Enzyme Linked Immuno Mass Spectrometric Assay (ELIMSA)

Florentinus-Mefailoski, Angelique; Safi, Frozan; Marshall, John

doi:10.1016/j.jprot.2013.11.022

Cited by 26 publications

(36 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previously, the alkaline phosphatase (AP) enzyme system was more sensitive than the HRP system that requires the strong oxidizing agent H 2 O 2 as a cofactor, 3 and so the development of the AP based ELIMSA was continued by looking for more effective substrates. The use of the alkaline phosphatase substrate PA5P was found to be a strong improvement on the previous substrate naphthol AS-MX phosphate in that the latter's hydrophobic naphthol substituent causes distinct tailing in the chromatographic peaks with the result that baseline to baseline separation is not always obtained at convenient injection intervals (≤5 min.)…”

Section: ■ Resultsmentioning

confidence: 99%

“…It was previously demonstrated that ELISA and ELIMSA gave results that were linear and consistent in the nanogram per well range by direct comparison to colorimetric assays. 3 It seems that ELIMSA may be used to absolutely quantify PSA proteins at picograms per well. Since all the normal samples analyzed here were no less than 33 pg per well, all samples were well within the quantitative range of the assay as measured against absolute PSA standards.…”

Section: ■ Discussionmentioning

confidence: 99%

“…The PSA ELIMSA was performed as previously described but with the substitution of the substrate PA5P instead of naphthol AS-MX phosphate. 3 The PA5P substrate, PSA standards and AP-SA probe were dissolved in water on ice, aliquoted, and freeze-dried for day-to-day consistency in the assay. The plates were coated with the capture antibody in bicarbonate coating buffer prior to blocking the plates in 1% albumin and 1% goat serum in 1× PBS pH 7.4 followed by incubating the plasma in 1× PBS, 0.3 M NaCl pH 7.4 for 2 h. After the wells were washed again three times with washing buffer, the substrate was reacted with the streptavidin− AP in substrate buffer (20 mM Tris pH 8.8).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…If mass spectrometry is log linear and normal for PA, then instead of using UV−vis spectroscopy to read the 96 well ELISA plate, the production of the colorless PA could be measured using LC−ESI-MS with a sensitive ion trap. 3 The capacity to measure zeptomole amounts of blood proteins may have great application to biomedical research and permits the common analysis of many ultra low abundance analytes.…”

Section: ■ Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Pyridoxamine-5-phosphate Enzyme-Linked Immune Mass Spectrometric Assay Substrate for Linear Absolute Quantification of Alkaline Phosphatase to the Yoctomole Range Applied to Prostate Specific Antigen

Florentinus-Mefailoski

Marshall

2014

Anal. Chem.

Self Cite

View full text Add to dashboard Cite

There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap.

show abstract

Section: ■ Resultsmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Pyridoxamine-5-phosphate Enzyme-Linked Immune Mass Spectrometric Assay Substrate for Linear Absolute Quantification of Alkaline Phosphatase to the Yoctomole Range Applied to Prostate Specific Antigen

Florentinus-Mefailoski

Marshall

2014

Anal. Chem.

Self Cite

View full text Add to dashboard Cite

show abstract

“…In a recently described technology, ELISA has been combined with MS-based quantification of the enzymatic products. The technology is called enzyme-linked immuno mass spectrometric assay and may provide increased analytical sensitivity, as compared to regular ELISA, by reducing the background [2] . Immunochemical assays may also be multiplexed in different ELISA-like formats.…”

Section: Introductionmentioning

confidence: 99%

Update on ultrasensitive technologies to facilitate research on blood biomarkers for central nervous system disorders

Andréasson

Blennow

Zetterberg

2016

Alz & Dem Diag Ass & Dis Mo

View full text Add to dashboard Cite

Most research on fluid biomarkers for central nervous system (CNS) disorders has so far been performed using cerebrospinal fluid (CSF) as the biomarker source. CSF has the advantage of being closer to the brain than serum or plasma with a relative enrichment of CNS-specific proteins that are present at very low concentrations in the blood and thus difficult to reliably quantify using standard immunochemical technologies. Recent technical breakthroughs in the field of ultrasensitive assays have started to change this. Here, we review the most established ultrasensitive quantitative technologies that are currently available to general biomarker laboratories and discuss their use in research on biomarkers for CNS disorders.

show abstract

An enzyme-linked immuno-mass spectrometric assay with the substrate adenosine monophosphate

Florentinus-Mefailoski

Soosaipillai

Dufresne

et al. 2014

Anal Bioanal Chem

View full text Add to dashboard Cite

An enzyme-linked immuno-mass spectrometric assay (ELIMSA) with the specific detection probe streptavidin conjugated to alkaline phosphatase catalyzed the production of adenosine from the substrate adenosine monophosphate (AMP) for sensitive quantification of prostate-specific antigen (PSA) by mass spectrometry. Adenosine ionized efficiently and was measured to the femtomole range by dilution and direct analysis with micro-liquid chromatography, electrospray ionization, and mass spectrometry (LC-ESI-MS). The LC-ESI-MS assay for adenosine production was shown to be linear and accurate using internal (13)C(15)N adenosine isotope dilution, internal (13)C(15)N adenosine one-point calibration, and external adenosine standard curves with close agreement. The detection limits of LC-ESI-MS for alkaline phosphatase-streptavidin (AP-SA, ∼190,000 Da) was tested by injecting 0.1 μl of a 1 pg/ml solution, i.e., 100 attograms or 526 yoctomole (5.26E-22) of the alkaline-phosphatase labeled probe on column (about 315 AP-SA molecules). The ELIMSA for PSA was linear and showed strong signals across the picogram per milliliter range and could robustly detect PSA from all of the prostatectomy patients and all of the female plasma samples that ranged as low as 70 pg/ml with strong signals well separated from the background and well within the limit of quantification of the AP-SA probe. The results of the ELIMSA assay for PSA are normal and homogenous when independently replicated with a fresh standard over multiple days, and intra and inter diem assay variation was less than 10 % of the mean. In a blind comparison, ELIMSA showed excellent agreement with, but was more sensitive than, the present gold standard commercial fluorescent ELISA, or ECL-based detection, of PSA from normal and prostatectomy samples, respectively.

show abstract

Enzyme Linked Immuno Mass Spectrometric Assay (ELIMSA)

Cited by 26 publications

References 38 publications

Pyridoxamine-5-phosphate Enzyme-Linked Immune Mass Spectrometric Assay Substrate for Linear Absolute Quantification of Alkaline Phosphatase to the Yoctomole Range Applied to Prostate Specific Antigen

Pyridoxamine-5-phosphate Enzyme-Linked Immune Mass Spectrometric Assay Substrate for Linear Absolute Quantification of Alkaline Phosphatase to the Yoctomole Range Applied to Prostate Specific Antigen

Update on ultrasensitive technologies to facilitate research on blood biomarkers for central nervous system disorders

An enzyme-linked immuno-mass spectrometric assay with the substrate adenosine monophosphate

Contact Info

Product

Resources

About