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ELISA Kit Catalog #KHS2021

sP-selectin Human sP-selectin

www.invitrogen.com Invitrogen Corporation

542 Flynn Road, Camarillo, CA 93012 Tel: 800-955-6288

E-mail: techsupport@invitrogen.com

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TABLE OF CONTENTS

Purpose ....................................................................................... 4 Introduction ................................................................................ 4 Principle of the Method .............................................................. 6 Reagents Provided ...................................................................... 7 Storage Instructions .................................................................... 8 Specimen Collection................................................................... 8 Supplies Required but not Provided ........................................... 9 Procedural Notes/Lab Quality Control ....................................... 9 Preparation of Reagents .............................................................. 11 Assay Method: Procedure and Calculations ............................... 15 Calculation of Results................................................................. 19 Limitations of the Procedure....................................................... 22 Performance Characteristics ....................................................... 23 Reagent Preparation Summary ................................................... 30 Assay Method Summary............................................................. 31 References .................................................................................. 32

Rev. B7 03/09/10 PR098

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PURPOSE

The sP-selectin ELISA is an enzyme-linked immunosorbent assay for the quantitative detection of soluble human P-selectin levels in cell culture supernatant, human serum, plasma, amniotic fluid, or other body fluids.

INTRODUCTION

P-selectin (CD62P, GMP-140, PADGEM) belongs to the selectin family of adhesion molecules (8). P-selectin acts as a receptor that supports binding of leukocytes to activated platelets and endothelium. P-selectin-mediated adhesive interactions operate in conjunction with cell-cell interactions directed by related molecules and are likely to be important in both hemostatic and inflammatory processes (15).

P-selectin is located in membranes of α granules in unstimulated platelets, and redistributes to the cell surface upon platelet activation (1,18). P-selectin is also present in endothelial cells in membranes of Weibel-Palade bodies and megakaryocytes. Surface appearance of P-selectin is very rapid, but declines to basal level within a short time following stimulation (5,7).

P-selectin is a 140 kDa protein that is highly glycosylated (9). The cDNA-derived amino acid sequence (10) predicts a molecule with a series of cysteine-rich domains. Like the other selectins, P-selectin contains an N-terminal Ca2+ dependent lectin-like domain and an EGF-like motif which is followed by nine consensus repeats, a transmembrane domain, and a short cytoplasmic tail.

The human gene for P-selectin is located on chromosome 1q21-24 (20).

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P-selectin is a receptor for neutrophils and monocytes, recognizing oligosaccharide structures on the target cells (4,6,12,16).

The physiological role of P-selectin might be the mediation of initial leukocyte adhesion to activated endothelium during acute inflammation. It may work in concert with E-selectin to direct early, regionally specific adherence of neutrophils and monocytes at sites of acute inflammation.

A soluble form of P-selectin found in serum and plasma (3,19) has been described which might represent a proteolytic fragment, or more likely a soluble splice variant lacking the transmembrane domain (11).

For Research Use Only. CAUTION: Not for human or animal therapeutic or diagnostic use.

READ ENTIRE PROTOCOL BEFORE USE.

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PRINCIPLE OF THE METHOD

An anti-sP-selectin monoclonal coating antibody is adsorbed onto microwells. sP-selectin present in the sample or standard binds to antibodies adsorbed to the microwells; an HRP-conjugated monoclonal anti-sP-selectin antibody is added and binds to sP-selectin captured by the first antibody. Following incubation, unbound enzyme conjugated with anti-sP-selectin is removed during a wash step and substrate solution reactive with HRP is added to the wells. A colored product is formed in proportion to the amount of soluble sP-selectin present in the sample or standard. The reaction is terminated by addition of acid, and absorbance is measured at 450nm. A standard curve is prepared from seven sP-selectin sample concentration determined.

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REAGENTS PROVIDED

Reagent

96 Test Kit

Aluminum pouch with 1 microwell plate coated with Monoclonal Antibody (murine) to human sP-selectin.

