{ "id": "ep1zze5wzm", "slug": "chemiluminescence-assay", "title": "Chemiluminescence assay", "updated": "2026-10-21", "topic_path": [ { "id": "life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/topics/life" }, { "id": "life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology" }, { "id": "life.biology.biochemistry", "label": "Biochemistry and metabolism", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.biochemistry" }, { "id": "life.biology.biochemistry.practice", "label": "Biochemistry field and methods", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.biochemistry.practice" }, { "id": "life.biology.biochemistry.practice.methods", "label": "Biochemical methods and techniques", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.biochemistry.practice.methods" }, { "id": "life.biology.biochemistry.practice.methods.detect", "label": "Detection methods and analytical reactions", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.biochemistry.practice.methods.detect" } ], "geo": [ { "id": "geo.nongeo.t1946.life.biology.biochemistry.practice.methods.detect", "label": "Non-geographic · 1946 to 2000: Detection methods and analytical reactions", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry.practice.methods.detect", "path": [ { "id": "geo.nongeo", "label": "Non-geographic", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo" }, { "id": "geo.nongeo.t1946", "label": "Non-geographic · 1946 to 2000", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946" }, { "id": "geo.nongeo.t1946.life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life" }, { "id": "geo.nongeo.t1946.life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology" }, { "id": "geo.nongeo.t1946.life.biology.biochemistry", "label": "Biochemistry and metabolism", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry" }, { "id": "geo.nongeo.t1946.life.biology.biochemistry.practice", "label": "Biochemistry field and methods", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry.practice" }, { "id": "geo.nongeo.t1946.life.biology.biochemistry.practice.methods", "label": "Biochemical methods and techniques", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry.practice.methods" }, { "id": "geo.nongeo.t1946.life.biology.biochemistry.practice.methods.detect", "label": "Detection methods and analytical reactions", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry.practice.methods.detect" } ] } ], "excerpt": "A chemiluminescence assay is a biochemical method that detects biomolecules like proteins and nucleic acids by measuring light from a chemical reaction, used in Western blots and clinical immunoassays.", "snippet": "A chemiluminescence assay is a biochemical method that detects biomolecules like proteins and nucleic acids by measuring light from a chemical reaction, used in Western blots and clinical immunoassays.", "node": "life.biology.biochemistry.practice.methods.detect", "markdown": "# Chemiluminescence assay\n\nA chemiluminescence assay is a biochemical detection method that quantifies or detects biomolecules such as proteins and nucleic acids by measuring light emitted from a chemical reaction. An enzyme label or a chemiluminescent tag on an antibody or probe converts analyte binding into photon output, which the instrument reports as relative light units (RLU) and which a standard curve converts into a calibrated concentration. Chemiluminescent detection underlies routine Western blotting, bench ELISAs, and the automated immunoassay analyzers used in clinical laboratories.