Blood Phosphate Content Assay Kit (AHM, Micro Method)

Cat. No.: B1507967

GRADE & PURITY BioReagent

Overview

Blood phosphorus mainly refers to inorganic phosphorus existing in the blood in the form of inorganic phosphate salts. The concentrations of calcium and phosphorus in plasma significantly influence bone salt formation and bone calcification. The relative stability of their contents depends on the balance between calcium/phosphorus absorption and excretion, and the equilibrium between calcification and decalcification. Blood phosphorus levels can serve as one of the detection indicators for various diseases, holding significant application value in the field of medical testing.

Detection Principle

After processing the serum sample to remove organic phosphorus, inorganic phosphorus reacts with ammonium molybdate to form phosphomolybdic acid. Upon reduction by ferrous sulfate, it turns blue. The phosphorus content in blood is calculated by measuring the absorbance at 620 nm.

Note: Before formal testing, it is recommended to perform a preliminary experiment with 2-3 samples expected to show significant differences.

Reagents, consumables and Equipments not provided

• Microplate reader or visible spectrophotometer (capable of measuring absorbance at 620 nm)

• 96-well plate or micro-volume glass cuvette, Adjustable pipettes and tips

• Centrifuge

• Deionized water

Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
Reagent Ⅰ	Ready-to-use. Equilibrate to room temperature before use.	Store at 4°C. Toxic and irritating odor; perform experiment in a fume hood.
Reagent Ⅱ	Ready-to-use. Equilibrate to room temperature before use.	Store at 4°C protected from light.
Working Reagent Ⅲ	Prepare immediately before use. Dissolve Reagent Ⅲ thoroughly in 11 mL deionized water, then add Reagent Ⅱ and mix well.	Use on the same day.
Standard	Ready-to-use. Equilibrate to room temperature before use.	Store at 4°C.

2. Sample Preparation

Serum Sample: Pipette 50 µL of serum, add 950 µL of Reagent Ⅰ, mix well, centrifuge at 8,000 rpm at room temperature for 10 minutes. Collect the supernatant for testing.

3. Experimental Steps

3.1 Preheat the microplate reader or visible spectrophotometer for 30 minutes and set the wavelength to 620 nm. Zero the visible spectrophotometer with deionized water.

3.2 Operation Table (perform in micro-volume glass cuvette / 96-well plate):

Reagent (µL)	Blank Well	Standard Well	Test Well
Sample Supernatant	0	0	50
Deionized Water	50	0	0
Standard	0	50	0
Reagent Ⅰ	50	50	50
Working Reagent Ⅲ	100	100	100

3.3 Mix well and incubate at room temperature for 10 minutes. Measure the absorbance at 620 nm, recorded as A<sub>Blank, A<sub>Standard, A<sub>Test. Calculate ΔA<sub>Test = A_Test - A<sub>Blank and ΔA<sub>Standard = A_Standard - A_Blank.

Note: The blank and standard wells only need to be measured once. It is recommended to perform a preliminary experiment with 2-3 samples showing expected large differences before formal testing. If ΔA_Test is too small, appropriately increase the sample amount. If ΔA_Test is greater than 1.0, further dilute the sample with Reagent Ⅰ and multiply the result by the dilution factor, or reduce the amount of sample used for extraction.

4. Calculation of Results

Note: We provide two formula sets: the derived calculation formulas and the simplified formulas. They are completely equivalent. The simplified formulas are recommended as the final calculation formulas.

Blood Phosphorus Concentration Calculation:

Blood Phosphorus Content (mmol/dL) = (C_{;Standard × ΔATest ÷ ΔAStandard) × Sample Dilution Factor = 2 × ΔATest ÷ ΔA;Standard}

Parameter Description:

C_Standard: 0.1 mmol/dL;

Sample Dilution Factor: (50 µL serum + 950 µL Reagent Ⅰ) ÷ 50 µL serum = 20.

5. Example Results

               Figure 1: Goat Blood Phosphorus Content

Notes

1. Working Reagent Ⅲ must be prepared immediately before use and is limited to use on the same day.

2. Hemolysis should be avoided during the assay procedure, as organic phosphoesters from red blood cells entering the serum can be hydrolyzed by enzymes, leading to increased serum inorganic phosphorus levels.

3. This product is for scientific research use only and is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.

Contents & Storage

B1507967	Component	96T	Storage
B1507967A	Reagent Ⅰ	65 mL×2	2-8℃
B1507967B	Reagent Ⅱ	1.6 mL	2-8℃. Store in the dark.
B1507967C	Reagent Ⅲ	1 EA	2-8℃
B1507967D	Standard	2 mL	2-8℃