1 plate

HRP-Conjugate anti-sP-selectin monoclonal (murine) antibody; 0.06 mL per vial.*

1 vial

sP-selectin Standard, lyophilized. Size and reconstitution instructions noted on vial label (80ng/mL).*

2 vials

Wash Buffer (20x concentrate) (PBS with 1% Tween 20); 50 mL per bottle.*

1 bottle

Assay Buffer (20x concentrate) (PBS with 1% Tween 20 and 10% BSA); 5 mL per vial.*

1 vial

Substrate Solution (TMB); 15 mL per vial. 1 vial

Stop Solution (1 M phosphoric acid); 15 mL per vial. 1 vial Blue-Dye, Green-Dye; 0.4 mL per vial.* 1 vial

Adhesive Plate Covers. 2

* reagents contain preservative (0.01% Proclin® 300).

Positive Control, Lyophilized 1 vial

Sample Diluent; 12 mL per vial 1 vial

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STORAGE INSTRUCTIONS

Store kit reagents at 2 to 8°C. Store Lyophilized Control at – 20°C .Immediately after use, remaining reagents should be returned to cold storage (2 to 8°C), controls to –20°C, respectively. Expiration date of the kit and reagents is stated on labels.

The expiration of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling.

SPECIMEN COLLECTION

Cell culture supernatant, human serum, plasma, amniotic fluid, or other body fluids are suitable for use in the assay. Remove the serum or plasma from the clot or red cells, respectively, as soon as possible after clotting and seperation.

Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens.

Samples must be stored frozen at -20°C to avoid loss of bioactive sP-selectin. If samples are to be run within 24 hours, they may be stored at 2 to 8°C. Avoid repeated freeze-thaw cycles. Prior to assay, frozen sera or plasma should be brought to room temperature, slowly and gently mixed by hand, and properly diluted with Assay Buffer (1:10).

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SUPPLIES REQUIRED BUT NOT PROVIDED

1. 5 mL and 10 mL graduated pipettes. 2. 10 μL to 1,000 μL adjustable single channel micropipettes with

disposable tips. 3. 50 μL to 300 μL adjustable multi-channel micropipette with

disposable tips. 4. Multi-channel micropipette reservoir. 5. Beakers, flasks, cylinders necessary for preparation of reagents. 6. Device for delivery of wash solution (multi-channel micropipette,

wash bottle or automatic wash system). 7. Microwell strip reader capable of reading at 450 nm (620 nm as

optional reference wavelength). 8. Glass-distilled or deionized water. 9. Statistical calculator with program to perform regression analysis. PROCEDURAL NOTES/LAB QUALITY CONTROL

1. Do not mix or interchange different reagent lots from various kit lots.

2. Do not use kit reagents beyond expiration date on label. 3. Do not expose kit reagents to strong light during storage or

incubation. 4. Do not pipette by mouth. 5. Do not eat or smoke in areas where kit reagents or samples are

handled. 6. Avoid contact of skin and mucous membranes with kit reagents or specimens. 7. Rubber or disposable latex gloves should be worn while handling kit reagents or specimens.

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8. Avoid contact of substrate solution with oxidizing agents and metal. 9. Avoid splashing or generation of aerosols. 10. In order to avoid microbial contamination or cross-contamination of reagents or specimens which may invalidate the test, use disposable pipette tips and/or pipettes. 11. Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagents. 12. Exposure to acids will inactivate the conjugate. 13. Glass-distilled water or deionized water must be used for reagent preparation. 14. Substrate solution must be at room temperature prior to use. 15. Decontaminate and dispose of specimens 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. 16. 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.

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PREPARATION OF REAGENTS

Wash Buffer and Assay Buffer should be prepared before starting the test procedure.

A. Wash Buffer

If crystals have formed in the Wash Buffer Concentrate, warm it gently until they have completely dissolved.

Pour entire contents (50 mL) of the Wash Buffer Concentrate into a clean 1,000 mL graduated cylinder. Bring final volume to 1,000 mL with glass-distilled or deionized water. Mix gently to avoid foaming. Adjust the pH of the final solution to 7.4.

