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Bilirubin (Total and Direct) Colorimetric Assay Kit

based on 1 citations in multiple journalsBilirubin (Total and Direct) Colorimetric Assay Kit14.1 4
Allows measurement of both Total and Direct or Conjugated Bilirubin
Catalog #: K553

Product Details

Cat # +Size K553-100
Size 100 assays
Detection Method Absorbance (600 nm for Total Bilirubin and 550 nm for Direct Bilirubin)
Applications Measurement of Bilirubin in Serum samples
Features & Benefits • Simple protocol, robust and reliable data
• This assay allows detection of both Total and Direct or conjugated Bilirubin in serum samples.
Kit Components • Bilirubin Reagent 1
• Bilirubin Reagent 2
• Catalyst
• Total Bilirubin Probe
• Direct Bilirubin Probe
• Bilirubin Standard (0.2 µg/µl)
• DMSO (Anhydrous)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.


Bilirubin, a degradation product of heme catabolism, is a non-polar molecule. There are two forms of bilirubin: water-soluble (conjugated or direct) and water-insoluble (unconjugated or indirect) bilirubin. Bilirubin is produced in the endoplasmic reticulum as unconjugated bilirubin, which binds to albumin in plasma and forms albumin-bilirubin complex. This complex is transported to the liver, where it is conjugated with glucuronic acid and forms conjugated bilirubin. Bilirubin has potent antioxidant, anti-inflammatory and autoimmune properties. Bilirubin concentration in human body depends on gender, drug intake, age, etc. Low serum bilirubin is directly correlated with pathological conditions including diabetes mellitus, metabolic syndrome, and cardiovascular diseases. However, high bilirubin indicates hemolysis, jaundice, Gilbert’s syndrome, hepatitis, drug toxicity, and possible blockage of bile ducts. BioVision’s Bilirubin Assay Kit utilizes the Jendrassik-Grof principle to detect bilirubin. Total bilirubin (unconjugated + conjugated) concentration is determined in the presence of a catalyst, where bilirubin reacts with a diazo- salt to form azobilirubin, which absorbs at 600 nm. Direct bilirubin (conjugated) is determined in the absence of catalyst (550 nm).

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What is the Standard used in the kit composed of – is it conjugated or unconjugated bilirubin?
The Standard is mostly unconjugated free bilirubin, not counting trace impurities of the alpha- and beta-isomers, which are not determined.
According to our data, total bilirubin value is higher than direct bilirubin value. Could you explain why this could be happening?
The calculations for measuring total and direct Bilirubin Concentration are shown in the datasheet. Both total and direct bilirubin measures the formation of azobilirubin, which is detected at 550/600 nm. The total bilirubin reaction contains the proprietary catalyst, which solubilize “indirect” bilirubin and accelerate the reaction. Direct bilirubin is a component of total bilirubin and therefore should always be smaller in value. It has been shown that the presence of immunoglobulins (2220 mg/dL) may falsely increase the direct, but not the total, bilirubin measurement. Hemolysis and lipemia can also interfere at high concentrations. Additionally, we suggest removing IgG from the samples.
Is this kit compatible with plasma sample?
Yes, it is possible to use plasma samples. However, we recommend using fresh and non-hemolysed samples. Frozen samples can be used too provided they haven’t undergone hemolysis during collection or multiple freeze thaw.
Can the kit detect all three isoforms of conjugated bilirubin?
The kit measures total conjugated bilirubin. It cannot distinguish between different isoforms.
What’s the analytical range of this kit (i.e. in which concentration range can the kit give accurate measurement)?
0.005 ug/ul (i.e. 0.25 ug in 50 ul sample; Final concentration (in sample): 0.5 mg/dl)
What solvent you would recommend to dissolve bilirubin powder at high concentrations (>= 10mg/ml)—I’ve tried chloroform, 0.01M NaOH and DMSO, DMSO seems to be the best, but the samples from DMSO dissolved bilirubin confused me when I tried to measure the total bilirubin as described above.
DMSO is the ‘standard’ solvent for bilirubin. Bilirubin is extremely hard to dissolve. NaOH might work too, however, the solution needs to be degassed. Bilirubin is extremely air and light sensitive.
Why did the DMSO-dissolved sample have the A530 value increase eventually—does DMSO reacting or inhibiting the react of bilirubin with the working reagent, or it’s just that DMSO-dissolved bilirubin needs more time to be completely dissolved in the culture medium?
DMSO acts a ‘catalyst’. He needs to make sure 50% DMSO is present in every well, as stated in the protocol.
What is the success rate and accuracy of the bilirubin assay kit when using a sample of plasma rather than serum?
Plasma and serum are often interchangeably used for total bilirubin content. It has been reported that mean total bilirubin results were 20% higher in the plasma samples when compared to serum samples.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Is the chloride reagent supposed to come in bright purple color? or should it be rather colorless?
The chloride reagent is bluish in colour to begin with. The final colour developed is going to be intense blue which can be detected at 620 nm.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration?
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Suh, Dong Ho et al. (2017) In vivo metabolomic interpretation of the anti-obesity effects of hyacinth bean (Dolichos lablab L.) administration in high-fat diet mice, Mol Nutr Food Res. 2017 Jan 20.
For more citations of this product click here