\n\n| Key fact | Detail |\n|---|---|\n| Measured output | RLU, which can correlate with the concentration of the analyte over a validated working range |\n| Core reactions | Luminol/HRP emitting at 425 nm [1](https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/chemiluminescent-western-blotting/); dioxetane/ALP phenolate emitting near 470 nm; acridinium ester flash chemistry [2](https://pmc.ncbi.nlm.nih.gov/articles/PMC6189797/) |\n| Sensitivity | Analytical detection limits down to one zeptomole (10\\(^{-21}\\) mol) in luminescent immunoassays [3](https://webstore.ansi.org/preview-pages/CLSI/preview_I+LA23-A.pdf); 0.01 attomole of alkaline phosphatase with AMPPD on membrane |\n| Dynamic range | RLUs from hundreds to millions on typical plate readers [4](https://www.alpco.com/resources/a-comparison-of-colorimetric-and-chemiluminescence-elisas); linear response reported over up to six orders of magnitude [5](https://www.sciencedirect.com/science/article/abs/pii/S0039914099002945) |\n| Kinetics | Flash systems peak within seconds or milliseconds; glow systems emit steadily for many minutes [6](https://www.labcluster.com/news4_4/ELISA_Episode3.pdf) |\n| Instrumentation | Luminometer or PMT-based plate reader, CCD imager, or X-ray film [7](https://www.scientificlabs.co.uk/handlers/libraryFiles.ashx?filename=Manuals_R_RPN2235_A.pdf) |\n| First immunoassay label | A stable chemiluminescent-labeled antibody for immunological assays, reported by J. S. A. Simpson and colleagues in Nature, 1979 [8](https://doi.org/10.1038/279646a0) |\n\n## How it works\n\nChemiluminescence is light produced by a chemical reaction whose energy release populates an excited electronic state; emission of blue light requires roughly 300 kJ·mol\\(^{-1}\\) and red light roughly 150 kJ·mol\\(^{-1}\\).[5](https://www.sciencedirect.com/science/article/abs/pii/S0039914099002945) Three reaction families dominate bench and clinical work.\n\n**Luminol with peroxidase.** [Horseradish peroxidase](https://www.edgepedia.org/horseradish-peroxidase) (HRP) oxidizes luminol in the presence of hydrogen peroxide to an excited 3-aminophthalate dianion, which emits a photon as it returns to the ground state [9](https://pmc.ncbi.nlm.nih.gov/articles/PMC5959035/):\n\n\\[ \\text{luminol} + \\mathrm{H_2O_2} \\rightarrow [\\text{3-APA}^{*}] \\rightarrow \\text{3-APA} + h\\nu \\]\n\nThe product emits at 425 nm.[1](https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/chemiluminescent-western-blotting/) Enhancers such as 4-[iodophenol](https://www.edgepedia.org/iodophenol) and 4-(imidazol-1-yl)phenol act as electron-transfer mediators; the phenoxyl radical they form intensifies the reaction and converts a flash into steady-state emission over many minutes.[10](https://cris.unibo.it/retrieve/aad89bc2-b061-4def-870f-e93a8dfb19f7/Trac%20post%20print.pdf)\n\n**Dioxetane phosphate substrates.** [Alkaline phosphatase](https://www.edgepedia.org/alkaline-phosphatase) (ALP) removes the phosphate protecting group from an adamantylidene-dioxetane such as AMPPD; the unstable phenolate then undergoes chemiexcitation (a CIEEL-type electron exchange) and emits near 470 nm.[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC6565457/) Published emission wavelengths for the phenolate product are given as 466 nm [1](https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/chemiluminescent-western-blotting/) and 470 nm. The glow is stable for up to hours, which permits long exposures.\n\n**Acridinium esters.** These labels emit light on oxidation by hydrogen peroxide in alkaline solution without any enzyme, reaching maximum intensity in 0.4 s.[2](https://pmc.ncbi.nlm.nih.gov/articles/PMC6189797/)\n\nIn each format, CL signal can correlate with the concentration of the analyte or reaction components over a validated working range, and concentration is determined from an assay-specific calibration curve.\n\n## How it is done\n\n**Chemiluminescent ELISA (CLEIA).** A representative four-step sandwich protocol for the drug atezolizumab coats plates with PD-L1 (50 µL at 1 µg/mL, 37 °C, 1 h), blocks with 2% BSA, binds sample for 0.5 h, adds HRP-conjugated IgG for 0.5 h, then adds the luminol-based CL substrate and reads at 425 nm after 60 s.[12](https://pubs.rsc.org/en/content/articlelanding/2024/ra/d4ra00202d) [Calibration](https://www.edgepedia.org/calibration) uses standards fitted with 4- or 5-parameter logistic curves to convert RLU to concentration [4](https://www.alpco.com/resources/a-comparison-of-colorimetric-and-chemiluminescence-elisas); detection limits are commonly computed as \\( \\mathrm{LOD} = 3.3 \\cdot \\mathrm{SD}_{a}/b \\) and \\( \\mathrm{LOQ} = 10 \\cdot \\mathrm{SD}_{a}/b \\), where \\( \\mathrm{SD}_{a} \\) is the standard deviation of the calibration intercept and \\( b \\) its slope.