Transfer to a clean wash bottle and store at 2 to 25°C. Please note that the Wash Buffer is stable for 30 days. Wash Buffer may be prepared as needed according to the following table:

Number Wash Buffer Distilled

of Strips Concentrate (mL) Water (mL) 1 - 6 25 475 1 - 12 50 950

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B. Assay Buffer

Mix the contents of the bottle well. Add contents of Assay Buffer Concentrate (5.0 mL) to 95 mL distilled or deionized water, and mix gently to avoid foaming. Store at 2 to 8°C. Please note that the Assay Buffer is stable for 30 days. Assay Buffer may be prepared as needed according to the following table:

C. Preparation of HRP-Conjugate

The HRP-Conjugate must be diluted 1:100 with Assay Buffer just prior to use in a clean plastic test tube.

Please note that the HRP-Conjugate should be used within 30 minutes after dilution. HRP-Conjugate may be prepared as needed according to the following table:

Number Assay Buffer Distilled of Strips Concentrate (mL) Water (mL) 1 - 6 2.5 47.5 1 - 12 5.0 95.0

Number HRP-Conjugate Assay Buffer of Strips (mL) (mL) 1 - 6 0.03 2.97 1 - 12 0.06 5.94

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D. Preparation of sP-selectin Standard

Reconstitute sP-selectin Standard by addition of distilled water. Reconstitution volume is stated on the label of the Standard vial. Swirl or mix gently to insure complete homogeneous solubilization.

E. TMB Substrate Solution

Use clean pipettes and containers known to be metal free. Warm to room temperature before use. Avoid direct exposure of TMB reagents to intense light and oxidizing agents during storage or incubation.

F. Control

Reconstitute by adding 100 µL distilled water to lyophilized control. Allow the reconstituted control to sit for exactly 10 minutes. Swirl or mix gently to ensure complete and homogeneous solubilization. Further treate the control like your samples in the assay. For control range please refer to vial label. Store reconstituted control aliquoted at –20°C. Avoid repeated freeze and thaw cycles.

G. Addition of color-producing reagents: Blue-Dye, Green-Dye

In order to help our customers avoid any mistakes in pipetting the ELISAs, we offer a tool that helps to monitor the addition of even very small volumes of a solution to the reaction well by giving distinctive colors to each step of the ELISA procedure.

This procedure is optional, does not in any way interfere with the test results, and is designed to help the customer with the performance of the test, but can also be omitted, just following the instruction booklet.

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Alternatively, the dye solutions from the stocks provided (Blue-Dye, Green-Dye) can be added to the reagents according to the following guidelines:

(1) Diluent: Before standard and sample dilution, add the Blue-Dye at a dilution of 1:250 (see table below) to Assay Buffer (1x) according to the test protocol. After addition of Blue-Dye, proceed according to the instruction booklet.

(2) HRP-Conjugate: Before dilution of the concentrated conjugate, add the Green-Dye at a dilution of 1:100 (see table below) to the Assay Buffer used for the final conjugate dilution. Proceed after addition of Green-Dye according to the instruction booklet, preparation of HRP-Conjugate.

5 mL Assay Buffer 20 μL Blue-Dye 12 mL Assay Buffer 48 μL Blue-Dye 50 mL Assay Buffer 200 μL Blue-Dye 60 mL Assay Buffer 240 μL Blue-Dye

3 mL Assay Buffer 30 μL Green-Dye 6 mL Assay Buffer 60 μL Green-Dye 12 mL Assay Buffer 120 μL Green-Dye

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ASSAY METHOD: PROCEDURE AND CALCULATIONS

Be sure to read the Procedural Notes/Lab Quality Control section before carrying out the assay.

Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.

1. 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 coated with monoclonal antibody (murine) to human sP-selectin from holder and store in foil bag with the desiccant provided at 2 to 8°C, sealed tightly.

2. Wash the Microwell Strips three times with approximately 300 μL Wash Buffer per well with thorough aspiration of microwell contents between washes. Take care not to scratch the surface of the microwells.

3. After the last wash, empty wells and tap Microwell Strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the Microwell Strips immediately after washing or place upside down on a wet absorbent paper for not longer than 15 minutes. Do not allow wells to dry. 4. Add 100 μL of Assay Buffer*, in duplicate, to all standard wells.

