Thermo Fisher Scientific Human sCD44var User Guide
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USER GUIDE
Human sCD44var (v6) ELISA Kit
Enzyme-linked Immunosorbent Assay for quantitative detection of human CD44var (v6)
Catalog Number
BMS210
Pub. No.
MAN0016547
Rev.
A.0 (30)
WARNING!
Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
The Human sCD44var (v6) ELISA Kit is an enzyme-linked immunosorbent assay for the quantitative detection of human
CD44var (v6).
Summary
CD44 (Pgp-1; Ly-24; ECMR III; F10-44-2; H-CAM; HUTCH-I; In(Lu)related p80; Hermes antigen; hyaluronan receptor) is a polymorphic glycoprotein that participates in a wide variety of cell-cell or cellmatrix interactions including lymphocyte homing, establishment of Band T-cell immune responses, tumor metastasis formation, and inflammation.
Three isoform categories of the CD44 molecule have been identified:
1. a predominant 80-90 kDa category, the so-called standard form named C44std,
2. an intermediate size category of 110-160 kDa, and
3. a category which includes very large isoforms of 250 kDa covalently modified by the addition of chondroitin sulfate.
This CD44-family of transmembrane receptor molecules is derived from a single gene located on chromosome 11. Alternative splicing of the mRNA gives rise to the different isoforms, containing inserts of varying sizes in the extracellular domain of the molecule (exons v2v10). All CD44 isoforms are variably glycosylated. In contrast to standard CD44 (CD44std) which is almost ubiquitously expressed, the variety of CD44 isoforms (CD44var) have a much more restricted distribution, e.g., on keratinocytes (exons v3-v10), ephitelial cells
(exons v8-v10), activated lymphocytes and macrophages (exon v6).
A splice variant of CD44 (exons v4-v7) confers metastatic behaviour in a rat carcinoma model; aberrant expression of splice variants has been detected on a variety of human tumor cell lines as well as primary and metastatic human tumors, including lymphomas, carcinomas (colon, thyroid, mamma, bladder), and gliomas. Detection of abnormal regulation of CD44 splicing thus could be helpful in cancer diagnosis and disease evaluation.
The sCD44var (v6) ELISA detects all circulating CD44 isoforms comprising the sCD44var (v6) sequences.
Determination of sCD44var (v6) will provide more detailed insight into different pathological modifications during cancer and other diseases.
For literature update refer to our website.
Principles of the test
An anti-human CD44var (v6) coating antibody is adsorbed onto microwells.
Coating Antibody
Fig. 1 Coated microwell
Human CD44var (v6) present in the sample or standard binds to antibodies adsorbed to the microwells and the HRP-conjugated antihuman CD44var (v6) antibody is added and binds to human CD44var
(v6) captured by the first antibody.
Standard or Sample
HRP-Conjugate
Fig. 2 First incubation
Following incubation unbound HRP-conjugated anti-human CD44var
(v6) is removed during a wash step, and substrate solution reactive with HRP is added to the wells.
For Research Use Only. Not for use in diagnostic procedures.
Substrate
Fig. 3 Second incubation
A colored product is formed in proportion to the amount of human
CD44var (v6) present in the sample or standard. The reaction is terminated by addition of acid and absorbance is measured at 450 nm.
A standard curve is prepared from 6 human CD44var (v6) standard dilutions and human CD44var (v6) concentration determined.
- Reacted Substrate
Fig. 4 Stop reaction
Reagents provided
Reagents for human CD44var (v6) ELISA BMS210 (96 tests)
1 aluminum pouch with a Microwell Plate (12 strips of 8 wells each) coated with monoclonal antibody to human CD44var (v6)
2 vials (10 µL) HRP-Conjugate anti-human CD44var (v6) monoclonal antibody
2 vials human CD44var (v6) Standard lyophilized, 20 ng/mL upon reconstitution
1 vial (50 mL) Sample Diluent
1 vial (5 mL) Assay Buffer Concentrate 20x (PBS with 1% Tween ™
10% BSA)
20,
1 bottle (50 mL) Wash Buffer Concentrate 20x (PBS with 1% Tween ™
20)
1 vial (15 mL) Substrate Solution (tetramethyl-benzidine)
1 vial (15 mL) Stop Solution (1M Phosphoric acid)
2 Adhesive Films
Storage instructions – ELISA kit
Store kit reagents between 2°C and 8°C. Immediately after use remaining reagents should be returned to cold storage (2°C to 8°C).
