Chemiluminescent Alkaline Phosphatase ELISA Systems

Catalog nos. C10552, C10553, C10554, C10555, C10556

Table 1. Contents and storage information.

Amount
Material Composition Storage†
C10552 C10553 C10554 C10555 C10556‡

10X assay buffer 200 mM Tris (pH 9.8),

100 mL 100 mL 100 mL 100 mL 100 mL
(Component A) 10 mM MgCl2

Blocking reagent Highly purified casein,

7.5 g 7.5 g 7.5 g 7.5 g 7.5 g
(Component B) dry powder

CSPD® substrate/
Sapphire‑II™ enhancer 100 mL – – – 25 mL
(Component C) • 2–6°C
CSPD® substrate/ • Dessicate
0.4 mM ready‑to‑use • Protect
Emerald-II™ enhancer CSPD® or CDP‑Star® – 100 mL – – 25 mL
(Component C) from light
substrate containing
CDP-Star® substrate/ 10% (v/v)Sapphire‑II™
Sapphire‑II™ enhancer or Emerald‑II™ – – 100 mL – 25 mL
(Component C) enhancer

CDP-Star® substrate/
Emerald-II™ enhancer – – – 100 mL 25 mL
(Component C)

†These storage conditions are appropriate when storing the entire kit upon receipt. For optimal storage conditions for each component,
see labels on the individual vials. When stored as directed, this kit is stable for at least 1 year.
‡C10556 is a sampler kit that contains 25 mL of each of the four substrate/enhancer combinations (Components C–F).

Number of assays: Sufficient material is supplied for 1,000 assays based on the protocol below.

Introduction

Description of the System
The Chemiluminescent Alkaline Phosphatase ELISA detection system incorporates CSPD® or
CDP-Star® 1,2-dioxetane substrates for alkaline phosphatase with Sapphire-II™ or Emerald‑II™
enhancer in a system designed for rapid and ultrasensitive analyte detection in enzyme-linked
immunoassays.1–7 Light emission from alkaline phosphatase-activated CSPD® or CDP-Star®
substrate is in the form of a “glow”. Maximum light emission is reached in 5 to 60 minutes,
depending on the temperature and the substrate chosen. Enzymatic dephosphorylation
of substrate occurs at a constant rate proportional to enzyme concentration; the resulting
anion decomposes with a finite half-life. Light emission can be quantitated with a variety of
luminometers without the need for solution injection. Enzyme-linked immunosorbent assays
(ELISAs) can be formatted in several configurations on a variety of solid supports including

Revised: 12–June–2009  |  MP 10552

 microplate wells, tubes, polystyrene beads, or ferrite particles.

The high sensitivity obtained with 1,2-dioxetane substrates is demonstrated in a sandwich

immunoassay format ELISA that employs a biotinylated detector antibody and streptavidin-
alkaline phosphatase conjugate for quantitating recombinant human IL-6 (rhIL-6). The
results obtained with CSPD® substrate/Sapphire-II™ enhancer (Figure 1) show a significant
improvement in signal-to-noise performance at all concentrations of rhIL-6 and a much
wider assay dynamic range compared to those obtained with the fluorescent substrate
4-methylumbelliferyl phosphate (4-MUP), and the colorimetric substrate, p-nitrophenyl
phosphate (pNPP). This benefit can be expected when any colorimetric ELISA is converted to
1,2-dioxetane/enhancer chemiluminescence.

10,000

1,000

CSPD/Sapphire II
S/N

100 pNPP
4-MUP

10

1
0.001 0.01 0.1 1 10 100 1,000 10,000

rhIL-6 Conc (pg/mL)

Figure 1. Comparison of Chemiluminescent, Fluorescent, and Colorimetric Detection for ELISA Quantitation of rhIL-6..

