Salmonella typhi IgM ELISA

Catalog Number SE120112

Storage Temperature 2–8 °C

TECHNICAL BULLETIN

Product Description Components

Salmonella typhi is the causative agent of typhoid fever,
a contagious infection of the intestines that affects the Materials Provided 96 Tests
whole body. In developing countries, typhoid often Microwells coated with Salmonella
occurs in epidemics. Most people in the United States 12 x 8 x 1
typhi antigen
get typhoid as a result of visiting another country where Sample Diluent: 2 bottle (ready to use) 25 mL
the food or water supply has been contaminated. Calibrator: 1 vial (ready to use) 1 mL
Symptoms usually start 1 to 3 weeks after exposure to Positive Control: 1 vial (ready to use) 1 mL
the bacteria. Symptoms include high fever, headache, Negative Control: 1 vial (ready to use) 1 mL
sore throat, vomiting, diarrhea, skin rash, and Enzyme conjugate: 1 bottle (ready to
weakness. The symptoms may take 2 weeks or more to 12 mL
use)
go away. Typhoid is spread when a person drinks or TMB Substrate: 1 bottle (ready to use) 12 mL
eats food and water contaminated by human waste
Stop Solution: 1 bottle (ready to use) 12 mL
(stool or urine) containing Salmonella typhi bacteria. A
Wash concentrate 20x: 1 bottle 25 mL
person who no longer has symptoms may still transmit
the bacteria as a carrier. Testing for immunoglobulin G
(IgG), IgA, and IgM antilipopolysaccharide (LPS) of Reagents and Equipment Required but Not
Salmonella typhi antibodies by Enzyme-Linked Provided.
Immunosorbent Assay (ELISA) showed the levels of all 1. Distilled or deionized water
three classes of immunoglobulin anti-LPS of S. typhi 2. Precision pipettes
were higher in typhoid patients than in healthy or febrile 3. Disposable pipette tips
nontyphoidal groups. The ELISA assay was much more 4. ELISA reader capable of reading absorbance at
sensitive and specific than any combination of the 450 nm
Widal test, and hence it could be a useful tool for the 5. Absorbent paper or paper towel
serologic diagnosis of typhoidal fever with a single 6. Graph paper
blood sample.
Precautions and Disclaimer
The Salmonella IgM ELISA Kit is intended for the This product is for R&D use only, not for drug,
detection of IgM antibody to Salmonella in human household, or other uses. Please consult the Safety
serum or plasma. Diluted serum (serum diluent Data Sheet for information regarding hazards and safe
contains sorbent to remove rheumatoid factor and handling practices.
human IgG interference) is added to wells coated with
purified antigen. IgM specific antibody, if present, binds Preparation Instructions
to the antigen. All unbound materials are washed away Sample Preparation
and the Enzyme Conjugate is added to bind to the 1. Collect blood specimens and separate the serum.
antibody-antigen complex, if present. Excess Enzyme 2. Specimens may be refrigerated at 2–8 °C for up to
Conjugate is washed off and TMB Substrate is added. seven days or frozen for up to six months. Avoid
The plate is incubated to allow the oxidation of the repetitive freezing and thawing.
Substrate by the enzyme. The intensity of the color
generated is proportional to the amount of IgM specific 20x Wash Buffer Concentrate
antibody in the sample. Prepare 1x Wash Buffer by adding the contents of the
bottle (25 mL, 20x) to 475 mL of distilled or deionized
water. Store at room temperature (18–26 °C).
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Storage/Stability Results
Store the kit at 2–8 °C. Calculations
1. Check Calibrator Factor (CF) value on the
Procedure calibrator bottle. This value might vary from lot to
Notes: The components in this kit are intended for use lot. Make sure the value is checked on every kit.
as an integral unit. The components of different lots 2. Calculate the cut-off value: Calibrator OD ×
should not be mixed. Calibrator Factor (CF).
3. Calculate the Ab (Antibody) Index of each
Optimal results will be obtained by strict adherence to determination by dividing the O.D. value of each
the test protocol. Precise pipetting as well as following sample by cut-off value.
the exact time and temperature requirements is
essential. Example of typical results:
Calibrator mean OD = 0.8
The test run may be considered valid provided the Calibrator Factor (CF) = 0.5
following criteria are met: Cut-off Value = 0.8 x 0.5 = 0.400
Positive control O.D. = 1.2
1. If the O.D. of the Calibrator is >0.250. Ab Index = 1.2/0.4 = 3
2. The Ab index for Negative control should be <0.9. Patient sample O.D. = 1.6
3. The Ab index for Positive control should be >1.2. Ab Index = 1.6/0.4 = 4.0

