Description
Bilirubin Assay Kit (BA0031) (BA0031)
The Bilirubin Assay Kit (SKU: BA0031) is designed to measure bilirubin in blood specimens in either 96-well or cuvette format. Bilirubin is a degradation product of haemoglobin formed when red blood cells die, existing as insoluble unconjugated (indirect) bilirubin and soluble conjugated (direct) bilirubin. It is a key diagnostic indicator; high levels result when too much haemoglobin is broken down or its removal is impaired, and the accumulation of bilirubin causes jaundice. This kit uses an improved Jendrassik-Grof method in which bilirubin reacts with diazotised sulfanilic acid to form a red-coloured product, with the intensity measured at 510-550 nm providing an accurate measure of the bilirubin level. Total bilirubin is assessed using caffeine benzoate to split bilirubin from the unconjugated bilirubin-protein complex. The single working-reagent, ten-minute procedure is readily automated for high-throughput work.
| Product Name: | Bilirubin Assay Kit (BA0031) |
| SKU: | BA0031 |
| Detection Method: | Colorimetric; modified Jendrassik-Grof reaction with diazotised sulfanilic acid measured at 530 nm |
| Detection Range: | Detection limit 0.16 mg/dL bilirubin |
| Sample Type: | Serum |
| Species Reactivity: | All |
| Assay Time: | 10 min incubation |
| Kit Size: | 180 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all reagents at 4°C. |
| Shelf Life: | 12 months after receipt. |
| Shipping: | Room Temperature |
A colorimetric assay for the quantitative determination of total and direct bilirubin in serum. The improved Jendrassik-Grof method uses the reaction of bilirubin with diazotised sulfanilic acid to form a red product; the intensity measured at 510-550 nm is proportional to the bilirubin level. Detection limit is 0.16 mg/dL bilirubin.
- Sensitive and accurate: detection limit of 0.16 mg/dL bilirubin in the 96-well plate assay.
- Simple and high-throughput: a single working reagent and a 10-minute incubation; readily automated for thousands of samples per day.
- Total and direct bilirubin in serum.
- Pharmacology: effects of drugs on bilirubin metabolism.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Hemolysis interferes with the assay; avoid exposing samples to light. Samples can be stored at -20°C for up to 3 months or at 2-8°C for 4 days. |
| 2 | Prepare fresh Working Reagent per well (at least 200 µL): Total = 50 µL A + 20 µL B + 130 µL C; Direct = 50 µL A + 20 µL B + 130 µL Saline; Blank = 50 µL A + 130 µL Saline + 20 µL H2O. |
| 3 | Calibrator: transfer 50 µL H2O and 50 µL Calibrator into two wells of a clear-bottom plate and add 200 µL H2O (250 µL per well). |
| 4 | Samples: transfer 50 µL sample into separate wells, add 200 µL of the respective Working Reagent (total and/or direct) and 200 µL Blank Reagent to the sample blank wells. |
| 5 | Incubate 10 min and read OD530nm (510 to 550 nm). |
Bilirubin = [(ODsample - ODblank) / (ODcalibrator - ODH2O)] x 5 (mg/dL), where the OD values are read at 530 nm for the sample, sample blank, calibrator and water, and 5 mg/dL is the equivalent bilirubin concentration of the calibrator.
| Component | Quantity | Storage |
| Reagent A | 30 mL | 4°C |
| Reagent B | 10 mL | 4°C |
| Reagent C | 30 mL | 4°C |
| Saline | 50 mL | 4°C |
| Calibrator (equivalent to 5 mg/dL Bilirubin) | 2 mL | 4°C |