Sandwich Immunoassay A direct sandwich ELISA is used for the detection of large molecules with multiple antigenic
sites, usually proteins. In this format, a solid support is coated with a capture antibody
that immunoadsorbs the antigen from the sample; a detector antibody conjugated to
alkaline phosphatase, specific for a second site on the antigen, is then added. Alternatively,
an unlabeled or hapten-labeled detector antibody (not from the same species as the
capture antibody) can be used, followed by a secondary antibody, anti-hapten antibody, or
streptavidin-alkaline phosphatase conjugate. The enzyme-generated signal is proportional to
the amount of antigen.

Sandwich immunoassay formats with 1,2-dioxetane substrates have been used for the
quantitation of a variety of animal and human proteins from plasma and tissue extracts. 2,3,6–13
In a related assay format, antigen-coated solid support has been used with 1,2-dioxetanes for
detection of HTLV-I antibodies14 and calculation of antibody binding constants.15 CDP‑Star®
substrate with Sapphire-II™ enhancer or Emerald-II™ enhancer has become widely used for
immunoassay protein detection applications such as detection of plasma proteins16 and
viral antigens in both clinical serum samples17 and in cell culture media as a viral infection
assay.18,19

Competitive Immunoassay Competitive ELISA formats, in which a competition for available antibody binding sites
occurs between a labeled and an unlabeled antigen, are typically used for detection of
small molecules. 1,2-Dioxetanes have been utilized in these assays for detection of peptides
and hormones.20–22 Competitive assays generate an inverse standard curve; for increasing
concentrations of antigen, a decrease in signal is observed. Because the standard curve
in a competitive ELISA exhibits maximum signal at the lowest analyte concentration, it
may be necessary to adjust reagent concentrations to optimize detection of low analyte
concentrations. The sensitivity of chemiluminescent detection enables the use of lower
concentrations of capture antibody and competing antigen to generate a standard curve
which spans a much lower concentration range compared to colorimetric methods.

Chemiluminescent Alkaline Phosphatase ELISA Systems  |  2

 Whole-Cell ELISA
Protein Detection Applications
Nucleic Acid and Nucleic Acid-
Protein Interaction Detection
Applications
Before Starting
Materials Required but Not
Provided
Caution
Immunoassay detection of a surface antigen on whole cells has been demonstrated with
1,2-dioxetane chemiluminescent detection.23 β-Galactosidase enzyme conjugates can also
be used with Galacton-Star® substrate with Sapphire-II™ enhancer for chemiluminescent
immunoassay detection, particularly for whole cell ELISA applications that may exhibit high
levels of cellular alkaline phosphatase.24
Anti-phosphopeptide immunoassays with CSPD® or CDP-Star® substrates and Sapphire‑II™
or Emerald-II™ enhancers have been developed for quantitating several protein kinase
activities, including PKA, PKC, CAM-KII, receptor interacting protein and src kinases,25
WaaP protein tyrosine kinase and sugar kinase,26 and p38 kinase.27 In addition, a receptor
binding assay of a neurotrophic factor to a tyrosine kinase receptor,28 as well as viral foci
imaging with immunodetection,29 has been demonstrated. Quantitation of protein-protein
interactions with an ELISA assay30 and quantitation of siRNA-mediated protein knockdown31
have been demonstrated using the CDP-Star® substrate with Sapphire‑II™ enhancer.
DNA probe hybridization assays, DNA-protein interaction assays, and DNA aptamer binding
assays are often formatted in microplate wells or on other solid phases. Alkaline phosphatase
(AP)-labeled probes, hapten-labeled probes, or antibodies to DNA-DNA or DNA-RNA
duplexes32 can be used to detect hybridization or binding with AP-conjugated detection
reagents and 1,2-dioxetane substrates. Chemiluminescent enzyme-linked oligonucleotide
assay (ELONA) has been used to quantitate DNA aptamer binding to protein.33 CDP-Star®
substrate is used in microplate-based assay systems for detection of viral RNA or DNA by
immunodetection,34,35 quantitative detection of labeled PCR products,36 and ELISA-PCR for
mRNA quantitation.37 In addition, detection of chemical-DNA adducts in mammalian tissues
has been demonstrated with CDP‑Star® substrate with Emerald-II™ enhancer.38
• Appropriate antibodies, andtigens, antibody- or antigen-alkaline phosphatase conjugates,
or biotinylated antigens
• Phosphate buffered saline (PBS)
• Tween®-20 detergent
• Deionized water
• 96-well white (opaque) luminometer microplates
• Microplate luminometer
The 10X assay buffer (Component A) and the CSPD® or CDP-Star® substrates with
Sapphire‑II™ or Emerald-II™ enhancers (Components C–F) are irritating to eyes and skin.
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
Wear suitable protective clothing and eye/face protection.
Chemiluminescent Alkaline Phosphatase ELISA Systems  |  3
 General Guidelines • Ultrasensitive immunoassay detection is often limited by non-specific binding of enzyme
conjugates to the plates; high quality, purified conjugates are critical.25
• Detailed protocols for immunoassay formats and microplate coating are available in
published literature.26
• The light produced from the alkaline phosphatase catalyzed decomposition of CSPD® or
CDP-Star® substrate is a glow-type emission, and the incubation time required to achieve
maximum light emission is a function of alkaline phosphatase activity and temperature.
• The chemiluminescent assay for alkaline phosphatase activity is subject to interference by
hemoglobin and by high concentrations of protein (greater than 11.6 g/L). Bilirubin (up to
173 μmol/L) and lipid (2–100 g/L) do not interfere with the assay.1
• Determine the optimal blocking reagents and conditions for the individual assay. Avoid
any materials which may contain contaminating alkaline phosphatase.
• Perform chemiluminescent ELISAs, which are read in a microplate luminometer, in
opaque white plates.
• Perform all steps at room temperature, unless otherwise indicated.
• Recommended volumes and terminology used in the protocols below are based upon use
of microplates.