Prepare standard dilutions by pipetting 100 μL of sP-selectin Standard (refer to PREPARATION OF REAGENTS, section D), in duplicate, into wells A1 and A2 (see Figures 1 and 2). Mix the contents thoroughly by repeated aspiration and ejection and transfer 100 μL to wells B1 and B2, respectively. Take care not to

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scratch the inner surface of the microwells. Continue this procedure five times, creating two rows of sP-selectin Standard dilutions ranging from 40 to 0.63 ng/mL. Discard 100 μL of the contents from the last microwells used (G1, G2).

*Sample diluent may be used instead.

Figure 1. Preparation of sP-selectin Standard dilutions:

t ransfer 100 µl

100 µl Assay Buffer

discard100 µl

A1 B1 C1 D1 F1-

sP-selectinStandard 200 µl

sP-selectin Standard 100 μL

100 μL Assay Buffer

Transfer 100 μL

Discard 100 μL

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Figure 2. Diagram depicting an example of the arrangement of blanks, standards and samples in the Microwell Strips:

5. Add 100 μL of Assay Buffer, in duplicate, to the blank wells. 6. Add 90 μL of Assay Buffer, in duplicate, to the sample wells. 7. Add 10 μL of each Sample, in duplicate, to the designated wells. 8. Prepare HRP-Conjugate. (refer to PREPARATION OF

REAGENTS, section C). 9. Add 50 μL of diluted HRP-Conjugate to all wells, including the

blank wells. 10. Cover with a Plate Cover and incubate at room temperature (18 to

25°C) for 2 hours on a rotator set at 100 rpm, if available.

1 2 3 4 A Standard 1 Standard 1 Sample 1 Sample 1 (40 ng/mL) (140 ng/mL) B Standard 2 Standard 2 Sample 2 Sample 2 (20 ng/mL) (70 ng/mL) C Standard 3 Standard 3 Sample 3 Sample 3 (10 ng/mL) (35 ng/mL) D Standard 4 Standard 4 Sample 4 Sample 4 (5 ng/mL) (17.5 ng/mL) E Standard 5 Standard 5 Sample 5 Sample 5 (2.5 ng/mL) (8.75 ng/mL) F Standard 6 Standard 6 Sample 6 Sample 6 (1.25 ng/mL) (4.38 ng/mL) G Standard 7 Standard 7 Sample 7 Sample 7 (0.63 ng/mL) (2.19 ng/mL) H Blank Blank Sample 8 Sample 8

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11. Remove Plate Cover and empty wells. Wash Microwell Strips 3 times according to point c. of the test protocol. Proceed immediately to the next step.

12. Pipette 100 μL of TMB Substrate Solution into all wells, including the blank wells.

13. Incubate the Microwell Strips at room temperature (18 to 25°C) for about 15 minutes on a rotator set at 100 rpm, if available. Avoid direct exposure to intense light. The point at which the substrate reaction is stopped is often determined by the ELISA reader being used. Many ELISA readers record absorbance only up to 2.0 O.D. Therefore the color development within individual microwells must be watched by the person running the assay, and the substrate reaction stopped before positive wells are no longer properly recordable.

14. Stop the enzyme reaction by quickly pipetting 100 μL of Stop Solution into each well, including the blank wells. 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 to 8°C in the dark.

15. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wavelength (optionally 620 nm as the reference wavelength; 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 sP-selectin Standards.

Note: In case of incubation without shaking, the obtained O.D. values may be lower than indicated below. Nevertheless the results are still valid.

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CALCULATION OF RESULTS

1. Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20 percent of the mean.

2. Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the sP-selectin concentration on the abscissa. Draw a best fit curve through the points of the graph.

3. To determine the concentration of circulating sP-selectin 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 sP-selectin concentration.

4. For samples which have been diluted according to the instructions given in this manual, 1:10, the concentration read from the standard curve must be multiplied by the dilution factor (x10).