Expiry of the kit and reagents is stated on labels.
Expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, this reagent is not contaminated by the first handling.
Sample collection and storage instructions
Cell culture supernatant, serum, plasma (EDTA, citrate, heparin) and urine were tested with this assay. Other biological samples might be suitable for use in the assay. Remove serum or plasma from the clot or cells as soon as possible after clotting and separation.
Pay attention to a possible Hook Effect due to high sample
concentrations (see “Calculation of results“ on page 4).
Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic samples.
Samples should be aliquoted and must be stored frozen at –20°C to avoid loss of bioactive human CD44var (v6). If samples are to be run within 24 hours, they may be stored at 2°C to 8°C (for sample stability
refer to “Sample stability“ on page 6).
Avoid repeated freeze-thaw cycles. Prior to assay, the frozen sample should be brought to room temperature slowly and mixed gently.
Materials required but not provided
• 5 mL and 10 mL graduated pipettes
• 5 µL to 1000 µL adjustable single channel micropipettes with disposable tips
• 50 µL to 300 µL adjustable multichannel micropipette with disposable tips
• Multichannel micropipette reservoir
• Beakers, flasks, cylinders necessary for preparation of reagents
• Device for delivery of wash solution (multichannel wash bottle or automatic wash system)
• Microwell strip reader capable of reading at 450 nm (620 nm as optional reference wave length)
• Glass-distilled or deionized water
• Statistical calculator with program to perform regression analysis
Precautions for use
• All reagents should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice.
• Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures.
• Do not mix or substitute reagents with those from other lots or other sources.
• Do not use kit reagents beyond expiration date on label.
• Do not expose kit reagents to strong light during storage or incubation.
• Do not pipet by mouth.
• Do not eat or smoke in areas where kit reagents or samples are handled.
• Avoid contact of skin or mucous membranes with kit reagents or samples.
• Rubber or disposable latex gloves should be worn while handling kit reagents or samples.
• Avoid contact of substrate solution with oxidizing agents and metal.
• Avoid splashing or generation of aerosols.
• To avoid microbial contamination or cross-contamination of reagents or samples that may invalidate the test, use disposable pipette tips and/or pipettes.
• Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagent.
• Exposure to acid inactivates the conjugate.
• Glass-distilled water or deionized water must be used for reagent preparation.
• Substrate solution must be at room temperature prior to use.
• Decontaminate and dispose samples and all potentially contaminated materials as if they could contain infectious agents.
The preferred method of decontamination is autoclaving for a minimum of 1 hour at 121.5°C.
• Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0% sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite.
2 Human sCD44var (v6) ELISA Kit User Guide
Preparation of reagents
1.
Buffer Concentrates should be brought to room temperature and should be diluted before starting the test procedure.
2.
If crystals have formed in the Buffer Concentrates, warm them gently until they have completely dissolved.
Wash buffer (1x)
1.
Pour entire contents (50 mL) of the Wash Buffer Concentrate (20x) into a clean 1000 mL graduated cylinder. Bring to final volume of
1000 mL with glass-distilled or deionized water.
2.
Mix gently to avoid foaming.
3.
Transfer to a clean wash bottle and store at 2°C to 25°C. Please note that Wash Buffer (1x) is stable for 30 days.
4.
Wash Buffer (1x) may also be prepared as needed according to the following table:
Number of Strips
1–6
1–12
Wash Buffer
Concentrate (20x)
(mL)
25
50
Distilled Water (mL)
475
950
Assay buffer (1x)
1.
Pour the entire contents (5 mL) of the Assay Buffer Concentrate
(20x) into a clean 100 mL graduated cylinder. Bring to final volume of 100 mL with distilled water. Mix gently to avoid foaming.
2.
Store at 2°C to 8°C. Please note that the Assay Buffer (1x) is stable for 30 days.
3.
Assay Buffer (1x) may also be prepared as needed according to the following table:
Number of Strips
1–6
1–12
Assay Buffer
Concentrate (20x)
(mL)
2.5
5.0
Distilled Water (mL)
47.5
95.0
HRP-Conjugate
Note: The HRP-Conjugate should be used within 30 minutes after dilution.
1.
Dilute the HRP-Conjugate just prior to use by adding 760 µL
Assay Buffer (1x) to the tube containing the HRP-Conjugate concentrate. Mix the contents of the tube well.