Luminometers We recommend using a single-mode luminometer or a multi-mode detection instrument set

for luminescence measurement to measure light emission from 96- or 384-well microplates.
Measure light emission for 1 second per well, or as appropriate for the instrument.

Preparing Working Solutions Prepare all solutions with deionized water. Keep 10X PBS sterile at room temperature

1X Assay Buffer
1.1 Dilute 10X assay buffer (Component A) 1:10 in deionized water before use. Store at 4°C.

Blocking Buffer
1.2 Dilute 3 mL of 10X PBS 1:10 in 27 mL of deionized water, and microwave for 40 seconds.
Do not boil.

1.3 Add 0.06 g of blocking reagent (Component B) and allow the solution to cool.

1.4 After the solution has cooled, add 15 μL of Tween®-20 detergent. Cool to room temperature
before use. The solution will remain opaque, but particles will be dissolved.

Wash Buffer
1.5 Add 50 μL of Tween®-20 detergent to 10 mL of 10X PBS, and mix well.

1.6 Add deionized water to 100 mL.

Coating Microplates Empirically determine the optimal coating buffer and protein concentration (usually 0.2 to
10 μg/mL).

2.1 Add 50–100 μL coating solution per well, seal plate, and incubate for 2 hours at 37°C or
overnight at room temperature or 4°C.

Chemiluminescent Alkaline Phosphatase ELISA Systems  |  4

Experimental Protocols

Direct Sandwich ELISA Direct sandwich immunoassays are the most sensitive format for detecting soluble antigen.
We recommend this format for antigens for which two distinct antibodies are available. Many
variations on this format are possible, including monoclonal antibody screening of hybridoma
culture supernatants.

3.1 Coat plate with capture antibody (see step 2.1), then wash the coated plate 3 times with wash
buffer (steps 1.5–1.6) by filling wells with wash buffer from a squirt bottle. Shake buffer into
a sink after each wash. After the last wash, remove residual liquid by tapping plate face down
on clean paper towels.

Note: If you are using automated 96-well microplate washer instrumentation, fill the wells to
slightly below well capacity.

3.2 Incubate wells with blocking buffer (steps 1.2–1.4) for 1 hour at room temperature or
overnight at 4°C.

3.3 Wash blocked plate 3 times with wash buffer following the same wash procedure as in
step 3.1.

3.4 Dilute antigen samples in blocking buffer, and add 100 μL of the diluted antigen into each
well. Incubate for 1 hour with shaking at room temperature.

