Human PTX3/Pentraxin 3
ELISA
Catalog # LF-EK50795 (1 kit)
Catalog # LF-EK50796 (4 kits bundle)

Sandwich Enzyme-Linked Immunosorbent Assay for Quantitative Detection ofhuman PTX3/Pentraxin 3

For research use only
Not for diagnostic or therapeutic purposes1

Contents
1.

Introduction ····························································

3

2.

Principles of Method ··················································

3

3.

Intended Use ····························································

4

4.

Storage and Stability ··················································

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5.

Chemical Hazard ······················································

4

6.

Kit Contents ····························································

5

7.

Materials Required But Not Provided ·····························

6

8.

Reagent preparation ··················································

61) Sample Preparation and Storage ···········································

62) Sample Dilution Guideline ·················································

73) Reagent Preparation and Storage ··········································

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Assay Procedure ·······················································

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10. Characteristics ·························································

101) Typical result ·······························································

102) Sensitivity ···································································

103) Detection range ······························································

104) Specificity ···································································

115) Specificity ···································································

116) Precision ···································································

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11. Troubleshooting ························································

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12. Reference ································································

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9.

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1. Introduction
PTX3(Pentraxin 3) is a member of the pentraxin superfamily. This super family characterized
by cyclic multimeric structure. The PTX3 gene is mapped to 3q25.32. The predicted 381-aminoacid PTX3 protein has homology to the pentraxin protein family. Significant levels of PTX3
were detected in plasma of neutropenic patients with systemic A. PTX3 is effective inpreventing CMV infection and reactivation, as well as subsequent Aspergillus infection. PTX3
activates the classical pathway of complementactivation and facilitates pathogen recognition bymacrophages and DCs.

2. Principles of Method
Abfrontier’s human PTX3 ELISA Kit was based on standard sandwich enzyme-linked
immune-sorbent assay technology. A monoclonal antibody from mouse specific for PTX3 hasbeen precoated onto 96-well plates. Standards(NSO, E18-S381) and test samples are added to
the wells, a biotinylated detection polyclonal antibody from goat specific for PTX3 is added
subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase
Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP
substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to
produce a blue color product that changed into yellow after adding acidic stop solution. The
density of yellow is proportional to the human PTX3 amount of sample captured in plate.

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3.

Intended Use

The AbFrontier human PTX3 ELISA kit is to be used for the in vitro quantitative
determination ofhuman PTX3 in cell culture supernates, serum, plasma(EDTA) and saliva.

The assay will recognize both native and recombinant human PTX3.

This kit has been configured for research use only and is not to be used indiagnostic procedures.

4. Storage and Stability℃All kit components of this kit are stable at 2 to 8 . Any unused reconstituted standard should
be discarded or frozen at -20℃. Standard can be frozen and thawed one time only without lossof immunoreactivity.

5. Chemical Hazard- Stop solution: This reagent is an irritant to eyes, skin and mucous membranes. Avoid contact
with eyes, skin and clothing. Wear suitable protective clothing, gloves and eye protection. In
the event of contact with eyes or skin, wash immediately with plenty of water.

- All reagents containing Sodium Azide also contain Thimerosal as a preservative. Thimerosal
contains Hg thus should be handled with great care.

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6. Kit Contents
Contents

NumberVolume1 (in aluminum foil bag with desiccant)96 Well Plate
Standard Protein

2

20 ng/tube

Secondary Antibody

1

130 µl (dilution 1:100)1

130 µl (dilution 1:100)

Sample diluent Buffer

1

30 ml

Antibody diluent buffer

1

12 ml

ABC diluent buffer

1

12 ml

TMB color developing agent

1

10 ml

TMB stop solution

1

10 ml

Avidin-Biotin-Peroxidase Complex(ABC)① 96 Well Plate: Human PTX3 microtiter plate, one plate of 96 wells.
A plate using break-apart strips coated with a monoclonal antibody specific to Human
PTX3.

② Standard Protein: Recombinant Human PTX3.

③ Secondary Antibody: Biotin labeled anti Human PTX3 antibody.