5. It is suggested that each testing facility establish a control sample of known sP-selectin concentration and runs this additional control with each assay. If the values obtained are not within the expected range of this control, the assay results may be invalid.

6. A representative standard curve is shown in Figure 3. This curve cannot be used to derive test results. Every laboratory must prepare a standard curve for each group of Microwell Strips assayed.

Note: Calculation of samples with an O.D. exceeding 2.0 may result in incorrect, low sP-selectin levels. Such samples require further dilution e.g., 1:20 to 1:40 with Assay Buffer in order to precisely quantitate the actual sP-selectin level.

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Figure 3. Representative standard curve for sP-selectin ELISA. Recombinant sP-selectin was diluted in serial two-fold steps in Assay Buffer, symbols represent the mean of three parallel titrations.

Do not use this standard curve to derive test results. A standard curve must be run for each group of Microwell Strips assayed.

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Typical data using the P-selectin ELISA

Measuring wavelength: 450 nm Reference wavelength: 620 nm

Standard

sP-selectin concentration

(ng/mL)

O.D. Mean

C.V. (%)

1 40 2.710 3.5 2 20 1.431 7.3 3 10 0.714 3.0 4 5 0.433 3.7 5 2.5 0.253 4.9 6 1.25 0.156 2.8 7 0.63 0.111 0.6

Blank 0 0.066 2.1

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LIMITATIONS OF THE PROCEDURE

1. Since exact conditions may vary from assay to assay, a standard curve must be established for every run.

2. Bacterial or fungal contamination of either samples or reagents or cross-contamination between reagents may cause erroneous results.

3. Disposable pipette tips, flasks or glassware are preferred. Reusable glassware must be washed and thoroughly rinsed of all detergents before use.

4. Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells before dispensing fresh Wash Buffer, fill with Wash Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods.

5. The use of radioimmunotherapy has significantly increased the number of patients with human anti-mouse IgG antibody (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 mouse monoclonal antibodies of irrelevant specificity) are added to the Assay Buffer.

For Research Use Only. CAUTION: Not for human or animal therapeutic or diagnostic use.

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PERFORMANCE CHARACTERISTICS

SENSITIVITY

The limit of detection of sP-selectin defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus two standard deviations) was determined to be less than 0.2 ng/mL (mean of 6 independent assays).

REPRODUCIBILITY

1. Intra-Assay

Reproducibility within the assay was evaluated in three independent experiments. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of sP-selectin. Two standard curves were run on each plate. Data below show the mean sP-selectin concentration and the coefficient of variation for each sample. The overall intra-assay coefficient of variation has been calculated to be 7.8%.

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b. Inter-assay

Assay to assay reproducibility within one laboratory was evaluated in three independent experiments by three technicians. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of sL-selectin. Two standard curves were run on each plate. Data below show the mean sL-selectin concentration and the coefficient of variation calculated on 18 determinations of each sample. The calculated overall coefficient of variation was 4.2%.

Positive Sample

Experiment

sP-selectin Concentration (ng/mL)

Coefficient of Variation (%)

1 1 188.60 6.8 2 172.56 9.7 3 177.60 6.7 2 1 182.58 9.4 2 189.48 9.8 3 208.08 3.9 3 1 131.37 10.8 2 141.99 12.7 3 146.63 4.1 4 1 60.49 10.4 2 62.25 12.0 3 61.62 5.8 5 1 68.18 8.8 2 66.87 7.2 3 68.72 3.9 6 1 47.95 9.9 2 51.62 7.0 3 49.33 3.0 7 1 11.43 5.5 2 13.40 13.9 3 13.18 9.5 8 1 8.59 4.7 2 10.77 8.9 3 9.72 4.0

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2. Inter-Assay

Assay-to-assay reproducibility within one laboratory was evaluated in three independent experiments by three technicians. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of sP-selectin. Two standard curves were run on each plate. Data below show the mean sP-selectin concentration and the coefficient of variation calculated on 18 determinations of each sample. The calculated overall coefficient of variation was 5.4 %.