[12](https://pubs.rsc.org/en/content/articlelanding/2024/ra/d4ra00202d)\n\n**ECL Western blotting.** After incubating the membrane with HRP-conjugated secondary antibody, the substrate (for example 20X LumiGLO plus peroxide diluted to 1X) is applied for about 1 min, and the wrapped membrane is exposed to X-ray film for seconds to minutes.[13](https://www.cellsignal.com/products/wb-ip-reagents/20x-lumiglo-reagent-and-20x-peroxide/7003) Enhanced substrates such as Amersham ECL Select are applied at 0.1 mL/cm\\(^{2}\\) for 5 min and imaged on a CCD camera or film, starting with a 1-minute exposure; low-picogram targets tolerate primary antibodies diluted 1:5000 to 1:30000.[7](https://www.scientificlabs.co.uk/handlers/libraryFiles.ashx?filename=Manuals_R_RPN2235_A.pdf)\n\n**Cell-based luminol/HRP assay.** To measure extracellular hydrogen peroxide, about 1 × 10\\(^{6}\\) cells are suspended in 1 mL buffer with 1 µM beta-lapachone, 10 µg/mL HRP, and 10 µM luminol, with or without 500 units/mL catalase, and read in a luminometer at 37 °C for 30 min. Because added HRP does not cross cell membranes, the signal reports extracellular H\\(_2\\)O\\(_2\\) flux, and catalase-inhibitable signal confirms specificity.[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC5959035/) Absolute values require a standard curve of known H\\(_2\\)O\\(_2\\) amounts.[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC5959035/)\n\n## Origin\n\nAlbrecht reported the chemiluminescence of luminol (5-amino-2,3-dihydrophtalazine-1,4-dione) in Zeitschrift für Physikalische Chemie in 1928 [14](https://doi.org/10.1515/zpch-1928-13625), and this luminol/H\\(_2\\)O\\(_2\\) reaction seeded later work on dioxetane, acridinium ester, and luminol labels.[10](https://cris.unibo.it/retrieve/aad89bc2-b061-4def-870f-e93a8dfb19f7/Trac%20post%20print.pdf) Kopecky and Mumford synthesized 3,3,4-trimethyl-1,2-dioxetane and described its luminescent thermal decomposition in the Canadian Journal of Chemistry in 1969.[15](https://doi.org/10.1139/v69-114) The decisive step for bioanalysis came when Schaap, Sandison, and Handley reported alkaline phosphatase-catalyzed chemiluminescence from an aryl phosphate-substituted dioxetane in Tetrahedron Letters in 1987, creating triggerable dioxetanes.[16](https://doi.org/10.1016/s0040-4039%2800%2995314-0) Bronstein, Edwards, and Voyta then synthesized AMPPD and AMPGD, dioxetane substrates activated at 470 nm by alkaline phosphatase and β-D-galactosidase respectively, in the Journal of Bioluminescence and [Chemiluminescence](https://www.edgepedia.org/chemiluminescence) in 1989, and Schaap, Akhavan, and Romano applied such substrates to ultrasensitive immunoassays and DNA probes in Clinical Chemistry the same year.[17](https://doi.org/10.1093/clinchem/35.9.1863) For the peroxidase side, Thomas P. Whitehead and colleagues introduced the enhanced luminescence procedure in Nature in 1983 [18](https://doi.org/10.1038/305158a0), and J. S. A. Simpson and colleagues had reported a stable chemiluminescent-labeled antibody in Nature in 1979.[8](https://doi.org/10.1038/279646a0) Adam, Reinhardt, and Saha-Möller later retraced how AMPPD was rationally designed from firefly bioluminescence principles in [The Analyst](https://www.edgepedia.org/the-analyst) in 1996, noting that in sensitivity it surpassed hazardous radioactive immunoassay probes.[19](https://doi.org/10.1039/an9962101527)\n\n## Variants\n\n**CLIA (chemiluminescent immunoassay).** Acridinium-ester labels dominate automated clinical immunoassays because of their exceptional stability and attomole-range detection limits.[10](https://cris.unibo.it/retrieve/aad89bc2-b061-4def-870f-e93a8dfb19f7/Trac%20post%20print.pdf) An automated ACS:180 analyzer produced up to 130 results per hour. Nanoparticle-based formats speed the workflow: a GoldMag nanoparticle sandwich CLIA for HE4 ran in under 1 hour, versus 2 to 3 h for microplate CLEIA.