2.
Make a further 1:100 dilution with Assay Buffer (1x) in a clean plastic tube or reagent reservoir.
3.
The second dilution (1:100) of the HRP-Conjugate may be prepared as needed according to the following table:
Number of Strips
1–6
1–12
Prediluted HRP-
Conjugate (mL)
0.03
0.06
Assay Buffer (1x)
(mL)
2.97
5.94
Human CD44var (v6) standard
1.
Reconstitute human CD44var (v6) standard by addition of distilled water.
2.
Reconstitution volume is stated on the label of the standard vial.
Swirl or mix gently to insure complete and homogeneous solubilization (concentration of reconstituted standard =
20 ng/mL).
3.
Allow the standard to reconstitute for 10 to 30 minutes. Mix well prior to making dilutions.
The standard has to be used immediately after reconstitution and cannot be stored.
External standard dilution
1.
Label 6 tubes, one for each standard point: S1, S2, S3, S4, S5, S6.
2.
Prepare 1:2 serial dilutions for the standard curve as follows:
Pipette 225 µL of Sample Diluent into each tube.
3.
Pipette 225 µL of reconstituted standard (concentration of standard = 20 ng/mL) into the first tube, labeled S1, and mix
(concentration of standard 1 = 10 ng/mL).
4.
Pipette 225 µL of this dilution into the second tube, labeled S2, and mix thoroughly before the next transfer.
5.
Repeat serial dilutions 4 more times thus creating the points of
the standard curve (see Figure 5).
Sample Diluent serves as blank.
Transfer 225 µL
S1 S2 S3
Reconstituted
Human CD44var (v6)
Standard
Sample Diluent
225 µL
Fig. 5 Dilute standards - tubes
S4 S6
Discard
225 µL
Test protocol
Note: In case of incubation without shaking, the obtained O.D. values may be lower than indicated below. Nevertheless the results are still valid.
1.
Predilute your samples before starting with the test procedure.
Dilute serum, plasma and urine samples 1:20 with Sample
Diluent according to the following scheme: 10 µL sample +
190 µL Sample Diluent.
2.
Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Each sample, standard, blank and optional control sample should be assayed in duplicate. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2°C to 8°C sealed tightly.
3.
Wash the microwell strips twice with approximately 400 µL Wash
Buffer per well with thorough aspiration of microwell contents between washes. Allow the Wash Buffer to sit in the wells for about 10–15 seconds before aspiration. Take care not to scratch the surface of the microwells.
After the last wash step, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the microwell strips immediately after washing. Alternatively, microwell strips can be placed upside down on a wet absorbent paper for not longer than 15 minutes. Do not allow wells to dry.
Human sCD44var (v6) ELISA Kit User Guide 3
4.
Standard dilution on the microwell plate (alternatively, the
standard dilution can be prepared in tubes, see “External standard dilution“ on page 3):
Add 100 µL of Sample Diluent in duplicate to standard wells
A1/2–F1/2. Pipette 100 µL of prepared standard (see “Human
CD44var (v6) standard“ on page 3, concentration = 20.00 ng/mL)
in duplicate into well A1 and A2 (see Table 1). Mix the contents of
wells A1 and A2 by repeated aspiration and ejection
(concentration of standard 1, S1 = 10.00 ng/mL), and transfer
100 µL to wells B1 and B2, respectively (see Figure 6). Take care
not to scratch the inner surface of the microwells. Continue this procedure 4 times, creating two rows of human CD44var (v6) standard dilutions ranging from 10.00 to 0.32 ng/mL. Discard
100 µL of the contents from the last microwells (F1, F2) used.
Transfer 100 µL
S1 S2 S3 S4 S6
Reconstituted
Human CD44var (v6)
Standard
Sample Diluent
100 µL
Fig. 6 Dilute standards - microwell plate.
Discard
100 µL
(S1–S6) in the standard wells according to Table 1.
Table 1 Example of the arrangement of blanks, standards, and samples in the microwell strips.
A
B
C
D
1
Standard 1
10.00 ng/mL
Standard 2
5.00 ng/mL
Standard 3
2.50 ng/mL
Standard 4
1.25 ng/mL
2
Standard 1
10.00 ng/mL
Standard 2
5.00 ng/mL
Standard 3
2.50 ng/mL
Standard 4
1.25 ng/mL
3
Sample 2
Sample 3
Sample 4
Sample 5
4
Sample 2
Sample 3
Sample 4
Sample 5
E
F
Standard 5
0.63 ng/mL
Standard 6
0.32 ng/mL
Blank
Standard 5
(0.63 ng/mL)
Standard 6
0.32 ng/mL
Blank
Sample 6
Sample 7
Sample 6
Sample 7
G Sample 8 Sample 8
H Sample 1 Sample 1 Sample 9 Sample 9
5.
