Inorganic Phosphate Content Assay Kit (UV Micro Method)

Cat. No.: I1510382

GRADE & PURITY BioReagent

Descripción general

Inorganic Phosphorus in Serum primarily consists of two phosphate anions, H₂PO₄⁻ and HPO₄²⁻, which can rapidly interconvert depending on the pH environment. In serum at pH 7.4, the concentration ratio of these two anions is approximately 1:4. Under acidosis, the ratio is about 1:1, while under alkalosis, it shifts to 1:9. In urine at pH 4.5, the concentration ratio is about 100:1. The conventional detection method recommended by the WHO is colorimetry. The National Center for Clinical Laboratories of China recommends the ferrous sulfate molybdenum blue method and the metol molybdenum blue method as routine methods. UV spectrophotometry can also be employed.

Detection Principle

Without the need for sample pretreatment, inorganic phosphorus reacts with ammonium molybdate to form ammonium phosphomolybdate. The absorbance is directly measured at 325–340 nm using a full-wavelength microplate reader, and the inorganic phosphorus content is calculated according to the formula. Advantages of the UV method include simplicity, speed, and stability. Its disadvantages include susceptibility to interference from hemolysis, icterus, and lipemia. This kit is intended for research purposes only and is not suitable for clinical diagnosis or other applications.

Reagents, consumables and Equipments not provided

•  Full-wavelength microplate reader (capable of measuring absorbance at 340 nm)
• Precision balance, centrifuge tubes or test tubes, centrifuge, UV-compatible microplate
• Deionized water

Procedure (For Reference Only)

1. (Optional) Sample Preparation

1.1 Plasma/Serum Samples

Plasma or serum samples prepared by conventional methods can be used directly for assay with this kit. Store at -20°C for Pi detection.

1.2 Cell or Tissue Samples

Homogenize an appropriate amount of cells or tissue. After low-speed centrifugation, collect the supernatant and store at -20°C for Pi detection.

1.3 High-Concentration Samples

If the sample contains a high concentration of Pi, dilute it with ddH₂O. Regular distilled water is not recommended for dilution.

Note: (Optional) After sample preparation, the protein concentration can be measured using a BCA Protein Assay Kit to facilitate subsequent calculation of Pi content per unit protein weight in tissues or cells. The Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Assay Kit is recommended.

2. Preparation of Phosphorus Standard Working Solution

Take an appropriate amount of the Phosphorus Standard (1 mg/mL) and dilute it with the Phosphorus Standard Diluent at a ratio of 1:39 (Phosphorus Standard: Diluent) to obtain the Phosphorus Standard Working Solution (25 μg/mL = 0.807 mmol/L). Store at 4°C for Pi detection.

3. Preparation of Ammonium Molybdate Chromogenic Solution

Weigh an appropriate amount of Ammonium Molybdate Powder. Mix and dissolve it with Ammonium Molybdate Diluent (2×) and deionized water at a ratio of 12 mg : 10 mL : 10 mL (Powder : 2× Diluent : H₂O). Alternatively, weigh 120 mg of Ammonium Molybdate Powder and add it to 100 mL of deionized water. After complete dissolution, aliquot into 10 mL or other appropriate volumes. Mix an aliquot with an equal volume of Ammonium Molybdate Diluent (2×) before use. Store at -20°C to extend the shelf life. The prepared Ammonium Molybdate Chromogenic Solution can be stored at 4°C for up to 2 weeks. Discard if the solution becomes turbid or the color darkens. Please note that this product provides more Ammonium Molybdate Powder than the required amount.

4. Pi Loading

Set up blank wells, standard wells, and sample wells according to the table below. Add solutions in the specified order, avoiding bubble formation, and mix gently. If the inorganic phosphorus concentration in the sample is too high, reduce the sample volume or dilute appropriately before assay. It is best to set up duplicate wells for samples and use the average value.

Component (μL)	Blank Well	Standard Well	Sample Well
ddH₂O	5	—	—
Phosphorus Std. Working Sol. (25 μg/mL)	—	5	—
Sample	—	—	5
Ammonium Molybdate Chromogenic Sol.	200	200	200

5. Pi Measurement

Mix well and incubate at room temperature for 5 minutes. Use the blank well to zero the microplate reader. Measure the absorbance at 340 nm and record the values for the standard well (A_standard) and sample well (A_sample).

6. Calculation

6.1 Formula for Inorganic Phosphorus in Serum/Plasma:

Pi (mmol/L) = (A_sample / A_standard) × 0.807 × N

Pi (mg/L) = (A_sample / A_standard) × 25 × N

6.2 Formula for Inorganic Phosphorus in Urine:

Pi (mmol/L) = (A_sample / A_standard) × 0.807 × N

Pi (mg/L) = (A_sample / A_standard) × 25 × N

6.3 Formula for Inorganic Phosphorus in Tissues:

Pi (mmol/mg prot) = (A_sample / A_standard) × 0.807 × N / c

Parameter Description:

A_sample: Absorbance of the sample well

A_standard: Absorbance of the standard well

N: Sample dilution factor

c: Protein concentration of the sample (mg/L)

Unit Conversion:

1 mg/dL = 10 mg/L = 1 mmol/L × 0.323

Reference Interval

Serum phosphorus concentration in healthy adults: 0.9 – 1.34 mmol/L (2.76 – 4.16 mg/dL)

Precautions

1. Assays should be performed within 5 minutes to 2 hours. The absorbance of the standard tube remains unchanged after 3 hours, while the absorbance of the sample tube increases over time.

2. Hemolyzed samples interfere with the detection; avoid using them whenever possible.

3. For icteric or lipemic samples, a sample blank should be prepared.

4. This method can be adapted for endpoint detection on automated biochemical analyzers.

5. The prepared Ammonium Molybdate Chromogenic Solution can be stored at 4°C for up to 2 weeks. Discard if the solution becomes turbid or the color darkens.

6. If the sample concentration is too high, dilute with distilled water and repeat the assay. Multiply the result by the dilution factor.

7. The absorbance measured at 340 nm is 82% of that measured at 325 nm.

8. For your safety and health, please wear a lab coat and disposable gloves during operation.

Appendix

Standard Curve Preparation: Under room temperature conditions, follow the instructions and measure the absorbance at 340 nm for a series of phosphorus standards (5, 10, 15, 20, 25, 30, 35, 40 μg/mL) using a UV spectrophotometer. A sample standard curve is shown below (for reference only):

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Room temperature

Enviado en

Wet ice

Estabilidad y almacenamiento

Each component has a shelf life of 1 year under corresponding storage conditions.

Contents & Storage

I1510382	Component	100T	Storage
I1510382A	Phosphorus Standard (1 mg/mL)	1 mL	2-8℃.
I1510382B	Phosphorus Standard Diluent	2 mL	RT.
I1510382C	Ammonium Molybdate Powder	1 EA	RT.
I1510382D	Ammonium Molybdate Diluent (2×)	10 mL	RT.