[2](https://pmc.ncbi.nlm.nih.gov/articles/PMC6189797/)\n\n**Enhanced luminol ECL Western blotting.** HRP-conjugated antibodies with phenol-enhanced luminol substrate are the standard protein-gel readout, captured on film or CCD.[1](https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/chemiluminescent-western-blotting/) CCD imagers offer high sensitivity, broad dynamic range, and better quantification than X-ray film.[7](https://www.scientificlabs.co.uk/handlers/libraryFiles.ashx?filename=Manuals_R_RPN2235_A.pdf)\n\n**Dioxetane ALP substrates.** AMPPD (disodium salt) is the substrate most commonly paired with alkaline phosphatase, and AMPGD the analogous galactosidase substrate.[20](https://biomeda.com/ELISA-Introduction/elisa-detection-strategies/elisa-chemiluminescent-assay.html) Later variants in the same lineage, such as CDP-Star, are used as ALP substrates; CDP-Star carries drawbacks of high purification cost and low stability.[10](https://cris.unibo.it/retrieve/aad89bc2-b061-4def-870f-e93a8dfb19f7/Trac%20post%20print.pdf)\n\n**Chemiluminescent nucleic acid detection.** CL labels for DNA analysis include acridinium esters, enzymes, nanoparticles, DNAzymes, and luminol systems.\n\n**Recent developments.** Replacing the spiro-adamantyl unit with a spiro-cyclobutyl-dioxetane accelerated chemiexcitation and lowered the β-galactosidase LoD 125-fold relative to the adamantyl analog, and adding an electron-withdrawing group to the phenolate donor gave a 3000-fold increase in quantum yield under physiological conditions.[10](https://cris.unibo.it/retrieve/aad89bc2-b061-4def-870f-e93a8dfb19f7/Trac%20post%20print.pdf) A fully automated paper-based smartphone CL immunoassay using 4-IMP-enhanced HRP-luminol-H\\(_2\\)O\\(_2\\) detection reached a rabbit IgG LoD of 62.4 pg/mL (3.53 pM) with a 30-second exposure.[21](https://livrepository.liverpool.ac.uk/3191480/1/2025%20Jihong%20ACA%20344013.pdf)\n\n## Applications\n\nChemiluminescent detection is routine in bench ELISAs and Western blots, in automated clinical immunoassay analyzers, and in DNA probe assays.[17](https://doi.org/10.1093/clinchem/35.9.1863) Representative performance: the atezolizumab CLEIA reached an LoD of 12.5 pg/mL and a working range of 25 to 800 pg/mL [12](https://pubs.rsc.org/en/content/articlelanding/2024/ra/d4ra00202d), and the HE4 nanoparticle CLIA covered 0.25 to 50 ng/mL with a detection limit of 0.084 ng/mL.[2](https://pmc.ncbi.nlm.nih.gov/articles/PMC6189797/) [Western blot](https://www.edgepedia.org/western-blot) detection of picogram amounts is routine, and femtogram amounts may be detectable with optimization.[1](https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/chemiluminescent-western-blotting/)\n\n## Limitations and alternatives\n\n**Failure modes.** Luminescent substrates hydrolyze spontaneously, so background rises with time and blank wells must be subtracted at each time point.[22](https://content.abcam.com/content/dam/abcam/product/documents/233/ab233466/Alkaline-Phosphatase-Assay-Kit-protocol-book-v2a-ab233466%20%28website%29.pdf) Home-brew luminol systems require pH stabilized near 8.5 to balance peroxidase activity (optimal at pH 5.5) against light emission (optimal at pH 12.0).[6](https://www.labcluster.com/news4_4/ELISA_Episode3.pdf) Acridinium esters form a non-emissive pseudobase; adding 0.1 M HNO\\(_3\\) before NaOH raised RLU 5 to 10 fold by inhibiting this.[2](https://pmc.ncbi.nlm.nih.gov/articles/PMC6189797/) Schaap-type dioxetanes are quenched by water, limiting CL efficiency in aqueous solution.[10](https://cris.unibo.it/retrieve/aad89bc2-b061-4def-870f-e93a8dfb19f7/Trac%20post%20print.pdf) Film's limited dynamic range makes film-based ECL data less quantitative than CCD imaging [23](https://content.abcam.com/content/dam/abcam/product/documents/133/ab133409/Optiblot-ECL-Ultra-Substrate-Kit-protocol-book-ab133409%20%28website%29.pdf), and chemiluminescent Western blots cannot be multiplexed, only stripped and re-probed.[1](https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/chemiluminescent-western-blotting/)\n\n**Kinetics and quantification.