Download Candida Albicans (Ab) IgM (EIA-1743) DRG International Inc., USA

Candida Albicans (Ab) IgM (EIA-1743)
Intended Use
The Candiquant-IgM Test measures Candida albicans specific serum IgM antibodies. It is intended for in vitro diagnostic
use only.
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Summary And Explanation
Systemic Candidiasis is a fungal infection resulting from the overgrowth and spread of Candida (yeast) into body tissues (1,2).
It is an important cause of mortality among patients who are compromised immunologically or undergoing prolonged antibiotic
therapy (3,4,5). The passage of C. albicans pathogens through the gastrointestinal (GI) mucosa into the host's bloodstream is
believed to be an important mechanism leading to systemic Candidiasis (6,7,8). Symptoms are varied and may include migraine
headaches, depression, urogenital dysfunction, intestinal gas and allergies. In more extreme cases, the patient is totally
incapacitated (1,2,11,12,13).
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Studies indicate that a number of fungal pathogens can spread systemically from intestinal lumen to visceral organs (8,9,10).
Although there are over 100 species of Candida, only seven species are isolated with any frequency from medical specimens.
Two of these, C. albicans and C. tropicalis account for more than 80% of isolated yeast species. C. albicans is a part of the
human microflora and it is commonly known that its overgrowth can upset delicate balance and may manifest in the form of
oral and/or vaginal thrush. It may release mycotoxins or other products that affect the host's cellular immune system (14,15).
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Candida cytoplasmic antigens have been detected in patients with systemic Candidiasis, cancer, and those receiving therapies
which alter the host’s defense mechanisms. Diagnosis of this condition has been limited to medical history, diet diaries and
questionnaires (1,14,16). Many have used blood cultures as a diagnostic tool, but have found that it is unreliable with 40-60%
false negatives
as confirmed by visceral organ infection. Positive blood cultures become evident only at terminal stages. Throat and/or vaginal
cultures are commonly found to be positive (14,15), and thus patients with polysystemic Candidiasis cannot be identified easily.
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The passage of C. albicans and/or its cytoplasmic antigens through the GI mucosa into the host's blood system will stimulate
the host's humoral immune system, resulting in the host’s production of anti-C. albicans antibodies in their circulatory system.
In general, soon after antigen-crossover, the host’s immune response will produce IgM antibodies specific to C. albicans,
followed by an increase in specific IgG antibodies. Infection of the mucosal membranes may lead to the presence of a high
titer of IgA antibodies specific to C. albicans. The presence of an abnormally high titer of any anti-Candida antibody is a
good indicator of host immune response to C. albicans infection.
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The Candiquant Test uses ELISA technology and is designed for the measurement of specific antibodies: IgM, IgG and IgA
produced by the host against the C. albicans antigen. A high titer of Candida specific IgM may indicate the early onset of
Candida infection. A moderate titer of Candida specific IgG may indicate that the patient has experienced an overgrowth of
Candida in the past. A high titer of Candida specific IgG may indicate an active or prolonged infection. A high titer of
Candida specific IgA may indicate an active infection of the mucosal membranes (17).
Three quantitative procedures are available from DRG for the detection of C. albicans: Specific IgG, specific IgM, and
specific IgA. These test procedures may also be used to monitor the effectiveness of therapy. The following applies to the
detection and quantification of C. albicans specific serum IgM.
Principle Of The Test
C. albicans cytoplasmic antigens are purified and then immobilized onto microwells. The Calibrators, Controls, and a dilution of
the Serum Sample are added into the appropriate wells. The human antibodies in the serum sample are allowed to react
immunologically at room temperature with the immobilized C. albicans antigens. After washing off the unbound materials, the
bound antibodies are quantified by an enzyme-labelled second antibody specific to human IgM. A substrate is added, and the
color generated is read spectrophotometrically. The intensity of the color is directly proportional to the concentration (or titer)
of the C. albicans-specific serum IgM.