④ AV-HRP: Avidin-Biotin-Peroxidase Complex (ABC)⑤ Substrate (Stabilized chromogen): Tetramethylbenzidine (TMB) solution⑥ Stop Solution: 1 N solution of sulfuric acid (H2SO4)
Notice for Application of Kit
1． To inspect the validity of experiment operation and the appropriateness of sample
dilution proportion, pilot experiment using standards and a small number ofsamples is recommended.
2． The TMB Color Developing agent is colorless and transparent before using,contact us freely if it is not the case.
3． Before using the Kit, spin tubes and bring down all components to the bottom oftubes.
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4． Duplicate well assay is recommended for both standard and sample testing.
5． Don’t let 96-well plate dry, for dry plate will inactivate active components onplate.
6． Don’t reuse tips and tubes to avoid cross contamination.
7． To avoid to use the reagents from different batches together.
8． In order to avoid marginal effect of plate incubation due to temperature difference(reaction may be stronger in the marginal wells), it is suggested that the diluted
ABC and TMB solution will be pre-warmed in 37℃ for 30 min before using.

7. Materials Required But Not Provided① Microtiter plate reader in standard size.
② Automated plate washer.
③ Adjustable pipettes and pipette tips. Multichannel pipettes are recommended in thecondition of large amount.

④ Clean tubes and Eppendorf tubes⑤ Washing buffer (neutral PBS or TBS).
 Preparation of 0.01M TBS: Add 1.2 g Tris, 8.5 g NaCl; 450 ul of purified acetic acidor 700 ul of concentrated hydrochloric acid to 1000 ml H2O and adjust pH to 7.2-7.6.
Finally, adjust the total volume to 1 L.
 Preparation of 0.01 M PBS: Add 8.5 g sodium chloride, 1.4 g Na2HPO4 and 0.2 g
NaH2PO4 to 1000 ml distilled water and adjust pH to 7.2-7.6. Finally, adjust the total
volume to 1L.

8. Reagent Preparation
1) Sample Preparation and Storage
Store samples to be assayed within 24 hours at 2-8°C. For long-term storage, aliquot andfreeze samples at -20°C. Avoid repeated freeze-thaw cycles.
oCell culture supernates: Remove particulates by centrifugation, analyze
immediately or aliquot and store at -20°C

oSerum: Allow the serum to clot in a serum separator tube (about 4 hours) at room
temperature. Centrifuge at approximately 1000 X g for 15 min. Analyze the serum
immediately or aliquot and store frozen at -20°C.

6oPlasma: Collect plasma using EDTA as an anticoagulant. Centrifuge for 15 min at
1500 x g within 30 min of collection. Assay immediately or aliquot and store
samples at -20°C.

oSaliva: Collect saliva using a collection device without any protein binding or filtering capabilities
such as a Salivette or aliquot and store samples at -20°C.

2) Sample Dilution Guideline
The user needs to estimate the concentration of the target protein in the sample and select aproper dilution factor so that the diluted target protein concentration falls near the middle ofthe linear regime in the standard curve. Dilute the sample using the provided diluent buffer.
The following is a guideline for sample dilution. Several trials may be necessary in practice.
The sample must be well mixed with the diluents buffer.
oHigh target protein concentration (200-2000 ng/ml). The working dilution is
1:100. i.e. Add 1µl sample into 99 µl sample diluent buffer.

oMedium target protein concentration (20-200 ng/ml). The working dilution is
1:10. i.e. Add 10µl sample into 90 µl sample diluent buffer.

o

Low target protein concentration (312-20,000 pg/ml). The working dilution is
1:2. i.e. Add 50µl sample to 50 µl sample diluent buffer.

oVery Low target protein concentration (≤312 pg/ml). No dilution necessary, or
the working dilution is 1:2.

3) Reagent Preparation and Storage：

A. Reconstitution of the human PTX3 standard PTX3 standard solution should be
prepared no more than 2 hours prior to the experiment. Two tubes of PTX3 standard(20ng per tube) are included in each kit. Use one tube for each experiment.
a. 20,000pg/ml of human PTX3 standard solution: Add 1ml sample diluent
buffer into one tube, keep the tube at room temperature for 10 min and mix
thoroughly.
b. 10,000pg/ml→312pg/ml of human PTX3 standard solutions: Label 6

Eppendorf tubes with 10,000pg/ml, 5000pg/ml, 2500pg/ml, 1250pg/ml,
625pg/ml, 312pg/ml respectively. Aliquot 0.3ml of the sample diluent buffer
into each tube. Add 0.3ml of the above 20,000pg/ml PTX3 standard solution
into 1st tube and mix. Transfer 0.3ml from 1st tube to 2nd tube and mix.
Transfer 0.3ml from 2nd tube to 3rd tube and mix, and so on.
Note: The standard solutions are best used within 2 hours. The 20ng/ml standard
solution should be stored at 4°C for up to 12 hours, or at -20°C for up to 48 hours.
Avoid repeated freeze-thaw cycles.
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B. Preparation of biotinylated anti-human PTX3 antibody working solution: The solution
should be prepared no more than 2 hours prior to the experiment.
a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing
0.1-0.2 ml more than total volume)
b. Biotinylated anti-human PTX3 antibody should be diluted in 1:100 with theantibody diluent buffer and mixed thoroughly. (i.e. Add 1µl Biotinylated antihuman PTX3 antibody to 99µl antibody diluent buffer.)C.

Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution: The
solution should be prepared no more than 1 hour prior to the experiment.
a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing
0.1-0.2 ml more than total volume)
b. Avidin- Biotin-Peroxidase Complex (ABC) should be diluted in 1:100 with the
ABC dilution buffer and mixed thoroughly. (i.e. Add 1µl ABC to 99µl ABC
diluent buffer.)

9. Assay Procedure
The ABC working solution and TMB color developing agent must be kept warm at 37°C
for 30 min before use. When diluting samples and reagents, they must be mixed
completely and evenly. Standard PTX3 detection curve should be prepared for eachexperiment. The user will decide sample dilution fold by crude estimation of PTX3 amountin samples.
1. Aliquot 0.1ml per well of the 20,000pg/ml, 10,000pg/ml, 5000pg/ml, 2500pg/ml,
1250pg/ml, 625pg/ml, 312pg/ml human PTX3 standard solutions into the precoated 96well plate. Add 0.1ml of the sample diluent buffer into the control well (Zero well). Add
0.1ml of each properly diluted sample of human cell culture supernates, serum,plasma(EDTA) or saliva to each empty well. See “Sample Dilution Guideline” above
for details. It is recommended that each human PTX3 standard solution and each sample
be measured in duplicate.
2. Seal the plate with the cover and incubate at 37°C for 90 min.
3. Remove the cover, discard plate content, and blot the plate onto paper towels or otherabsorbent material. Do NOT let the wells completely dry at any time.
4. Add 0.1ml of biotinylated anti-human PTX3 antibody working solution into each welland incubate the plate at 37°C for 60 min.
5. Wash plate 3 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer
stay in the wells for 1 min. Discard the washing buffer and blot the plate onto paper
towels or other absorbent material. (Plate Washing Method: Discard the solution in the8plate without touching the side walls. Blot the plate onto paper towels or other absorbent
material. Soak each well with at least 0.3 ml PBS or TBS buffer for 1~2 minutes. Repeat
this process two additional times for a total of THREE washes. Note: For automatedwashing, aspirate all wells and wash THREE times with PBS or TBS buffer, overfillingwells with PBS or TBS buffer. Blot the plate onto paper towels or other absorbent
material.)
6. Add 0.1 ml of prepared ABC working solution into each well and incubate the plate at
37°C for 30 min.
7. Wash plate 5 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer
stay in the wells for 1-2 min. Discard the washing buffer and blot the plate onto paper
towels or other absorbent material. (See Step 5 for plate washing method).
8. Add 90µl of prepared TMB color developing agent into each well and incubate plate at
37°C in dark for 15-20 min (Note: For reference only, the optimal incubation timeshould be determined by end user. And the shades of blue can be seen in the wells withthe four most concentrated human PTX3 standard solutions; the other wells show no
obvious color).
9. Add 0.1 ml of prepared TMB stop solution into each well. The color changes into yellow
immediately.
10. Read the O.D. absorbance at 450nm in a microplate reader within 30 min after adding
the stop solution.
For calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of Zerowell). The standard curve can be plotted as the relative O.D.450 of each standard solution(Y) vs. the respective concentration of the standard solution (X). The human PTX3
concentration of the samples can be interpolated from the standard curve.
Note: if the samples measured were diluted, multiply the dilution factor to theconcentrations from interpolation to obtain the concentration before dilution.

Summary
1. Add samples and standards and incubate the plate at 37°C for 90 min. Do not wash.
2. Add biotinylated antibodies and incubate the plate at 37°C for 60 min. Wash plate 3times with 0.01M TBS.
3. Add ABC working solution and incubate the plate at 37°C for 30 min. Wash plate 5times with 0.01M TBS.
4. Add TMB color developing agent and incubate the plate at 37°C in dark for 15-20
min.
5. Add TMB stop solution and read.