** Flash luminescence peaks within seconds or milliseconds and must be triggered and read inside the instrument at a constant timing interval, whereas glow reactions can be started outside the reader; one bulletin recommends a 2-minute stabilization before reading glow plates.[6](https://www.labcluster.com/news4_4/ELISA_Episode3.pdf) Published signal-duration figures differ: one manufacturer states emission is maximal immediately after substrate exposure and continues up to 3 hours [13](https://www.cellsignal.com/products/wb-ip-reagents/20x-lumiglo-reagent-and-20x-peroxide/7003), while another reports substrate lasting up to 24 hours in some cases but only minutes at high analyte concentrations.[1](https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/chemiluminescent-western-blotting/)\n\n**Alternatives.** Radioisotopic immunoassays (RIA/IRMA) are limited to analyte concentrations above roughly 10\\(^{7}\\) molecules/mL, and enzymes catalyzing chemiluminescent reactions serve as labels of higher effective specific activity.[24](https://onlinelibrary.wiley.com/doi/10.1002/bio.1170040113) Colorimetric ELISAs are read near 450 nm with optical density capped between 0 and 4, whereas chemiluminescent RLUs range from hundreds to millions, giving broader dynamic range.[4](https://www.alpco.com/resources/a-comparison-of-colorimetric-and-chemiluminescence-elisas) Supplier claims of 10,000-fold sensitivity advantage over absorbance and 1,000-fold over fluorescence [20](https://biomeda.com/ELISA-Introduction/elisa-detection-strategies/elisa-chemiluminescent-assay.html) conflict with a head-to-head study of colorimetric, chemiluminescence, time-resolved fluorescence, and electrochemiluminescence platforms across five assay formats, which found that no detection platform consistently performed better than all the others and that vendor-claimed improvements in sensitivity or dynamic range were not observed.[25](https://www.sciencedirect.com/science/article/abs/pii/S002217591100144X) [Instrumentation](https://www.edgepedia.org/instrumentation) is a luminometer or PMT-based plate reader, a CCD imager, or film; PMT detectors need a 1 to 2 kV power supply and are fragile and costly, which motivates CMOS and smartphone readout.[21](https://livrepository.liverpool.ac.uk/3191480/1/2025%20Jihong%20ACA%20344013.pdf)\n\n## References\n\n1. [Chemiluminescent Western blotting (Jackson ImmunoResearch technical guide)](https://www.jacksonimmuno.com/secondary-antibody-resource/immuno-techniques/chemiluminescent-western-blotting/)\n2. [Preparation of an Acridinium Ester-Labeled Antibody and Its Application in GoldMag Nanoparticle-Based, Ultrasensitive Chemiluminescence Immunoassay for the Detection of Human Epididymis Protein 4](https://pmc.ncbi.nlm.nih.gov/articles/PMC6189797/)\n3. [CLSI I/LA23-A: Assessing the Quality of Immunoassay Systems (Radioimmunoassays and Enzyme, Fluorescence, and Luminescence Immunoassays)](https://webstore.ansi.org/preview-pages/CLSI/preview_I+LA23-A.pdf)\n4. [A Comparison of Colorimetric and Chemiluminescence ELISAs](https://www.alpco.com/resources/a-comparison-of-colorimetric-and-chemiluminescence-elisas)\n5. [Chemiluminescence as diagnostic tool. A review](https://www.sciencedirect.com/science/article/abs/pii/S0039914099002945)\n6. [Selecting the Detection System - Colorimetric, Fluorescent, Luminescent Methods - ELISA Technical Bulletin No. 5](https://www.labcluster.com/news4_4/ELISA_Episode3.pdf)\n7. [Amersham ECL Select Western Blotting Detection Reagent instructions (RPN2235)](https://www.scientificlabs.co.uk/handlers/libraryFiles.ashx?filename=Manuals_R_RPN2235_A.pdf)\n8. [J. S. A. SIMPSON and colleagues (1979). A stable chemiluminescent-labelled antibody for immunological assays. Nature.](https://doi.org/10.1038/279646a0)\n9. [A Highly Sensitive Chemiluminometric Assay for Real-Time Detection of Biological Hydrogen Peroxide Formation](https://pmc.ncbi.nlm.nih.gov/articles/PMC5959035/)\n10. [TrAC review on chemiluminescent new labels (post-print, 2024)](https://cris.unibo.it/retrieve/aad89bc2-b061-4def-870f-e93a8dfb19f7/Trac%20post%20print.pdf)\n11. [Development