Sample

sP-selectin Concentration (ng/mL)

Coefficient of Variation (%)

1 179.59 4.6 2 193.38 6.8 3 140.00 5.6 4 61.46 1.5 5 67.92 1.4 6 49.63 3.7 7 12.67 8.5 8 9.69 11.2

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RECOVERY

The spike recovery was evaluated by spiking three levels of recombinant sP-selectin into human serum. As shown below, recoveries were determined in three independent experiments with 6 replicates each. The unspiked serum was used as blank in these experiments. Average recovery ranged from 58% to 93% with an overall mean recovery of 75%.

Sample matrix Spike high %

Spike medium %

Spike low %

Serum 69 81 79

Plasma (EDTA) 58 61 70

Plasma (citrate) 85 83 93

Plasma (heparin) 83 68 66

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DILUTION PARALLELISM

Four serum samples with different levels of sP-selectin were analyzed at serial two-fold dilutions with 4 replicates each. In the table below, the percent recovery of expected values is listed. Recoveries ranged from 81.3% to 108.9% with an overall mean recovery of 91.6%.

Sample

Dilution

Expected Value

Observed Value

% Recovery of Exp. Value

1 1:10 -- 126.9 -- 1:20 63.45 56.21 88.6 1:40 31.73 26.70 84.2

2 1:10 -- 159.74 -- 1:20 79.87 78.93 98.8 1:40 39.93 40.06 100.3

3 1:10 -- 217.00 -- 1:20 108.50 97.05 89.4 1:40 54.25 45.44 83.8

4 1:10 -- 102.12 -- 1:20 51.06 49.01 96.0 1:40 25.53 24.66 96.6

sP-selectin Concentration (ng/mL)

1:80 15.86 12.90 81.3

1:80 19.97 21.75 108.9

1:80 27.12 22.21 81.9

1:80 12.76 11.41 89.4

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SAMPLE STABILITY

1. Freeze-Thaw Stability

Aliquots of serum samples (unspiked or spiked with sP-selectin) were stored at -20°C and thawed and frozen five times, and the sP-selectin levels determined. There was no significant loss of sP-selectin concentrations between 0 and 5 freeze-thaw cycles.

2. Storage Stability

Aliquots of a serum sample (spiked and unspiked with sP-selectin) were stored at -20°C, 2 to 8°C, room temperature (RT) and at 37°C and the sP-selectin level determined after 24 hours. There was no significant loss of sP-selectin immunoreactivity during storage under above conditions.

COMPARISON OF SERUM AND PLASMA

From two individuals each, serum as well as EDTA, citrate, and heparin plasma was obtained at the same time and tested for P-selectin. Concentrations were not significantly different and therefore all these blood preparations are suitable for use in the assay. It is nevertheless highly recommended to assure the uniformity of blood preparations.

SPECIFICITY

This assay recognizes both natural and recombinant human sP-selectin. To define the specificity of this ELISA, several polypeptides were tested for cross-reactivity. There was no cross-reactivity determined for any of the test proteins, most notably there was no interference of sE-selectin and sL-selectin in this assay.

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EXPECTED VALUES

A panel of 40 sera from apparently healthy blood donors (male and female) was tested for sP-selectin.

Sample matrix Minimum (ng/mL)

Maximum (ng/mL)

Mean (ng/mL)

Serum 67 233 126

Plasma (EDTA) 50 165 106

Plasma (citrate) 92 212 135

Plasma (heparin) 60 188 129

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REAGENT PREPARATION SUMMARY

A. Wash Buffer Add 50 mL Wash Buffer Concentrate (20x) to 950 mL distilled water

B. Assay Buffer Number of Strips

Assay Buffer Concentrate (mL)

Distilled Water (mL)

1 - 6 2.5 47.5

1 - 12 5.0 95.0

C. HRP-Conjugate Number of Strips

HRP-Conjugate (mL)

Assay Buffer (mL)

1 - 6 0.03 2.97

1 - 12 0.06 5.94

D. sP-selectin Standard

Reconstitute sP-selectin Standard by addition of distilled water. Reconstitution volume is stated on the label of the standard vial.

