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

Total Iron Binding Capacity (TIBC)

ELISA Kit

Instruction Manual

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

Experimental Principle

The Total Iron Binding Capacity (TIBC) level in the sample is determined using a double-antibody sandwich ELISA method. The microtiter plate is pre-coated with purified human TIBC antibodies to form a solid-phase antibody. After adding the sample, TIBC binds to the immobilized antibody, followed by incubation with HRP-labeled TIBC antibody. This forms an antibody-antigen-enzyme-labeled antibody complex. After washing, the substrate TMB is added, and a color change occurs due to the catalytic action of HRP. The final color turns from blue to yellow upon acid addition. The intensity of the color correlates directly with the TIBC concentration in the sample. The absorbance is measured at 450 nm using a microplate reader, and the TIBC concentration is calculated based on a standard curve.

Kit Composition

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

2. Stop Solution – 6ml × 1 bottle

3. Enzyme Standard Reagent – 6ml × 1 bottle

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

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

6. Standard Dilutions – 1.5ml × 1 bottle

7. Sample Diluent – 6ml × 1 bottle

8. Instructions – 1 copy

9. Reagent A – 6ml × 1 bottle

10. Developer B – 6ml × 1 bottle

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 freeze-thaw cycles.

2. Samples containing NaN3 are not suitable for testing, as it may inhibit HRP activity.

Kit Procedure

1. Standard Dilution: Prepare dilutions according to the provided chart.

2. Loading: Add 50 μl of standard, 40 μl of sample diluent, and 10 μl of sample to each well (final 5x dilution).

3. Incubation: Seal and incubate at 37°C for 30 minutes.

4. Washing: Use 20× diluted washing solution. Wash 5 times, then dry.

5. Enzyme Addition: Add 50 μl of enzyme reagent to all wells except blank.

6. Incubation: Repeat incubation at 37°C for 30 minutes.

7. Washing: Repeat the washing procedure.

8. Color Development: Add 50 μl of TMB developer and incubate at 37°C for 10 minutes.

9. Stop Reaction: Add 50 μl of stop solution to terminate the reaction.

10. Measurement: Read OD values at 450 nm within 15 minutes of stopping the reaction.

Calculation

Plot the standard curve using concentrations vs. OD values. Determine the sample concentration from the curve or use linear regression. Multiply by the dilution factor (5x) to obtain the actual sample concentration.

Notes and Precautions

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

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

3. Use a pipette and calibrate regularly. Keep loading time under 5 minutes if possible.

4. Always run a standard curve and consider sample dilution if OD is too high.

5. Use a new sealing film for each test to prevent cross-contamination.

6. Keep the substrate away from light.

7. Follow instructions strictly. Rely on microplate reader results.

8. Treat all samples, washes, and waste as biohazardous materials.

9. Do not mix reagents from different batches.

Storage Conditions & Expiry

1. Store the kit at 2–8°C.

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