and Applications of Bioluminescent and Chemiluminescent Reporters and Biosensors](https://pmc.ncbi.nlm.nih.gov/articles/PMC6565457/)\n12. [A novel ultrasensitive chemiluminescence enzyme immunoassay ... for the quantitation of atezolizumab (RSC Advances, 2024)](https://pubs.rsc.org/en/content/articlelanding/2024/ra/d4ra00202d)\n13. [20X LumiGLO Reagent and 20X Peroxide (Cell Signaling Technology)](https://www.cellsignal.com/products/wb-ip-reagents/20x-lumiglo-reagent-and-20x-peroxide/7003)\n14. [Herbert Otto Albrecht (1928). Über die Chemiluminescenz des Aminophthalsäurehydrazids. Zeitschrift für Physikalische Chemie.](https://doi.org/10.1515/zpch-1928-13625)\n15. [Karl R. Kopecky, Cedric Mumford (1969). Luminescence in the thermal decomposition of 3,3,4-trimethyl-1,2-dioxetane. Canadian Journal of Chemistry.](https://doi.org/10.1139/v69-114)\n16. [Chemical and enzymatic triggering of 1,2-dioxetanes. 3: alkaline phosphatase-catalyzed chemiluminescence from an aryl phosphate-substituted dioxetane (Tetrahedron Letters, 1987)](https://doi.org/10.1016/s0040-4039%2800%2995314-0)\n17. [A P Schaap, H Akhavan, L J Romano (1989). Chemiluminescent substrates for alkaline phosphatase: application to ultrasensitive enzyme-linked immunoassays and DNA probes.. Clinical Chemistry.](https://doi.org/10.1093/clinchem/35.9.1863)\n18. [Thomas P. Whitehead and colleagues (1983). Enhanced luminescence procedure for sensitive determination of peroxidase-labelled conjugates in immunoassay. Nature.](https://doi.org/10.1038/305158a0)\n19. [Waldemar Adam, Dirk Reinhardt, Chantu R. Saha-Möller (1996). From the firefly bioluminescence to the dioxetane-based (AMPPD) chemiluminescence immunoassay: a retroanalysis. The Analyst.](https://doi.org/10.1039/an9962101527)\n20. [ELISA Chemiluminescent Assay detection strategies (Biomeda)](https://biomeda.com/ELISA-Introduction/elisa-detection-strategies/elisa-chemiluminescent-assay.html)\n21. [A Fully Automated Paper-Based Smartphone-assisted chemiluminescence immunoassay](https://livrepository.liverpool.ac.uk/3191480/1/2025%20Jihong%20ACA%20344013.pdf)\n22. [Alkaline Phosphatase Assay Kit protocol book v2a ab233466 (website) (content.abcam.com)](https://content.abcam.com/content/dam/abcam/product/documents/233/ab233466/Alkaline-Phosphatase-Assay-Kit-protocol-book-v2a-ab233466%20%28website%29.pdf)\n23. [Optiblot ECL Ultra Substrate Kit protocol book ab133409 (website) (content.abcam.com)](https://content.abcam.com/content/dam/abcam/product/documents/133/ab133409/Optiblot-ECL-Ultra-Substrate-Kit-protocol-book-ab133409%20%28website%29.pdf)\n24. [Ekins (1989), High specific activity chemiluminescent and fluorescent markers, Journal of Bioluminescence and Chemiluminescence](https://onlinelibrary.wiley.com/doi/10.1002/bio.1170040113)\n25. [Comparison of four distinct detection platforms using multiple ligand binding assay formats](https://www.sciencedirect.com/science/article/abs/pii/S002217591100144X)\n\n---\n*Topic: Encyclopedia › Life and health › Biological foundations › Biochemistry and metabolism › Biochemistry field and methods › Biochemical methods and techniques › Detection methods and analytical reactions*\n\n*Initially written Sep 29, 2026 · Reviewed: Sep 30, 2026 · Edited: Sep 30, 2026; Oct 15, 2026; Oct 21, 2026 · Last review: Sep 30, 2026*\n\n*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*\n\nLicense: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license\n", "same_as": [], "url": "https://www.edgepedia.org/chemiluminescence-assay", "markdown_url": "https://www.edgepedia.org/chemiluminescence-assay.md", "license": { "name": "Edgepedia Community License 1.0", "url": "https://www.edgepedia.org/edgepedia/license", "summary": "Free with credit, commercial use included. 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Edgepedia Community License 1.0.", "speakable": "A chemiluminescence assay is a biochemical method that detects biomolecules like proteins and nucleic acids by measuring light from a chemical reaction, used in Western blots and clinical immunoassays." }