3.5 Wash wells 3 times with wash buffer following the same wash procedure as in step 3.1.

3.6 Dilute detector antibody-alkaline phosphatase (AP) conjugate in blocking buffer, and add
100 μL of the dilution into each well. Incubate for at least 1 hour with shaking at room
temperature.

Note: Determine the optimal dilution of antigen samples empirically.

3.7 Wash wells 4 times with wash buffer, then twice with 1X assay buffer (prepared in step 1.1).

3.8 Add 100 μL of substrate/enhancer solution (Component C) into each well and incubate for
10 minutes at room temperature.

3.9 Measure chemiluminescence in a luminometer at 10 minute intervals until light emission has
reached plateau.

Competitive ELISA using

Antigen-Coated Plates This assay format is useful for detection of small antigen molecules, such as therapeutic
drugs, for which a specific antibody and milligram quantities of antigen are available.

4.1 Coat plate with antigen (see step 2.1), then wash the coated plate 3 times with wash buffer
(steps 1.5–1.6) by filling wells with wash buffer from a squirt bottle. Shake buffer into a sink
after each wash. After the last wash, remove residual liquid by tapping plate face down on
clean paper towels.

Note: If you are using automated 96-well microplate washer instrumentation, fill the wells to
slightly below well capacity.

4.2 Incubate wells with blocking buffer (steps 1.2–1.4) for 1 hour at room temperature or
overnight at 4°C.

4.3 Wash blocked plate 3 times with wash buffer following the same wash procedure as in
step 4.1.

Chemiluminescent Alkaline Phosphatase ELISA Systems  |  5

 4.4 Dilute standard or test antigen samples separately in blocking buffer to 2X final
concentration.

4.5 Dilute antibody-alkaline phosphatase (AP) conjugate in blocking buffer to 2X final

concentration.

4.6 Add equal volumes of antigen dilution (from step 4.4) and antibody-(AP) conjugate
dilution (from step 4.5) to wells (50–75 μL/well). Incubate for 2 hours with shaking at room
temperature.

4.7 Wash wells 4 times with wash buffer, then once with 1X assay buffer (prepared in step 1.1).

4.8 Add 100 μL of substrate/enhancer solution (Component C) into each well and incubate for
10 minutes at room temperature.

4.9 Measure chemiluminescence in a luminometer at 10 minute intervals until light emission has
reached plateau.

Competitive ELISA using

Antibody-Coated Plates This assay format, which is recommended for small antigens that can be modified without
affecting antibody affinity, requires a biotinylated or alkaline phosphatase-conjugated antigen.

5.1 Coat plate with capture antibody (see step 2.1), then wash the coated plate 3 times with wash
buffer (steps 1.5–1.6) by filling wells with wash buffer from a squirt bottle. Shake buffer into
a sink after each wash. After the last wash, remove residual liquid by tapping plate face down
on clean paper towels.

Note: If you are using automated 96-well microplate washer instrumentation, fill the wells to
slightly below well capacity.

5.2 Incubate wells with blocking buffer (steps 1.2–1.4) for 1 hour at room temperature or
overnight at 4°C.

5.3 Wash blocked plate 3 times with wash buffer following the same wash procedure as in
step 5.1.

5.4 Dilute standard or test antigen samples separately in blocking buffer, and 100 μL of the
dilution per well. Incubate for 10 minutes with shaking at room temperature.

5.5 Dilute biotinylated antigen or antigen-alkaline phosphatase (AP) conjugate in blocking buffer,
and add 50 μL per well containing the diluted standard or test antigen samples (step 5.4).

5.6 Wash wells 4 times with wash buffer, then once with 1X assay buffer (prepared in step 1.1).

5.7 Add 100 μL of substrate/enhancer solution (Component C) into each well and incubate for
10 minutes at room temperature..

