Human Soluble protein-100

(S-100)ELISA Kit

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Catalog No. MBS703472
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(96T)
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This immunoassay kit allows for the in vitro quantitative determination of human
S-100 concentrations in serum, plasma and other biological fluids.

Expiration date six months from the date of manufacture

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an antibody
specific to S-100. Standards or samples are then added to the appropriate
microtiter plate wells with a biotin-conjugated polyclonal antibody preparation
specific for S-100 and Avidin conjugated to Horseradish Peroxidase (HRP) is
added to each microplate well and incubated. Then a TMB (3,3',5,5'
tetramethyl-benzidine) substrate solution is added to each well. Only those
wells that contain S-100, biotin-conjugated antibody and enzyme-conjugated
Avidin will exhibit a change in color. The enzyme-substrate reaction is
terminated by the addition of a sulphuric acid solution and the color change is
measured spectrophotometrically at a wavelength of 450 nm 2 nm. The

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concentration of S-100 in the samples is then determined by comparing the
O.D. of the samples to the standard curve.

DETECTION RANGE .c
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0.16 ng/ml-10 ng/ml. The standard curve concentrations used for the ELISAs
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were 10 ng/ml, 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.62 ng/ml, 0.32 ng/ml, 0.16
ng/ml.
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SPECIFICITY
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This assay recognizes recombinant and natural human S-100. No significant

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cross-reactivity or interference was observed.

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SENSITIVITY
The minimum detectable dose of human S-100 is typically less than 0.04
ng/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as
the lowest protein concentration that could be differentiated from zero.
MATERIALS PROVIDED

Reagent Quantity
Assay plate 1
Standard 2
Sample Diluent 1 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120l
HRP-avidin 1 x 120l
1 x 20 ml

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Wash Buffer
(25concentrate)
TMB Substrate 1 x 10 ml
Stop Solution .c 1 x 10 ml
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STORAGE
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1. Unopened test kits should be stored at 2-8C upon receipt and the
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microtiter plate should be kept in a sealed bag. The test kit may be used
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throughout the expiration date of the kit, provided it is stored as prescribed

above. Refer to the package label for the expiration date.
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2. Opened test plate should be stored at 2-8C in the aluminum foil bag with
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desiccants to minimize exposure to damp air. The kits will remain stable
until the expiring date shown, provided it is stored as prescribed above.
3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical
density range of 0-3 OD or greater at 450nm wavelength is acceptable for
use in absorbance measurement.
REAGENT PREPARATION
Bring all reagents to room temperature before use.

1. Wash Buffer If crystals have formed in the concentrate, warm up to

room temperature and mix gently until the crystals have completely
dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or
distilled water to prepare 500 ml of Wash Buffer.
2. Standard Centrifuge the standard vial at 6000-10000rpm for 30s.
Reconstitute the Standard with 1.0 ml of Sample Diluent. This
reconstitution produces a stock solution of 10ng/ml. Allow the standard to
sit for a minimum of 15 minutes with gentle agitation prior to making serial

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dilutions. The undiluted standard serves as the high standard (10 ng/ml).
The Sample Diluent serves as the zero standard (0 ng/ml). Prepare fresh

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for each assay. Use within 4 hours and discard after use.
3. Biotin-antibody Centrifuge the vial before opening. Dilute to the working
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concentration using Biotin-antibody Diluent(1:100), respectively.
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4. HRP-avidin Centrifuge the vial before opening. Dilute to the working

concentration using HRP-avidin Diluent(1:100), respectively.
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Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye,
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hand, face, and clothing protection when using this material.

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OTHER SUPPLIES REQUIRED

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Microplate reader capable of measuring absorbance at 450 nm, with the

correction wavelength set at 540 nm or 570 nm.

Pipettes and pipette tips.

Deionized or distilled water.

Squirt bottle, manifold dispenser, or automated microplate washer.

An incubator which can provide stable incubation conditions up to
37C0.5C.

SAMPLE COLLECTION AND STORAGE

Serum Use a serum separator tube (SST) and allow samples to clot for
30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum
and assay immediately or aliquot and store samples at -20C. Centrifuge
the sample again after thawing before the assay. Avoid repeated
freeze-thaw cycles.

