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Human IL-15 ELISA Kit, High Sensitivity (Serum, Plasma, TCM)

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Product Info

Matrix Compatibility Serum, Plasma, Tissue Culture Media
Assay Range 0.51 - 32.8 pg/ml
LLOQ

0.51 pg/ml

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Assay Length 4 hours
Specificity Human Interleukin 15
CVs

Inter-Assay: < 10%


Intra-Assay: < 10%


Spike recovery: 103%

 

This ELISA measures IL-15 in tissue culture media (TCM), serum and plasma. It measures both IL-15 alone and IL-15/IL-15Rα complexes and quantitates IL-15 in healthy donor matrices. This assay’s standard is the IL-15 receptor heterodimer, but the standard curve is calibrated to the IL-15 portion of the complex since the antibodies used are IL-15 specific.

 

This kit quantitates free human IL-15 and bound IL-15/IL-15R in serum, plasma and TCM using a sandwich immunoassay. The kit is based on an ELISA with biotinylated-detection antibody and streptavidin-conjugated horseradish peroxidase (HRP). Tetramethyl-benzidine (TMB) is the substrate.

Specifications

CVs and Spike Recovery

Inter-Assay: < 10%


Intra-Assay: < 10%


Spike recovery: 103%

Cross-reactivity

No cross-reactivity detected with

  • Human IL-2, IL-4, IL-7, IL-9, IL-21, IFN-α, IFN-β1a, IFN-γ, IFN-ω
  • Cynomolgus IFN-α2
  • Cynomolgus/Rhesus IFN-α
  • Mouse IL-15, IFN-αA, IFN-β, IFN-γ
  • Rat IL-15, IFN-α1, IFN-α14, IFN-β, IFN-γ
  • Bovine IFN-τ2
  • Pig IFN-α
Storage 2-8 °C
Expiration Date One year from the date of manufacture
Shipping Condition Wet Ice

 

 

Materials Provided

  • Pre-Coated Micro-Titer Stripwell Plate/Sealers
  • Wash Solution Concentrate
  • Human Interleukin-15 Standard (410 pg/ml)
  • Assay Buffer
  • Dilution Buffer
  • Antibody Concentrate
  • HRP Conjugate Concentrate
  • Concentrate Diluent
  • HRP Diluent
  • TMB Substrate Solution
  • Stop Solution

 

Additional Materials Required (Not Provided) 

  • Microplate reader capable of reading an OD at a wavelength of 450 nm
  • Variable volume microtiter pipettes
  • Adjustable multichannel pipette (50-300 μl)
  • Reagent reservoirs
  • Wash bottle or plate washing system
  • Distilled or deionized water
  • Serological pipettes (1, 5, 10 or 25 ml)
  • Disposable pipette tips (polypropylene)
  • Plate shaker

Tech Info & Data

 

Representative Standard Curve in Standard Diluent

 

Human IL-15 standard curves were prepared in Standard Diluent (SD) and assayed in triplicate over 9 runs. The figure below shows dose response and absorbance values. Error bars indicate standard deviation.

 

 

Intra-Assay Precision

Sample 1 2 3 4 5 6
n 26 26 26 26 26 26
Mean (pg/ml) 4.17 4.42 4.20 2.81 3.85 3.87
Std. Dev. 0.18 0.09 0.18 0.10 0.13 0.13
CV (%) 4.4 1.9 4.3 3.6 3.3 3.3

 

 

Inter-Assay Precision

Sample 1 2 3 4 5 6 7
Mean (pg/ml) 4.52 3.55 4.47 2.87 3.54 12.55 3.68
Std. Dev. 0.24 0.16 0.17 0.16 0.19 0.61 0.13
CV (%) 5.3 4.6 3.7 5.4 5.4 4.8 3.4

 

