Chloride Assay Kit, BioAssay™

Chloride is the major extracellular anion in human body fluids. Chloride plays a key role in maintaining proper water distribution, osmotic pressure and electrolyte balance. Low chloride concentrations may be found with prolonged vomiting, extensive burns, metabolic acidosis, Addisonia crisis and renal diseases. Elevated chloride concentrations are associated with dehydration, congestive heart failure, hyperventilation and urinary obstructions. Determination of chloride in sweat is useful in diagnosing cystic fibrosis.

Simple, direct and automation-ready procedures for measuring chloride concentration in biological samples are becoming popular in Research and Drug Discovery. Chloride assay kit is designed to measure chloride directly in biological samples without any pretreatment. The improved Fried method utilizes mercuric 2,4,6-tripyridyl-s-triazine, which forms a colored complex specifically with chloride. The intensity of the color, measured at 610nm, is directly proportional to the chloride concentration in the sample. The optimized formulation substantially reduces interference by substances in the raw samples.

Sensitive and accurate. Use as little as 5ul samples. Linear detection range 0.7 mg/dL (0.2mM) to 35 mg/dL (10mM) Cl- in 96-well plate assay. Simple and high-throughput. The procedure involves addition of a single working reagent and incubation for 5 min. Can be readily automated as a high-throughput assay for thousands of samples per day. Improved reagent stability and versatility. The optimized formulation has greatly enhanced reagent and signal stability. Cuvet or 96-well plate assay. Low interference in biological samples. No pretreatments are needed. Assays can be directly performed on raw biological samples i.e., in the presence of lipid, protein and minerals such as magnesium, iron and zinc.

Direct Assays: Cl- in serum, plasma, urine, saliva, sweat etc.
Drug Discovery/Pharmacology: effects of drugs on chloride metabolism.
Food and Beverages: chloride determination.
Environment: chloride determination in water and soil.

Kit Contents: (250 tests in 96-well plates)
Reagent: 50ml Chloride standard: 1ml 35 mg/dL Cl-
Storage conditions. The kit is shipped at room temperature. Store Reagent and Standard at 4°C. Shelf life: 12 months after receipt. Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Important: bring reagents to room temperature and shake well before use.

1. Dilute standards in distilled water as shown in the table. Serum, plasma, urine and milk samples should be diluted 20-fold in water. Transfer 5ul diluted standards and samples to wells of a clear bottom 96-well plate. Store diluted standards at 4°C for future use.
No STD+H2O Vol (ul) Cl- (mg/dL)
1 100ul+0ul 100 35.0
2 80ul+20ul 100 28.0
3 60ul+40ul 100 21.0
4 40ul+60ul 100 14.0
5 30ul+70ul 100 10.5
6 20ul+80ul 100 7.0
7 10ul+90ul 100 3.5
8 0ul+100ul 100 0
2. Add 200ul working reagent and tap lightly to mix.
3. Incubate 5 min at room temperature and read optical density at 610nm (550-650nm nm).

1. Set up test tubes labeled Standards and Samples. Transfer 10ul diluted standards and samples to appropriately labeled tubes.
2. Add 1000ul working reagent and vortex to mix. Incubate 5 min, transfer to cuvet and read OD at 610nm.

Subtract blank OD (water, #8) from the standard OD values and plot the OD against Cl- standard concentrations. Determine the slope using linear regression fitting. Chloride concentration of the sample is calculated as

=
ODSAMPLE–ODBLANK
Slope
X n (mg/dL)

ODSAMPLE and ODBLANK are OD610nm values of sample and sample blank (water or buffer in which the sample was diluted). n is the dilution factor (n=20 for serum, plasma, milk, urine). Conversions: 1 mg/dL Cl- equals 282uM, 0.001% or 10 ppm.

Pipeting devices and accessories (e.g. 5ul), clear flat-bottom 96-well plates and plate reader, or spectrophotometer and cuvets.

Cl- (mg/dL)
1 324 ± 5
2 341 ± 3
3 127 ± 1
4 5.71 ± 0.09
5 0.27 ± 0.17
6 < 0.17
7 < 0.17
8 < 0.17
9 0.25 ± 0.11

1. Human serum. 2. Fresh human urine. 3. Commercial 2% reduced fat milk (Kirkland). Water samples: 4. Tap water (Hayward, CA). 5. Tap water (San Bruno, CA). Food and Beverages: 6. Crystal Geyser natural alpine spring water. 7. Coca-cola® classic coke. 8. Lipton Lemon iced tea. Environmental: 9. Soil extract. 5.6g of soil (Hayward, CA) was extracted with
10ml MilliQ water. The supernatant was centrifuged to remove any insoluble particles. Clear supernatant was assayed.

This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.