DRG International Inc., USA
Fax: (908) 233 0758 e-mail: [email protected]
Candida Albicans (Ab) IgM (EIA-1743)
Warnings And Precautions
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WARNING
Potential Biohazardous Material
The matrix of the Calibrators and reference sera is human serum. The human serum used has been found negative for
HIV antibodies as well as for Hepatitis B surface antigen when tested with FDA licensed reagents. However, as there
is no test method that can offer complete assurance that HIV, hepatitis B virus or other infectious agents are absent,
these reagents should be handled at the Biosafety Level 2, as recommended for any potentially infectious human serum
or blood specimen in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological
and Biomedical Laboratories," 1984.
The microwell strips do not contain viable C. albicans organisms. However, the strips should be considered potentially
infectious and handled at the Biosafety Level 2.
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Sodium Azide
The reagents contain sodium azide as a preservative. Sodium azide may react with lead, copper or brass to form explosive
metal azides. When disposing of these materials, always flush with large volumes of water to prevent azide buildup.
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Procedural Notes
1. Strict adherence to the specified time and temperature of incubations is essential for accurate results.
2. Do not freeze reagents.
3. Store all components of the test kit at 2-8°C at all times. Do not allow reagents to stand at room temperature for extended
periods of time.
4. Positive and Negative Controls must be run each time the test is performed.
5. Do not mix reagents from different lots.
6. Do not use expired reagents.
7. Use only clear sera as test specimens. The samples should not have gross turbidity, hemolysis or any microbial contamination.
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Additional Materials Required but Not Supplied
1. Micropipet with disposable tips to deliver 25 µl and 100 µl.
2. For washing, use either a microtiter plate washer (automated or manual), or a squeeze wash bottle.
3. Distilled or deionized water.
4. 5 ml pipet for sample dilution and 10 ml pipet for substrate buffer delivery.
5. A 500 ml and a 1000 ml graduated container.
6. Microtiter plate reader with 450 nm wavelength absorbance capability.
7. Absorbent paper towels.
8. Test tubes (13 x 100 mm) for serum dilution.
9. Parafilm or plastic wrap.
Kit Components
1. 12 x 8 microwell strips coated with inactivated C. albicans antigens. The strips are packaged in a twelve-strip holder, sealed
in a ziplock bag. Reseal carefully any unused strips.
2. Candida albicans IgM Calibrators 0 through 4, human serum base. (5 x 1.0 ml vials).
3. Candida IgM Negative Control (1 x 1.0 ml).
4. Candida IgM Positive Control (1 x 1.0 ml).
5. Anti Human IgM-HRP Conjugate (1 x 10.0 ml).
6. Substrate Solution A (1 x 8.0 ml).
DRG International Inc., USA
Fax: (908) 233 0758 e-mail: [email protected]
Candida Albicans (Ab) IgM (EIA-1743)
7.
8.
9.
10.
Substrate Solution B (1 x 8.0 ml).
Serum Sample Diluent (concentrate) (1 x 20.0 ml).
Washing Buffer (concentrate). Phosphate buffered saline with Tween (1 x 20.0 ml vial).
Stopping Solution (1N HCl) (1 x 6.0 ml bottle).
Serum diluent, calibrators and controls contain 0.01% sodium azide as a preservative.
Store kit at 2-8°C.
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Specimen Collection
Collect venous blood by venipuncture into a glass vaccutainer (red top) tube. Separate the serum by centrifugation. Serum
samples may be stored refrigerated (2-8°C) for up to 10 days. If the sample cannot be tested during this period, store it frozen
at -20°C. Excess hemolysis, lipemia, the presence of large clots, or visible indications of bacterial growth in the specimen, may
interfere with the performance and accuracy of the test.
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Reagent Preparation And Storage
1. Serum Sample Diluent Buffer (1:25 Dilution):
Add the entire contents of the vial into a 500 ml container with 480 ml of distilled water. Mix thoroughly. Label the
container as "Working Sample Diluent Buffer", and store at 2-8°C until use. The reconstituted reagent is stable for the
shelf-life of the kit when stored at 2-8°C. Use 5 ml for each patient sample. Rinse out any crystals from the vial which
may be present.
