Human total iron binding capacity (TIBC) ELISA kit instruction manual - Database & Sql Blog Articles

Total Iron Binding Capacity (TIBC) Assay

ELISA Kit

Kit Instruction Manual

This kit is for research use only. Not for human or animal diagnostic purposes.

Experimental Principle

The Total Iron Binding Capacity (TIBC) in the sample is determined using a double-antibody sandwich ELISA method. A solid-phase antibody, specific to TIBC, is coated onto a microwell plate. The sample is added, and TIBC binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody then binds to the captured TIBC, forming an immune complex. After washing, the substrate TMB is added, which changes color under HRP catalysis. The intensity of the color is proportional to the TIBC concentration in the sample. The absorbance is measured at 450 nm, and the TIBC concentration is calculated from a standard curve.

Kit Composition

1. 30× Washing Solution – 20 ml × 1 bottle

2. Enzyme Standard Reagent – 6 ml × 1 bottle

3. Enzyme-Labeled Coating Plate – 12 wells × 8 strips

4. Sample Diluent – 6 ml × 1 bottle

5. Reagent A – 6 ml × 1 bottle

6. Color Developer B – 6 ml × 1 bottle

7. Stop Solution – 6 ml × 1 bottle

8. Standard (2400 pg/ml) – 0.5 ml × 1 bottle

9. Standard Dilutions – 1.5 ml × 1 bottle

10. Instructions – 1 copy

11. Sealing Film – 2 sheets

12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C. Avoid repeated freezing and thawing.

2. Sodium azide (NaN₃) should not be present in the samples, as it may inhibit HRP activity.

Kit Procedure

1. Prepare standards by serial dilution according to the provided table.

2. Load 50 µL of standard and 40 µL of sample diluent into each well, followed by 10 µL of the sample (final dilution 5×).

3. Incubate at 37°C for 30 minutes.

4. Wash 5 times with 20× diluted washing solution.

5. Add 50 µL of enzyme-labeled reagent to all wells except blank controls.

6. Incubate again at 37°C for 30 minutes.

7. Wash 5 times again.

8. Add 50 µL of TMB developer and incubate at 37°C for 10 minutes.

9. Add 50 µL of stop solution to terminate the reaction.

10. Measure OD at 450 nm within 15 minutes of stopping the reaction.

Calculation

Plot the standard curve using known concentrations and their corresponding OD values. Use linear regression or interpolate from the curve to determine the sample concentration. Multiply by the dilution factor (5×) to obtain the actual TIBC level.

Important Notes

1. Allow the kit to reach room temperature before use. Store unopened plates in a sealed bag.

2. If the washing solution crystallizes, warm it gently in a water bath before use.

3. Use a pipette for accuracy. Ensure consistent timing during sample loading.

4. Always run a standard curve in duplicate. If the sample OD exceeds the highest standard, dilute the sample before testing.

5. Discard the sealing film after one use to prevent contamination.

6. Protect the substrate from light.

7. Follow instructions strictly. Results must be confirmed with a microplate reader.

8. Treat all samples and waste as biohazardous material.

9. Do not mix components from different batches.

Storage Conditions & Expiration

1. Store the kit at 2–8°C.

2. Shelf life: 6 months from the date of manufacture.