E. Control Add 100 µL distilled water to lyophilized control.

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ASSAY METHOD SUMMARY

1. Wash Microwell Strips twice with Wash Buffer. 2. Add 100 μL Assay Buffer, in duplicate, to standard wells. 3. Pipette 100 μL solubilized sP-selectin Standard, in duplicate, into

the first wells and create standard dilutions ranging from 40 to 0.63 ng/mL by transferring 100 μL from well to well. Discard 100 μL from the last wells.

4. Add 100 μL Assay Buffer to the blank wells. 5. Add 90 μL Assay Buffer to the sample wells. 6. Add 10 μL sample, in duplicate, to designated wells. 7. Prepare HRP-Conjugate. 8. Add 50 μL of diluted HRP-Conjugate to all wells including blank

wells. 9. Cover Microwell Strips and incubate 2 hours at room temperature

(18 to 25°C); shaking is recommended. 10. Empty and wash Microwell Strips 3 times with Wash Buffer. 11. Add 100 μL of TMB Substrate Solution to all wells. 12. Incubate the Microwell Strips for about 10 to 20 minutes at room

temperature (18 to 25°C). 13. Add 100 μL Stop Solution to all wells including blank wells. 14. Blank microwell reader and measure color intensity at 450 nm.

Note: For samples which have been diluted according to the instructions in this manual 1:10, the concentration read from the standard curve must be multiplied by the dilution factor (x10). Calculation of samples with an O.D. exceeding 2.0 may result in incorrect, low sP-selectin levels. Such samples require further dilution of 1:20 to 1:40 with Assay Buffer in order to precisely quantitate the actual sP-selectin level.

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REFERENCES

1. Berman, D.L, E.L. Yeo, J.D. Wencel-Drake, B.C. Furie, M.H. Ginsberg, and B. Furie (1986) A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. J. Clin. Invest. 78:130.

2. Boukerche, H., O. Berthier-Vergnes, E. Tabone, J.F. Dore, L.L.K. Leung, and J.L. McGregor (1989) Platelet-melanoma cell interaction is mediated by the glycoprotein IIb-IIIa complex. Blood 74:658.

3. Dunlop, L.C., M.P. Skinner, L.J. Bendall, E.J. Favaloro, P.A. Castaldi, J.J. Gorman, J.R. Gamble, M.A. Vadas, and M.C. Berndt (1992) Characterization of GMP-140 as a circulating plasma protein. J. Exp. Med. 175:1147.

4. Geng, J.G., M.P. Bevilacqua, K.L. Moore, T.M. McIntyre, S.M. Prescott, J.M. Kim, G.A. Bliss, G.A. Zimmerman, and R.P. McEver (1990) Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature 343:757.

5. George, J.N., E.B. Pickett, S. Saucerman, R.P. McEver, T.J. Kunicki, N. Kieffer, and P.J. Newman (1986) Platelet surface glycoproteins. Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J. Clin. Invest. 78:340.

6. Hamburger, S.A., and R.P. McEver (1990) GMP-140 mediates adhesion of stimulated platelets to neutrophils. Blood 75:550.

7. Hattori, R., K.K. Hamilton, R.D. Fugate, R.P. McEver, and

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P.J. Sims (1989) Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140. J. Biol. Chem. 264:7768.

8. Hogg, N. (1992) Roll, roll, roll your leukocyte gently down the vein. Immunol. Today 13:113.

9. Johnston, G.I., A. Kurosky, and R.P. McEver (1989) Structural and biosynthetic studies of the granule membrane protein, GMP-140, from human platelets and endothelial cells. J. Biol. Chem. 264:1816.

10. Johnston, G.I., R.G. Cook, and R.P. McEver (1989) Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell 56:1033.

11. Johnston, G.I., G.A. Bliss, P.J. Newman, and R.P. McEver (1990) Structure of the human gene encoding GMP-140, a member of the selectin family of adhesion receptors for leukocytes. J. Biol. Chem. 265:21381.

12. Larsen, E., A. Celi, G.E. Gilbert, B.C. Furie, J.K. Erban, R. Bonfanti, D.D. Wagner, and B. Furie (1989) PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell 59:305.

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