2. Washing Buffer (1:50 Dilution):
Add the entire contents of the vial into 980 ml of distilled or deionized water in a 1000 ml container. Mix thoroughly. Label
the container as "Working Washing Buffer" and store at 2-8°C until use. The "Working Washing Buffer" is stable for
the shelf-life of the kit when stored at 2-8°C. Rinse out any crystals from the vial which may be present.
3. Substrate Reagent:
Mix Substrate Solution A and Substrate Solution B in the ratio of 1:1 and label as Working Substrate. Prepare the Working
Substrate solution within one hour prior to use. Prepare only the required volume. For example for six microwell strips mix
2.5 ml of Substrate Solution A with 2.5 ml of Substrate Solution B.
4. Storage of Remaining Kit Components
C. albicans antigen coated strips, Calibrators and Controls, Substrate Solutions and Stopping Solution should be stored at 28°C and are stable until the kit expiration date.
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Assay Procedure
The test kit contains 12 microwell strips. Assemble the microwell strips (the number depends on the number of specimen
samples to be tested) into the left side of the strip holder. Return unused strips to the original ziplock bag and store at 2-8°C.
NOTE:
Sample Dilution: Accurately pipet 25 µl of each patient serum to 5 ml of Serum Sample Diluent Buffer (see #1 of the above)
in a 13 x 100 mm test tube (1:200 dilution). Mix thoroughly by inversion or vortexing. Appropriately label all tubes containing
diluted test samples.
Well Positions for the Assay:
A1,Bl
C1,D1
E1,F1
G1,H1
A2,B2
C2,D2
E2,F2
Calibrator 0
Calibrator 1
Calibrator 2
Calibrator 3
Calibrator 4
Negative Control
Positive Control
DRG International Inc., USA
Fax: (908) 233 0758 e-mail: [email protected]
Candida Albicans (Ab) IgM (EIA-1743)
G2,H2
A3,B3
Patient Sample #1
Patient Sample #2
1. Serum Incubation
Into the appropriate wells, as shown in the above diagram, dispense 100 µl of Calibrators 0 through 4, Negative Control,
Positive Control, and diluted patient serum samples in duplicate.
There should be 100 µl of solution in each microwell at this point.
Cover the plate with parafilm and let it stand for 1 hour at room temperature (22-26°C).
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2. Wash Procedure
After a one hour incubation, discard the contents of all the wells into the sink by quick decantation. Blot the plate dry with
a paper towel. If you use an automatic or manual plate washer, wash each well three times with about 300 µl of washing
buffer according to the instrument manufacturer's instructions. If you use a squeeze bottle, fill it with washing buffer.
Carefully fill the wells up one by one with the washing buffer by squeezing the bottle (avoid air bubbles in the well during
washing), discard the washing buffer, blot dry with a paper towel, and then repeat the procedure two more times, making
sure that the plate is blotted dry each time between washes.
3. Conjugate Incubation
Add 100 µl of Anti-Human IgM-HRP Enzyme Conjugate reagent to all the microwells.
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Cover the plate (as in step # 1c) and let it stand for 30 minutes at room temperature (22-26°C).
5. Substrate Incubation
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4. Wash (same as step #2).
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Add 100 µl of the "Working" Substrate Reagent (see "Reagent Preparation" Section, #3), into all the wells.
Cover the plate, as in step #1c and let it stand in the dark at room temperature (22-26°C) for 10 minutes.
6. Stop Reaction/Read Absorbance
After exactly 10 minutes, add 50 µl of Stopping Solution to all the wells.
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Set the microplate reader to read at a wavelength of 450 nm and measure the optical density (O.D.) of each well. The
plate should be read within one hour after addition of the stop solution.
Calculaton Of Data
1. Read the O.D. of the Calibrators, Controls and Patient Samples and record the data as shown in Table #1.
2. Calculate the mean O.D. of calibrators, controls and all the samples (see Table #1).
3. Construct a calibration curve on linear graph paper using the mean O.D. on the Y axis and the calibrator value on the X
axis.