910. Characteristics
1) Typical result
Typical Data Obtained from Human PTX3
(TMB reaction incubate at 37℃ for 15 min)Standard

Optical Density

Human PTX3 (pg/ml)(at 450nm)

0

0.072

312

0.260

625

0.394

1,250

0.647

2,500

1.029

5,000

1.399

10,0001.883

20,000

2.063

Typical Human PTX3 ELISA Kit Standard Curve
This standard curve was generated at AbFrontier for demonstration purpose only. A
standard curve must be run with each assay.

2) Sensitivity: < 10 pg/ml
3) Detection range: 312pg/ml-20,000pg/ml

104) Specificity: No detectable cross-reactivity with other relevant proteins5) Specificity: Natural and recombinant human PTX3
6) Precision
Intra-Assay Precision (Precision within an assay): Three samples of known
concentration were tested on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays): Three samples of known
concentration were tested in separate assays to assess inter-assay precision.
Intra-Assay PrecisionInter-Assay Precision

Sample

1

2

3

1

2

3

N

16

16

16

24

24

24

Mean(ng/ml)

3.5

7.8

15.7

3.86

8.1

15.3

Standard deviation

0.22

0.45

1.0

0.28

0.48

1.16

CV(%)

6.3

5.8

6.4

7.3

5.9

7.6

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11. Troubleshooting
ProblemPossible Cause

Solution• Insufficient washing• Increase number of washes• Increase time of soaking
between in wash

High signal and backgroundin all wells• Too much AV-HRP• Check dilution, titration• Incubation time too long

• Reduce incubation time

• Development time too long

• Decrease the incubation time
before the stop solution is
added• Reagent added in incorrect• Review protocol

order, or incorrectly preparedNo signal• Standard has gone bad• Check the condition of stored(If there is a signal in thestandard

sample wells)• Assay was conducted from an • Reagents allows to come toincorrect starting point

20~30℃ before performing

assay• Insufficient washing• Increase number of washes– unbound AV-HRP remaining

carefully

Too much signal – whole• Too much AV-HRP• Check dilution

plate turned uniformly blue

• Plate sealer or reservoir• Use fresh plate sealer andreused, resulting in presence

reagent reservoir for eachof residual AV-HRP

step• Plate not developed long

• Increase substrate solution

enoughincubation time

• Improper calculation of• Check dilution, make newstandard curve dilution

standard curve

No signal when a signal is• Sample matrix is masking• More diluted sample

expected, but standard curve

detection

Standard curve achieved but
poor discrimination between
point

recommended

looks fineSamples are reading too high,but standard curve is fine

Edge effect•Samples

containprotein • Dilute samples and run

levels above assay range

again• Uneven temperature around• Avoid incubating plate inwork surface

areas where environmental
conditions vary
• Use plate sealer

1212. Reference
1. Bozza, S., Bistoni, F., Gaziano, R., Pitzurra, L., Zelante, T., Bonifazi, P., Perruccio, K.,
Bellocchio, S., Neri, M., Iorio, A. M., Salvatori, G., De Santis, R., Calvitti, M., Doni, A.,
Garlanda, C., Mantovani, A., Romani, L. Pentraxin 3 protects from MCMV infection andreactivation through TLR sensing pathways leading to IRF3 activation. Blood 108: 33873396, 2006.
2. Emsley J, White HE, O'Hara BP, Oliva G, Srinivasan N, Tickle IJ, Blundell TL, Pepys MB,
Wood SP (January 1994). "Structure of pentameric human serum amyloid P component".
Nature 367 (6461): 338–45.
3. Garlanda C, Bottazzi B, Bastone A, Mantovani A (2005). "Pentraxins at the crossroads
between innate immunity, inflammation, matrix deposition, and female fertility". Annu. Rev.
Immunol. 23: 337–66.
4. Diniz SN, Nomizo R, Cisalpino PS, Teixeira MM, Brown GD, Mantovani A, Gordon S, Reis
LF, Dias AA (April 2004). "PTX3 function as an opsonin for the dectin-1-dependentinternalization of zymosan by macrophages". J. Leukoc. Biol. 75 (4): 649–56.

13◈ Ordering Information
For orders, please contact :Young In Frontier Co., Ltd.
Tel : +82-2-2140-3300
Fax: +82-2-2140-3310
E-mail: orders@younginfrontier.com
Address: 11F, Byucksan Digital Valley 5th, Gasan-dong 60-73, Geumcheon-gu, Seoul,
Korea (153-801)
Website: http://www.abfrontier.com
Or, your local distributor.

For technical advice, please contact:
E-mail : orders@younginfrontier.com
Website : http://www.abfrontier.com

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