Inter-Batch Precision

Sample 1 2 3 4 5 6 7
Lot 1 (pg/ml) 5.26 3.97 5.12 3.24 4.02 13.94 4.18
Lot 2 (pg/ml) 5.16 3.94 5.09 3.20 3.97 13.58 4.26
Lot 3 (pg/ml) 4.80 3.62 4.71 2.85 3.66 12.77 3.86
Mean (pg/ml) 5.07 3.84 4.97 3.10 3.88 13.43 4.10
Std. Dev. 0.24 0.19 0.22 0.21 0.20 0.60 0.21
CV(%) 4.7 5.0 4.5 6.8 5.1 4.5 5.1

 

Intermediate Precision

Sample 1 2 3 4 5 6 7
n 27 27 27 27 27 27 27
Mean (pg/ml) 5.07 3.84 4.97 3.10 3.88 13.43 4.05
Std. Dev. 0.63 0.49 0.57 0.38 0.45 1.30 0.40
CV (%) 12.5 12.9 11.5 12.4 11.6 9.7

9.9

 

Spike Recovery

TCM

Spike Sample 1 2 3
Target Conc. (pg/ml) 24.61 6.15 0.82
Mean Recovery 92.8 97.0 94.0
Range (%) 91.5 – 95.1 93.3 – 99.0 93.5 – 94.5

Disodium-EDTA Plasma

Spike Sample 1 2 3
Target Conc. (pg/ml) 24.61 6.15 0.82
Mean Recovery 98.1 99.9 82.1
Range (%) 78.4 – 104.5 85.1 – 108.1 64.9 – 95.3

Serum

Spike Sample 1 2 3
Target Conc. (pg/ml) 24.61 6.15 0.82
Mean Recovery 99.2 94.7 75.6
Range (%) 99.2 94.7 75.6

 

Representative Standard Curves in Various Matrices

 

Percent Recovery of IL-15 in Various Matrices

 

MDD/LOQ

Assay 1 2 3
n 80 80 80
Mean Blank (A450) 0.055 0.052 0.057
Std. Dev. 0.004 0.007 0.008
MDD (A450) 0.067 0.072 0.079
MDD (pg/ml) 0.104 0.173 0.175
LOQ (Abs450) 0.096 0.121 0.132
LOQ (pg/ml) 0.341 0.571 0.575

 

Linearity in Pooled Healthy Donor Serum (HDS)

 

Linearity in Tissue Culture Media (TCM)

 

Parallelism in Individual Healthy Donor Sera (HDS)

 

Parallelism in Healthy Donor Plasma (HDP)

 

IL-15 Knockout under Control (Blue) and Specific (Beige) Conditions

Samples treated with PBL’s IL-15 specific capture antibody (beige) displayed knockdown of the observed sample concentration when compared to untreated control samples (blue). Samples treated with the specific IL-15 capture antibody recorded IL-15 levels at or below the assay LOQ.

 

 

IL-15 Concentrations under Knockout Conditions in HDS and HDP Samples

Sample [IL-15] pg/ml Control [IL-15] pg/ml Specific [IL-15] pg/ml Isotype
1 13.12 0.59 6.99
2 4.92 <LOQ 4.4
3 6.38 <LOQ 3.87
4 4.61 <LOQ 4.66
5 4.38 <LOQ 4.55
6 4.61 <LOQ 4.66
7 3.98 <LOQ 3.51
8 3.27 <LOQ 2.95
9 2.52 <LOQ 2.46

 

 

Representative Standard Curves on PBL’s IL-15 ELISA (41702) and Competitors

PBL’s and three competitor’s standards were assayed on their respective ELISAs. Dose response curves and absorbance values of all IL-15 standards are shown below. PBL’s standard exhibited greater sensitivity than competitive IL-15 standards; PBL’s standard curve demonstrated an improvement of 1 – 2 log units.

 

Comparison of PBL’s HEK Standard on Commercial ELISAs

PBL’s HEK293 IL-15/IL-15R standard was assayed on three commercial IL-15 kits based on the suggested kit calibration range in order to investigate selectivity towards the glycosylated IL-15/IL-15R complex. Human cell-expressed IL-15 contains multiple glycosylation patterns and may be predominately expressed as a complex, as opposed to the free protein. Accurate and precise measurements may require recognition of this form of IL-155. Competitor A and C’s IL-15 ELISAs were not able to detect the glycosylated IL-15/IL-15R complex. Competitor B’s ELISA displayed an approximately 50% decrease in calibration dose response. The 41702 assay standard curve is presented for reference purposes.