4. Interpolate the patient values from the calibration curve.
DRG International Inc., USA
Fax: (908) 233 0758 e-mail: [email protected]
Candida Albicans (Ab) IgM (EIA-1743)
0.025
0.586
0.899
1.398
1.932
0.175
0.037
0.596
0.913
1.410
1.940
0.183
0.031
0.591
0.906
1.404
1.936
0.179
0.0
12.5
25.0
50.0
100.0
3.0
1.086
0.510
1.291
1.096
0.516
1.309
1.091
0.513
1.300
30.5
10.5
44.0
Quality Control
1. The O.D. of the Standard 0 must be < 0.20.
2. Negative Control should read less than 10 units/ml
3. Positive Control should read 25 - 50 units/ml.
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Value*
U/ml
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Calib. 0
Calib. 1
Calib. 2
Calib. 3
Calib. 4
Negative
Control
Positive Control
Pat. Spl. 1
Pat. Spl. 2
Mean
O.D.
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Table 1
Sample Calculations:
I.D.
O.D.
Duplicates
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Interpretation
Positive Result: Patient samples with values greater than or equal to 25 units/ml are considered to be positive for the presence
of IgM specific antibodies to C. albicans.
Negative Result: Patient samples with values less than or equal to 12.5 units/ml should be considered negative for the presence
of IgM specific antibodies to C. albicans.
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Equivocal Result: Patient samples with values falling between 12.5 and 25 units/ml are equivocal. Patients with equivocal
results should be redrawn after two weeks, and the second sample run together with the first sample.
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Expected Values
The antibodies to Candida albicans are found in 20-30% of the normal population. A clinical correlation must exist for the
test to be useful. The titer of the antibody along with the symptomatic evidence has been taken into consideration in
determining the cut-off point in Candiquant-IgM.
Some conditions predisposing to candidiasis are:
Pregnancy
Diabetes mellitus
Chronic debilitating diseases
Prolonged therapy with broad spectrum antibiotics
AIDS (virtually 100% of AIDS patients have very high titers of the antibodies)
Limitations
1. The Candiquant-IgM ELISA is a qualitative test to detect the presence of IgM specific antibodies to C. albicans and does
not indicate the titer of the antibody.
2. A positive test result should only be used as an aid to the detection of disease along with other clinical tests such as
Histology and Culture.
3. A negative test result does not preclude the absence of antibodies to C. albicans. The infection may be ther,e but may be
DRG International Inc., USA
Fax: (908) 233 0758 e-mail: [email protected]
Candida Albicans (Ab) IgM (EIA-1743)
in its very early stages, or the antibody titer may be too low for the test to detect.
4. If the clinical symptoms and test results are inconclusive, additional testing should be done on a subsequent sample.
5. The Candiquant-IgM ELISA test should be used only for patients reporting clinical signs and symptoms related to
candidiasis. The test should not be used for patients who are asymptomatic.
Precision
1. Intra-assay precision was determined by measuring the units/ml of 12 replicates of confirmed Low, Medium and High
antibody positive C. albicans patient samples.
3.00
7.9%
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1.18
5.7%
High
12
53.17
6.51
12.3%
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S.D.
C.V.
Medium
12
38.11
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Low
Number of determinations:12
Mean value:
20.67
2. Inter-assay variability of the test for sample results was calculated from values obtained from confirmed Low, Medium and
High antibody positive C. albicans patient samples over a period of five months, using three different operators.
High
10
50.54
9.25
18.31%
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Medium
10
38.54
5.91
15.3%
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Low
Number of determinations:10
Mean value:
20.61
S.D.
3.70
C.V.
17.9%
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Clinical Significance
A high IgG antibody titer against cytoplasmic antigens of C. albicans may be indicative of past or ongoing infection. A high
IgM titer indicates current infection. High IgG and IgM antibody titers against cytoplasmic antigens of C. albicans is an even
better indicator of current infection. High IgA antibody titers against cytoplasmic C. albicans are indicative of mucosal or
vaginal Candidiasis.