Bring all specimens and kit reagents to room Notes: To enhance sensitivity and specificity of this IgM
temperature (18–26 °C) and gently mix. test, the provided sample diluent has been formulated
to block IgG and Rheumatoid Factor (RF) interferences.
1. Place the desired number of coated strips into the
holder. Turbidity could be seen after diluting serum with sample
2. Negative control, positive control, and calibrator are diluent. This turbidity is due to the blocking of serum
ready to use. Prepare 101-fold dilution of test IgG and has shown no interference with test results. It
samples by adding 5 µL of the sample to 0.5 mL of can be removed by centrifugation.
Sample Diluent. Mix well.
3. Dispense 100 µL of diluted sera, calibrator, and In specimens with high RF and high autoimmune
controls into the appropriate wells. For the reagent antibodies, the possibility of eliminating the
interferences cannot be ruled out entirely.
blank, dispense 100 µL of Sample Diluent in 1A well
position. Tap the holder to remove air bubbles from
Lipemic or hemolyzed samples may cause erroneous
the liquid and mix well. Incubate for 20 minutes at
results.
room temperature (18–26 °C).
4. Remove liquid from all wells. Wash wells three
Interpretation
times with 300 µL of 1x Wash Buffer. Blot on The following is intended as a guide to interpretation of
absorbent paper or paper towel. Salmonella typhi IgM antibody index (Ab Index) test
5. Dispense 100 µL of Enzyme Conjugate to each well results; each laboratory is encouraged to establish its
and incubate for 20 minutes at room temperature own criteria for test interpretation based on sample
(18–26 °C). populations encountered.
6. Remove Enzyme Conjugate from all wells. Wash
wells three times with 300 µL of 1x Wash Buffer. <0.9 – No detectable antibody to S. typhi IgM by ELISA
Blot on absorbent paper or paper towel. 0.9–1.1 – Borderline positive. Follow-up testing is
7. Dispense 100 µL of TMB Substrate and incubate for recommend if clinically indicated.
10 minutes at room temperature (18–26 °C). >1.1 – Detectable antibody to S. typhi IgM by ELISA
8. Add 100 µL of Stop Solution.
9. Read O.D. at 450 nm using ELISA reader within
15 minutes. A dual wavelength is recommended
with reference filter of 600–650 nm.
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References
1. Quiroga, T. et al., Diagnosis of typhoid fever by two 3. Mekara, Y. et al., Determination of antibody from
serologic methods. Enzyme-linked immunosorbent typhoid patients against lipopolysaccharide and
assay of antilipopolysaccharide of Salmonella typhi protein antigens of Salmonella typhi. Asian Pac. J.
antibodies and Widal test. Diagn. Microbiol. Infect. Allergy Immunol., 1990; 8(2):95-101.
Dis., 1992; 15(8):651-6. 4. Sippel, J.E. et al., Serodiagnosis of typhoid fever in
2. Jesudason, M.V. et al., Diagnosis of typhoid fever pediatric patients by anti-LPS ELISA. Trans. R.
by the detection of anti-LPS & anti-flagellin Soc. Trop. Med. Hyg., 1987; 81(6):1022-6.
antibodies by ELISA. Indian J. Med. Res., 5. Vitale, G. et al., An ELISA method in the diagnosis
1998;107:204-7. of typhoid fever. J. Clin. Lab. Immunol., 1990;
31(4):195-9.

SG,CH,MAM 10/14-1

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