Plasma Collect plasma using citrate, EDTA, or heparin as an
anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of

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collection. Assay immediately or aliquot and store samples at -20C.
Centrifuge the sample again after thawing before the assay. Avoid
repeated freeze-thaw cycles. .c
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Note: Grossly hemolyzed samples are not suitable for use in this assay.
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ASSAY PROCEDURE
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Bring all reagents and samples to room temperature before use. It is recommended
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that all samples, standards, and controls be assayed in duplicate. All the reagents
should be added directly to the liquid level in the well. The pipette should avoid
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contacting the inner wall of the well.

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1. Add 100l of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 2 hours at 37C.
2. Remove the liquid of each well, dont wash.
3. Add 100l of Biotin-antibody working solution to each well. Incubate for 1
hour at 37C. Biotin-antibody working solution may appear cloudy. Warm
up to room temperature and mix gently until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times for a total
of three washes. Wash: Fill each well with Wash Buffer (200l) and let it
stand for 2 minutes, then remove the liquid by flicking the plate over a sink.
The remaining drops are removed by patting the plate on a paper towel.
Complete removal of liquid at each step is essential to good performance.
5. Add 100l of HRP-avidin working solution to each well. Cover the
microtiter plate with a new adhesive strip. Incubate for 1 hour at 37C.
6. Repeat the aspiration and wash three times as step 4.
7. Add 90l of TMB Substrate to each well. Incubate for 10-30 minutes at
37C. Keeping the plate away from drafts and other temperature
fluctuations in the dark.

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8. Add 50l of Stop Solution to each well when the first four wells containing
the highest concentration of standards develop obvious blue color. If color

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change does not appear uniform, gently tap the plate to ensure thorough
mixing.
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9. Determine the optical density of each well within 30 minutes, using a
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microplate reader set to 450 nm.

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CALCULATION OF RESULTS
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Using the professional soft "Curve Exert 1.3" to make a standard curve is
recommended, which can be downloaded from our web.
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Average the duplicate readings for each standard, control, and sample and
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subtract the average zero standard optical density. Create a standard curve by
reducing the data using computer software capable of generating a four
parameter logistic (4-PL) curve-fit. As an alternative, construct a standard
curve by plotting the mean absorbance for each standard on the y-axis against
the concentration on the x-axis and draw a best fit curve through the points on
the graph. The data may be linearized by plotting the log of the S-100
concentrations versus the log of the O.D. and the best fit line can be
determined by regression analysis. This procedure will produce an adequate
but less precise fit of the data. If samples have been diluted, the concentration
read from the standard curve must be multiplied by the dilution factor.

LIMITATIONS OF THE PROCEDURE

The kit should not be used beyond the expiration date on the kit label.

Do not mix or substitute reagents with those from other lots or sources.

It is important that the Standard Diluent selected for the standard curve be
consistent with the samples being assayed.

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If samples generate values higher than the highest standard, dilute the
samples with the appropriate Standard Diluent and repeat the assay.

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Any variation in Standard Diluent, operator, pipetting technique, washing
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technique, incubation time or temperature, and kit age can cause variation
in binding.
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This assay is designed to eliminate interference by soluble receptors,

binding proteins, and other factors present in biological samples. Until all
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factors have been tested in the Quantikine Immunoassay, the possibility of

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interference cannot be excluded.

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TECHNICAL HINTS
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Centrifuge vials before opening to collect contents.

When mixing or reconstituting protein solutions, always avoid foaming.

To avoid cross-contamination, change pipette tips between additions of
each standard level, between sample additions, and between reagent
additions. Also, use separate reservoirs for each reagent.

When using an automated plate washer, adding a 30 second soak period
 following the addition of wash buffer, and/or rotating the plate 180 degrees
between wash steps may improve assay precision.

To ensure accurate results, proper adhesion of plate sealers during
incubation steps is necessary.

Substrate Solution should remain colorless or light blue until added to the
plate. Keep Substrate Solution protected from light. Substrate Solution
should change from colorless or light blue to gradations of blue.

Stop Solution should be added to the plate in the same order as the
Substrate Solution. The color developed in the wells will turn from blue to
yellow upon addition of the Stop Solution. Wells that are green in color
indicate that the Stop Solution has not mixed thoroughly with the Substrate

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Solution.

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