Add 100 µL of Sample Diluent in duplicate to the blank wells.
6.
Add 80 µL of Sample Diluent to the sample wells.
7.
Add 20 µL of each sample in duplicate to the sample wells.
8.
Prepare HRP-Conjugate (see “HRP-Conjugate“ on page 3).
9.
Add 50 µL of HRP-Conjugate to all wells.
10.
Cover with an adhesive film and incubate at room temperature
(18°C to 25°C) for 3 hours on a microplate shaker.
11.
Remove adhesive film and empty wells. Wash microwell strips
3 times according to Step 3 of the test protocol. Proceed immediately to the next step.
12.
Pipette 100 µL of TMB Substrate Solution to all wells.
13.
Incubate the microwell strips at room temperature (18°C to 25°C) for about 10 minutes. Avoid direct exposure to intense light.
The color development on the plate should be monitored and the substrate reaction stopped (see next point of this protocol) before positive wells are no longer properly recordable. Determination of the ideal time period for color development has to be done individually for each assay.
It is recommended to add the stop solution when the highest standard has developed a dark blue color. Alternatively the color development can be monitored by the ELISA reader at 620 nm.
4
The substrate reaction should be stopped as soon as Standard 1 has reached an OD of 0.9–0.95.
14.
Stop the enzyme reaction by quickly pipetting 100 µL of Stop
Solution into each well. It is important that the Stop Solution is spread quickly and uniformly throughout the microwells to completely inactivate the enzyme. Results must be read immediately after the Stop Solution is added or within one hour if the microwell strips are stored at 2°C to 8°C in the dark.
15.
Read absorbance of each microwell on a spectro-photometer using 450 nm as the primary wave length (optionally 620 nm as the reference wave length; 610 nm to 650 nm is acceptable). Blank the plate reader according to the manufacturer's instructions by using the blank wells. Determine the absorbance of both the samples and the standards.
Calculation of results
• Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20% of the mean value.
• Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the human
CD44var (v6) concentration on the abscissa. Draw a best fit curve through the points of the graph (a 5-parameter curve fit is recommended).
• To determine the concentration of circulating human CD44var
(v6) for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding human CD44var (v6) concentration.
• If instructions in this protocol have been followed, samples have been diluted 1:100 (1:20 external predilution, 1:5 dilution on the plate: 20 µL sample + 80 µL Sample Diluent) and the concentration read from the standard curve must be multiplied by the dilution factor (x 100).
• Calculation of samples with a concentration exceeding standard 1 may result in incorrect, low human CD44var (v6) levels (Hook
Effect). Such samples require further external predilution according to expected human CD44var (v6) values with Sample
Diluent in order to precisely quantitate the actual human
CD44var (v6) level.
• It is suggested that each testing facility establishes a control sample of known human CD44var (v6) concentration and runs this additional control with each assay. If the values obtained are not within the expected range of the control, the assay results may be invalid.
Human sCD44var (v6) ELISA Kit User Guide
•
A representative standard curve is shown in Figure 7.
Note: Do not use this standard curve to derive test results. Each laboratory must prepare a standard curve for each group of microwell strips assayed.
0.01
0.1
Fig. 7 Representative standard curve for human CD44var (v6)
ELISA. Human CD44var (v6) was diluted in serial 2-fold steps in
Sample Diluent.
Table 2 Typical data using the human CD44var (v6) ELISA.
Measuring wavelength: 450 nm
Reference wavelength: 620 nm
Standard
Human CD44var
(v6)
Concentration
(ng/mL)
O.D. at
450 nm
Mean O.D. at
450 nm
C.V. (%)
1
2
3
4
5
6
Blank
10.00
5.00
2.50
1.25
0.63
0.32
0.00
1.749
2.019
1.246
1.159
0.638
0.617
0.334
0.311
0.168
0.159
0.087
0.082
0.010
0.015
1.884
1.203
0.628
0.323
0.164
0.085
0.013
7.2
3.6
1.7
3.6
2.8
3.0
16.5
The OD values of the standard curve may vary according to the conditions of assay performance (e.g., operator, pipetting technique, washing technique, or temperature effects).