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THIS IS A SCREENING TEST ONLY AND LEVELS OF ANTI-CANDIDA SERUM IgM SHOULD BE
ASSESSED IN THE LIGHT OF THE PATIENT'S MEDICAL HISTORY. Moreover, in chronically ill patients,
assessment for total immune function is recommended. Immuno-suppressive drugs, steroids and fungiostatic
medications may alter test results.
References
1. Crook, W.G. The Yeast Connection: A Medical Breakthrough. P.O. Box 3494, Jackson, TN 38301. Professional Books,
1983.
2. Truss, C.O. The Missing Diagnosis. P.O. Box 26508, Birmingham, AL 35226, 1983.
3. Meunier-Carpenter, F., T.E. Kiehn, D. Armstrong. Fungaemia in the immunocompromised host: Changing patterns of
antigenema high mortality. Amer. J. Med., 71:363-370, 1981.
4. Ray, T.L. Fungal infections in the immunocompromised host. Med. Clin. No. Amer., 64:955-968, 1980.
5. Wingard, J.R., W.G. Merz, R. Saral. Candida tropicalis: A major pathogen in immunocompromised patients. Ann. Int.
Med., 91:539- 543, 1979.
DRG International Inc., USA
Fax: (908) 233 0758 e-mail: [email protected]
Candida Albicans (Ab) IgM (EIA-1743)
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6. Krause, W., H. Mathesis, K. Wulf. Fungaemia and Funguria after oral administration of Candida albicans, i:598-599,
1969.
7. Stone, H.H., C.E. Geheber, L.D. Kolb, W.R. Kitchens. Alimentary tract colonization by Candida albicans. J. Surg.
Res,. 14:273- 276, 1973.
8. Stone, H.H., L.D. Kolb, C.H. Currie, C.E. Geheber, J.F. Cuzzell. Candida sepsis; pathogenesis and principles of
treatment. Ann. Surg., 179:697-711, 1974.
9. Wingard, J.R., J.D. Dick, W.G. Merz, G.R. Sanford, A. Saral, W.H. Burns. Pathogenicity of Candida tropicalis and
Candida albicans after gastrointestinal inoculation in mice. Infect. Immuno.,. 29:808-813, 1980.
10. Haupt, H.M., W.G. Merz, W.E. Bochorner, W.P. Vaughan, R. Saral. Colonization and infection with Trichosporon species
in the immunosuppressed host. J. Infect. Dis., 147:199-203, 1983.
11. Palacios, H.J. Hypersensitivity as a cause of dermatologic and vaginal moniliasis resistant to topical therapy. Ann.
Allergy, 37:110-113, 1976.
12. Robinet, R.W. Asthma due to Candida albicans. Univ. Mich. Med. Ctr. Bull,. 34:12-15.
13. Iwata, K. A review of the literature on drunken symptoms due to yeast in the gastrointestinal tract. In "Proceedings of the
Second International Specialized Symposium on Yeast". Univ. of Tokyo Press, pp 260-268, 1972.
14. Witkins, S.S., I.R. Yu, W.J. Ledger. Inhibition of
Candida albicans: Induced lymphocytes proliferation by lympho-cytes and sera from woman with recurrent vaginitis.
Amer. J. Obstet. Gynecol., 147:809-811, 1983.
15. Witkins, S.S. Defective responses in patients with current Candidiasis infection. Medicine, May, 129, 1985.
16. Araj, G.F., R.L. Hopfer, S. Chestnut, V. Fainstein, G.P. Boday. Diagnostic value of ELISA for detection of Candida
albicans cytoplasmic antigens in sera of
cancer patients. J. Clin. Microbiol., 16:46-59, 1982.
17. Wojdani, A., M. Ghoueum, G. Cheung. Measurement of humoral and cellular immunity for the diagnosis of Candidiasis.
Clin. Ecol. 4:210, 1986.
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B.M./11-97
DRG International Inc., USA
Fax: (908) 233 0758 e-mail: [email protected]