7.8 Measure chemiluminescence in a luminometer at 10 minute intervals until light emission has
reached plateau.

Chemiluminescent Alkaline Phosphatase ELISA Systems  |  6

References

1. J Biolumin Chemilumin 4, 99 (1989); 2. Clin Chem 35, 1441 (1989); 3. Clin Chem 37, 1526 (1991); 4. Immunochemical Assays and Biosensor
Technology for the 1990s (Nakamura, RM, Kasahara, Y and Rechnitz, GA, eds), American Society for Microbiology, Washington, DC (1992),
p. 229‑250; 5. Clin Chem 40, 347 (1994); 6. Clin Chem 35, 2319 (1989); 7. Methods in Molecular Biology, Vol. 63: Recombinant Proteins: Detection
and Isolation Protocols (Tuan, R, ed), Humana Press, Inc, Totowa, NJ (1997), p. 71-76; 8. Clin Chem 37, 1639 (1991); 9. Bioluminescence and
Chemiluminescence: Current Status (Stanley, PE and Kricka, LJ, eds), John Wiley, Chichester, England (2001), p. 115-118; 10. J immunol Methods
168, 111 (1994); 11. J Neurosci Res 99, 622 (2002); 12. J Molecular Endocrinol 32, 145 (2004); 13. Respiratory Research 8, 3 (2007); 14. Clin Chem 36,
1090 (1990); 15. J Biol Chem 270, 12446 (1995); 16. Endocrinol 143, 1166 (2002); 17. J Clin Microbiol 41, 1901 (2003); 18. J Virol 74, 6893 (2000);
19. J Virol 81, 7048 (2007); 20. Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. AK Campbell, LJ Kricka and PE
Stanley, editors. John Wiley & Sons, Chichester, England (1994), p. 321-324; 21. Steroids 60, 686 (1995); 22. J Pharm Biom Anal 14, 1653 (1996); 23.
J immunolog Methods 138, 129 (1991); 24. Biochemistry 38, 1866 (1999); 25. Anal Biochem 244, 340 (1997); 26. J Biol Chem 277, 4722 (2002); 27.
Nature Biotech 21, 302 (2003); 28. Porc Natl Acad Sci USA 94, 6238 (1997); 29. J Virolog Methods 66, 311 (1997); 30. J Mol Biol 300, 1323 (2000);
31. Mol Neurodegen 2, 4 (2007); 32. Clin Chem 39, 1934 (1993); 33. Nature Biotech 14, 1021 (1996); 34. J Clin Microbiol 38, 2150 (2000); 35.
Nuc Acids Res 26, 5230 (1998); 36. BMC Genetics (http://www.biomedcentral.coom/1471-2156/5/6) (2004); 37. J Immunol 166, 3749 (2001); 38.
Carcinogenesis 23, 2043 (2002).

Product List Current prices may be obtained from our website or from our Customer Service Department.

Cat. no. Product Name Unit Size

C10552 Chemiluminescent Alkaline Phosphatase ELISA Kit #1 *with CSPD® Substrate/Sapphire-II™ Enhancer* *1000 assays* . . . . . . . . . . . . . . . 1 kit
C10553 Chemiluminescent Alkaline Phosphatase ELISA Kit #2 *with CSPD® Substrate/Emerald-II™ Enhancer* *1000 assays* . . . . . . . . . . . . . . . . 1 kit
C10554 Chemiluminescent Alkaline Phosphatase ELISA Kit #3 *with CDP-Star® Substrate/Sapphire-II™ Enhancer* *1000 assays* . . . . . . . . . . . 1 kit
C10555 Chemiluminescent Alkaline Phosphatase ELISA Kit #4 *with CDP-Star® Substrate/Emerald-II™ Enhancer* *1000 assays* . . . . . . . . . . . . 1 kit
C10556 Chemiluminescent Alkaline Phosphatase ELISA Sampler Kit *1000 assays* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kit

Chemiluminescent Alkaline Phosphatase ELISA Systems  |  7

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Chemiluminescent Alkaline Phosphatase ELISA Systems  |  8