Furthermore, shelf life of the kit may affect enzymatic activity and thus color intensity. Values measured are still valid.
Limitations
• Since exact conditions may vary from assay to assay, a standard curve must be established for every run.
• Bacterial or fungal contamination of either screen samples or reagents or cross-contamination between reagents may cause erroneous results.
• Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use.
Human sCD44var (v6) ELISA Kit User Guide
• Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Empty wells completely before dispensing fresh wash solution, fill with Wash
Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods.
• The use of radioimmunotherapy has significantly increased the number of patients with human anti-mouse IgG antibodies
(HAMA). HAMA may interfere with assays utilizing murine monoclonal antibodies leading to both false positive and false negative results. Serum samples containing antibodies to murine immunoglobulins can still be analyzed in such assays when murine immunoglobulins (serum, ascitic fluid, or monoclonal antibodies of irrelevant specificity) are added to the sample.
Performance characteristics
Sensitivity
The limit of detection of human CD44var (v6) defined as the analyte concentration resulting in an absorbance significantly higher than that of the dilution medium (mean plus 2 standard deviations) was determined to be 0.05 ng/mL (mean of 10 independent assays).
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Reproducibility
Intra-assay
Reproducibility within the assay was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of human CD44var (v6).
Two standard curves were run on each plate. Data below show the mean human CD44var (v6) concentration and the coefficient of
variation for each sample (see Table 3). The calculated overall intra-
assay coefficient of variation was 3.0%.
Table 3 The mean human CD44var (v6) concentration and the coefficient of variation for each sample.
Sample Experiment
1
2
3
4
5
6
7
8
Mean human CD44var (v6) concentration (ng/mL)
143
157
143
30
37
33
555
564
582
367
391
405
247
268
266
223
238
244
298
309
324
720
714
781
Coefficient of variation (%)
2.7
5.0
3.7
1.6
4.7
2.6
1.1
4.3
2.3
3.7
1.9
2.2
1.5
2.3
3.1
5.0
3.4
3.1
1.8
2.7
4.9
1.8
3.0
1.8
Inter-assay
Assay to assay reproducibility within one laboratory was evaluated in three independent experiments. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of human CD44var (v6). Two standard curves were run on each plate.
Data below show the mean human CD44var (v6) concentration and the coefficient of variation calculated on 18 determinations of each
5
4
5
6
7
8
1
2
3
sample (see Table 4). The calculated overall inter-assay coefficient of
variation was 4.2%.
Table 4 The mean human CD44var (v6) concentration and the coefficient of variation of each sample.
Sample
Mean human CD44var (v6) concentration (ng/mL)
148
33
567
388
260
235
310
739
Coefficient of variation
(%)
4.3
8.8
2.0
4.0
3.6
3.6
3.5
4.1
Spike recovery
The spike recovery was evaluated by spiking 4 levels of human
CD44var (v6) into serum. Recoveries were determined in 2 independent experiments with 6 replicates each. The amount of endogenous human CD44var (v6) in unspiked serum was subtracted from the spike values. The recovery ranged from 89–115% with an overall mean recovery of 106%.
Sample matrix Spike high (%)
Serum 115
Spike medium
(%)
107
Spike low (%)
97
Dilution parallelism
Serum samples with different levels of human CD44var (v6) were analyzed at serial 2-fold dilutions with four replicates each. The recovery ranged from 98% to 106% with an overall recovery of 102%.
Sample
1
2
3
4
Dilution
1:100
1:200
1:400
1:800
1:100
1:200
1:400
1:800
1:100
1:200
1:400
1:800
1:100
1:200
1:400
1:800
–
302
151
77
–
204
105
54
–
133
65
33
–
121
60
30
Expected human
CD44var (v6) concentration
(ng/mL)
603
302
153
75
408
211
108
57
265
130
66
33
242
120
61
32
Observed human
CD44var (v6) concentration
(ng/mL)
Recovery of expected human
CD44var (v6) concentration
(%)
–
100
101
97
–
103
103
105
–
98
101
101
–
99
102
106
Sample stability
Freeze-thaw stability
Aliquots of serum samples were stored at -20°C and thawed 5 times, and the human CD44var (v6) levels determined. There was no significant loss of human CD44var (v6) immunoreactivity detected by freezing and thawing.
