Insight into the "Signal Amplifier" of Life Activities: Exploring the Principle of the AMP Detection Kit

The core detection principle of the AMP detection kit is based on a clever two-step enzyme-linked reaction, with the ultimate goal of transforming AMP—which cannot be directly and sensitively detected—into a substance that is easy to detect and whose signal can be amplified through a series of conversions. The entire process is like a meticulously designed relay race.

MetabolismAMP Detection KitATPMedical Diagnosis

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Recent Advances
 
In fields such as life sciences, medical diagnosis, and food testing, accurately measuring intracellular energy molecules is key to understanding the state of life. Beyond the well-known ATP (Adenosine Triphosphate), the level of its direct precursor---AMP (Adenosine Monophosphate)---is also a crucial metabolic indicator. The AMP/ATP ratio serves as a "sensitive barometer" of cellular energy status, instantly reflecting cellular energy stress. The advent of AMP detection kits enables us to conveniently and highly sensitively quantify this key molecule. So, what kind of precise design lies behind it?
 
Core Principle: An Exquisite "Enzymatic Relay Race"
The core detection principle of the AMP detection kit is based on a clever two-step enzyme-linked reaction. Its ultimate goal is to transform AMP, which cannot be directly and sensitively detected, into a substance that is easy to detect and whose signal can be amplified, through a series of conversions. The entire process is like a meticulously designed relay race.
 
Step 1: From AMP to ADP -- Activating the "First Leg"
AMP itself cannot directly enter the general energy detection cycle. Therefore, the first step of detection is to "activate" it. This process is catalyzed by Adenylate Kinase (AK).
Reaction: AMP + ATP → 2 ADP
Function: AK uses one molecule of ATP as a phosphate group donor to phosphorylate one molecule of AMP, generating two molecules of ADP.
Significance: This step cleverly links the target AMP molecule to the energy currency ATP and converts it into ADP, an intermediate product in the energy metabolic pathway, preparing it for the next critical reaction.
 
Step 2: From ADP to ATP and Generating a Detectable Signal -- The Sprint and "Glow"
The generated ADP immediately enters a highly sensitive and established detection system. This system typically consists of Pyruvate Kinase (PK) and Lactate Dehydrogenase (LDH) forming a cyclic reaction, quantifying ATP (thus indirectly quantifying the original AMP) by monitoring the consumption of NADH.
Reaction Chain:
PK Reaction: ADP + Phosphoenolpyruvate (PEP) → ATP + Pyruvate
Pyruvate Kinase uses the high-energy phosphate bond of PEP to convert ADP back into ATP, simultaneously generating pyruvate.
LDH Reaction: Pyruvate + NADH + H⁺ → Lactate + NAD⁺
Lactate Dehydrogenase catalyzes the reduction of pyruvate to lactate, simultaneously consuming one molecule of NADH and converting it to NAD⁺.
Key to Signal Readout:
In this cycle, NADH is the "signal carrier." NADH has a strong absorption peak at a wavelength of 340 nm, while its oxidized product, NAD⁺, does not. Therefore, as the reaction proceeds, the concentration of NADH in the solution continuously decreases, and its absorbance at 340 nm decreases linearly accordingly.
The rate of this absorbance decrease is proportional to the amount of NADH consumed in the reaction. The amount of NADH consumed is proportional to the amount of ATP generated in the second step (i.e., the amount of ADP generated in the first step), and ultimately proportional to the initial AMP concentration in the sample.
 
Detection Workflow Overview (Using ANTBIO-Glo® AMP Assay Kit ANTBIO079024 as an example)

1. Enzyme Reaction Setup:
Perform the enzyme reaction in white opaque 96-well or 384-well plates. A reaction volume of 25 μL for 96-well plates and 5 μL for 384-well plates is recommended. Test compounds with concentration gradients can be added during the enzyme reaction.

Enzyme and substrate concentrations need optimization for different enzymatic reactions. An enzyme concentration within the linear signal response range can be used, depending on the required signal-to-noise ratio. Due to the high sensitivity of the AMP detection kit, enzyme usage can be significantly reduced.

If the enzyme reaction requires ATP, high-purity ATP should be used for optimal results. Some commercially available ATP contains ADP residues. Due to the high sensitivity of the AMP detection kit, ADP residues in ATP can cause high background. Therefore, high-purity ATP must be used in the enzyme reaction. The ATP provided with the kit or other high-purity ATP, such as Sigma-Aldrich ATP (Cat# A2383, purity ≥99%) or other higher purity ATP, can be used.
The enzyme reaction can use reaction buffers and cofactors reported in the literature or conditions optimized for the specific experiment.

The temperature and duration of the enzyme reaction should be set according to the specific enzyme. For high-throughput compound screening, optimizing the enzyme reaction at room temperature (22°C-25°C) is recommended to maintain temperature uniformity across the plate during the AMP detection process.

No additional reagents are needed to terminate the enzyme reaction after its completion. If specific experimental requirements necessitate a termination reagent, avoid using magnesium ion chelators like EDTA. The AMP detection reaction requires magnesium ions; the final magnesium ion concentration must be at least 5 mM.

2. Conversion of AMP to ADP and Removal of ATP after Enzyme Reaction
Take out the AMP GR Reagent and equilibrate it to room temperature (22°C-25°C). Mix gently by swirling [Notes 1, 2, 3].
If the enzyme reaction was performed at non-room temperature (e.g., 30°C), equilibrate the assay plate to room temperature [Note 4].
Add 25 µL of AMP GR Reagent to the 25 µL reaction in the 96-well plate (total volume 50 µL), or add 5 µL AMP GR Reagent to the 5 µL reaction in the 384-well plate (total volume 10 µL). Mix by orbital shaking [Note 5]
Incubate at room temperature for 40 minutes.

3. Enzyme Activity Measurement
Take out the AMP Detection Reagent and equilibrate it to room temperature. Mix gently by swirling [Note 4].
Add 50 µL of AMP Detection Reagent to the 50 µL reaction in the 96-well plate, or add 10 µL AMP Detection Reagent to the 10 µL reaction in the 384-well plate. Mix by orbital shaking. Incubate in the dark at room temperature for 30 minutes.
Luminescence signals can be read 30-180 minutes after adding the AMP Detection Reagent, or even longer [Note 6].
 
Notes
Upon first use, the reagent should be aliquoted and stored protected from light at -20°C or below to ensure stability.
AMP Detection Reagent stored long-term at -20°C may have slight precipitation upon thawing to room temperature. The supernatant can be used directly, or the precipitate can be removed by centrifugation before use.
Different batches are not recommended to be mixed.
The luciferase reaction in the AMP Detection Reagent is sensitive to temperature changes. Reagents and test samples/assay plates need to be equilibrated to room temperature (22°C-25°C), and the temperature should remain constant (±1°C) during the test.
Unless validated, changing the reagent volumes is not recommended. The volume ratio for the enzyme reaction, AMP GR Reagent, and AMP Detection Reagent should be 1:1:2.
The luminescence signal is very stable, with essentially no change in intensity within 3 hours.
This product is for research use only.
ANTBIO-Glo® AMP Assay Kit_ANTBIO079024_AntBio BIOSCIENCE Official Website
 

Product Name	Catalog Number	Detection Method	Sensitivity
ANTBIO-Glo® AMP Assay Kit	UA079024	Chemiluminescence	0.05 pmol