 

Healthy Donor Readability Comparison

Sample Type Quantifiable Mean (pg/ml) Range (pg/ml) % above LOQ % below LOQ %CV

PBL's ELISA

Serum 4.21 2.64– 13.75 100
(30/30)
0
(0/30)
3.0
EDTA Plasma 3.01 1.75– 4.17

100

(17/17)

0

(0/17)

2.4
Citrate Plasma 2.42 1.41– 3.99

100

(13/13)

0

(0/13)

3.1
Heparin Plasma 2.63 1.62– 3.87

100

(13/13)

0

(0/13)

2.9

Competitor A's ELISA

Serum 91.51 17.84– 244.51

30

(9/30)

70

(21/30)

30.1
EDTA Plasma 35.13 8.89– 126.25

47.1

(8/17)

52.9

(9/17)

10.7
Citrate Plasma 22.00 10.56– 38.40

46

(6/13)

54

(7/13)

14.7
Heparin Plasma 58.48 40.31-76.66

20

(2/10)

80

(8/10)

5.0

Competitor B's ELISA

Serum 5.78 4.13– 8.11

10

(3/30)

90

(27/30)

6.1
EDTA Plasma 4.74 N/A

5.9

(1/17)

94.1

(16/17)

N/A
Citrate Plasma N/A N/A

0

(0/13)

100

(13/13)

N/A
Heparin Plasma N/A N/A

0

(0/10)

100

(10/10)

N/A

Competitor C's ELISA

Serum N/A N/A

0

(0/30)

100

(30/30)

N/A
EDTA Plasma N/A N/A

0

(0/18)

100

(18/18)

N/A
Citrate Plasma N/A N/A

0

(0/12)

100

(12/12)

N/A
Heparin Plasma N/A N/A

100

(0/10)

0

(10/10)

N/A

 

 

Background

 

Interleukin-15 (IL-15) is a member of the IL-2 family of cytokines which also includes IL-4, -7, -9 and -211,2,3. It has similar and unique functions compared to IL-2. Signaling by IL-15 can be through the cytokine alone, in complex with soluble IL-15RAlpha and transpresented bound to IL15RAlpha. It is produced largely by monocytic cells but also by dendritic cells, keratinocytes, fibroblasts, myocytes, and nerve cells. It seems to enhance the expansion of CD8 memory cells, NK and NK-T cells4. IL-15 plays a role in tumor biology and autoimmune diseases including psoriasis, rheumatoid arthritis, inflammatory bowel disease, celiac disease, alopecia areata, and systemic lupus erythematosus5,6.

 

 

Citations

References:

 

  1. Liao W, Lin JX, Leonard WJ. IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation. Curr Opin Immunol. 2011;23(5):598-604. (link)
  2. Grabstein KH, et al. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science. 1994 May;264(5161):965-8. (link)
  3. Burton, JD, et al. A lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. PNAS. 1994;91:4935-4939. (link)
  4. Ring, AM, et al. Mechanistic and structural insight into the functional dichotomy between IL-2 and IL-15. Nat Immunol. 2012;13(12):1187-95. (link)
  5. Waldmann, TA. The biology of IL-15: implications for cancer therapy and the treatment of autoimmune disorders. J Investig Dermatol Symp Proc. 2013 Dec;16(1):S28-30. (link)
  6. Clark DN, et al. Cytokine inhibition as a strategy for treating systemic lupus erythematosus. Clin Immunol. 2013;148(3):335-43. (link)

Documentation

Certificate of Analysis (CoA) & Protocol, Technical Data Sheet (TDS), and Safety Data Sheet (SDS)
41702-1 CoA & Protocol (One-Page)

41702-1 Certificate of Analysis (CoA) & Protocol (One-Page)

41702-1 Technical Data Sheet

41702-1 Technical Data Sheet

41702 SDS

41702 Safety Data Sheet