Storage stability
Aliquots of serum samples were stored at –20°C, 2°C to 8°C, room temperature, and at 37°C, and the human CD44var (v6) level determined after 24 hours. There was no significant loss of human
CD44var (v6) immunoreactivity detected during storage under above conditions.
Comparison of serum and plasma
Sera, as well as EDTA, citrate and heparin plasmas from 22 individuals were obtained at the same time point. All these blood preparations were found suitable for CD44var (v6) determinations, although
CD44var (v6) levels in citrate and EDTA plasmas were slightly lower than serum levels. It is, therefore, highly recommended to assure the uniformity of sample preparations.
Specificity
The assay detects both natural and recombinant human CD44var (v6).
The interference of circulating factors of the immune system was evaluated by spiking these proteins at physiologically relevant concentrations into positive serum. No cross-reactivity was detected, notably not with CD44-polypeptides lacking the protein sequence encoded by exon 6.
Expected values
A panel of 40 sera samples from randomly selected apparently healthy donors (males and females) was tested for human CD44var (v6). The detected human CD44var (v6) levels ranged between 119.3 and
354.9 ng/mL with a mean level of 230.4 ng/mL and a standard deviation of 67.9 ng/mL. The levels measured may vary with the sample collection used.
Reagent preparation summary
Wash buffer (1x)
Add Wash Buffer Concentrate 20x (50 mL) to 950 mL distilled water.
Number of Strips
1–6
1–12
Wash Buffer
Concentrate (mL)
25
50
Distilled Water (mL)
475
950
Assay buffer (1x)
Add Assay Buffer Concentrate 20x (5 mL) to 95 mL distilled water.
Number of Strips
1–6
1–12
Assay Buffer
Concentrate (mL)
2.5
5.0
Distilled Water (mL)
47.5
95.0
HRP-Conjugate
Make a predilution of the HRP-Conjugate by adding 760 µL Assay
Buffer (1x). Make a further 1:100 dilution in Assay Buffer (1x):
Number of Strips
1–6
1–12
Prediluted HRP-
Conjugate (mL)
0.03
0.06
Assay Buffer (1x) (mL)
2.97
5.94
Human CD44var (v6) standard
Reconstitute lyophilized human CD44var (v6) standard with distilled water. (Reconstitution volume is stated on the label of the standard vial.)
Test protocol summary
Note: If instructions in this protocol have been followed, samples have been diluted 1:100 (1:20 external predilution, 1:5 dilution on the plate:
20 µL sample + 80 µL Sample Diluent) and the concentration read from the standard curve must be multiplied by the dilution factor
(x 100).
1.
Predilute sample with Sample Diluent 1:20.
2.
Determine the number of microwell strips required.
3.
Wash microwell strips twice with Wash Buffer.
6 Human sCD44var (v6) ELISA Kit User Guide
4.
Standard dilution on the microwell plate: Add 100 µL Sample
Diluent, in duplicate, to standard wells A1/2 - F1/2. Pipette 100 µL prepared standard into the first wells and create standard dilutions by transferring 100 µL from well to well. Discard 100 µL from the last wells.
dilutions in the microwell strips.
5.
Add 100 µL Sample Diluent in duplicate to the blank wells.
6.
Add 80 µL Sample Diluent to sample wells.
7.
Add 20 µL sample in duplicate, to designated sample wells.
8.
Prepare HRP-Conjugate.
9.
Add 50 µL HRP-Conjugate to all wells.
10.
Cover microwell strips and incubate 3 hours at room temperature
(18°C to 25°C).
11.
Empty and wash microwell strips 3 times with Wash Buffer.
12.
Add 100 µL of TMB Substrate Solution to all wells.
13.
Incubate the microwell strips for about 10 minutes at room temperature (18°C to 25°C).
14.
Add 100 µL Stop Solution to all wells.
15.
Blank microwell reader and measure color intensity at 450 nm.
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• Worldwide contact telephone numbers
• Product support information
– Product FAQs
– Software, patches, and updates
– Training for many applications and instruments
• Order and web support
• Product documentation
– User guides, manuals, and protocols
– Certificates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and
Conditions of Sale at www.thermofisher.com/us/en/home/global/
terms-and-conditions.html. If you have any questions, please contact
Life Technologies at www.thermofisher.com/support.
Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, Austria
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
©2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. All other trademarks are the property of their respective owners.
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15 July 2019

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