cylinder to dilute Wash Concentrate to 400 mL.
XV. LIMITATIONS OF THE TEST
Stock bottle to store diluted Wash Solution.
This assay has been validated using urine samples only. Other samples (e.g., plasma, serum or body fluids) that may contain Legionella
antigen have not been tested.
Distilled or deionized water to dilute Wash Concentrate.
Urinary Antigen EIA test will not detect infections caused by other serogroups and L. micdadei or L. longbeachae; culture is recommended for suspected pneumonia to detect
XII. ASSAY PROCEDURE
causative agents other than L. pneumophila
serogroup 1 and to confirm infection.
Reserve well A1 for substrate blank.
The diagnosis of Legionnaires' Disease cannot be based on clinical or radiological evidence alone. There is no single satisfactory laboratory test for Legionnaires' Disease. Culture results, serol-
Pipet 100 µL of well-mixed Positive Control, Negative Control and patient specimens into the appropriately labelled duplicate wells.
ogy and antigen detection methods should all be used along with clinical findings for diagnosis.
Pipet 100 µL of rabbit anti-Legionella HRP Conjugate into all previously pipetted wells, except A1.
Excretion of Legionella
antigen in urine may vary depending on the individual patient and their underlying illness or treatment. Some individuals have been shown to excrete antigen for an
Gently tap the plate to mix reagents within wells, being careful to avoid splashing and cross-contamination of wells.
extended period of time, so that a history of a recent respiratory illness compatible with Legionnaires' Disease should be sought. Early treatment with appropriate antibiotics may also decreaseantigen excretion in some individuals. Antigen excretion may begin as early as 3 days after onset of symptoms and persist for up to 1 year afterwards (9).
Incubate at room temperature (20-25°C) for 2 hours.
Antigenuria may persist for prolonged periods and may therefore indicate previous infection rather than current infection.
After incubation, decant or aspirate sample and conjugate mixture from wells. If decanting, blot strips on absorbent toweling.
Add 250 µL per well of Wash Solution. Decant or aspirate. Repeat wash step for a minimum of three washes (see Section VI, Precautions: Performance
Testing diuretic urine may affect the ability of the test to detect antigen.
XVI. EXPECTED VALUES AND PERFORMANCE CHARACTERISTICS
Pipet 200 µL of Color Developer to all previously pipetted wells, including A1 (substrate blank).
1. Expected Values
Incubate in the dark at room temperature (20-25°C) for 15 minutes. Covering the plate is sufficient.
10. After incubation, stop color development by pipetting 50 µL of stop solution to each previously pipetted well, including A1.
One hundred twenty-five randomly selected, presumed negative, uncharacterized urine specimens were tested using the Legionella
Urinary Antigen EIA kit. One hundred twenty-one specimenstested negative for the presence of Legionella
urinary antigen. The four presumed negative samples giving positive results were tested twice more. In one assay, two of the four samples tested
11. Gently tap plate to mix reagents. NOTE: All the wells should appear yellow, not green. Green color indicates insufficient mixing.
low positive and two tested negative, and in the second assay, all samples tested negative.
12. Read absorbances immediately at 450 nm on a microtiter plate reader, blanking against well A1 (substrate blank).
Distribution of Expected Values
Ratio to Negative Control
Number of Samples
APPLICATION OF THE METHODS OF PHYSICAL REHABILITATION IN THE TREATMENT OF WOMEN WITH HYPERPROLACTINEMIA Gagara V.F., Mirnaya A.І., Sakhnenko H.P. Zaporozhye National Technical University Annotation. Purpose: The results of studies of the effectiveness effects on the body of women with reproductive dysfunction and hyperprolactinemia matched set of methods of physical rehabilitation. Material/Methods: The study
CODE OF PRACTICE FOR THE CARE & HANDLING OF PIGS: REVIEW OF SCIENTIFIC RESEARCH ON PRIORITY ISSUES JULY 2012 Pig Code of Practice Scientists' Committee Harold W. Gonyou B.Sc.(Agr), M.Sc., Ph.D. (Chair) Research Scientist Prairie Swine Centre and Adjunct Professor, University of Saskatchewan Nicolas Devillers Ph.D