Hi Janis Heyne,

First of all congratulations for your research. Please find some troubleshooting steps below:

1. Try chase buffers of different pH value

2. Coat different concentration of antibodies on control line (I hope, you already tried)

3. Try different size of NC membrane ( We got best result with 5-15µm size)

4. Block the membrane with 1% BSA or any other blocking agent before final run

I wish it will help you.

Best wishes,

Ishrat

To make a lateral flow control line more uniform, consider the following strategies:

1. Optimize Reagent Concentration:

- Ensure the concentration of the control line reagent (typically an antibody or antigen) is optimal. Too high or too low concentrations can affect line uniformity.

2. Improve Dispensing Technique:

- Use precise and consistent dispensing techniques, such as automated striping machines, to apply the control line reagent. Manual pipetting can introduce variability.

3. Use High-Quality Membranes:

- Select membranes with consistent pore size and high-quality manufacturing standards. Variability in membrane properties can lead to uneven control lines.

4. Optimize Buffer Conditions:

- Ensure that the buffer used for the control line reagent is optimized for proper flow and binding. Adjusting pH, ionic strength, and surfactant concentration can improve uniformity.

5. Control Drying Conditions:

- Standardize the drying process to avoid variations. Use controlled temperature and humidity conditions to ensure consistent drying of the control line.

6. Maintain Consistent Flow Rate:

- Ensure the flow rate of the sample across the membrane is consistent. Variations in flow rate can cause uneven deposition of the control line reagent.

7. Test for Batch Consistency:

- Regularly test batches of control line reagents and membranes for consistency. Variations between batches can lead to differences in line uniformity.

8. Implement Quality Control Checks:

- Regularly monitor the uniformity of the control lines during production. Implement quality control measures to catch and correct any deviations early.

By carefully optimizing and controlling these factors, you can achieve a more uniform control line in your lateral flow assays.

Thanks for the replies Motasim Mohammedahmed and Ishrat Jahan Saifi .

We've tried pretty much all of your suggestions. We're using the best quality papers and reagents. I read somewhere that this leading edge effect may be intrinsic to the affinity of the antigen/antibody combination we are using and cannot change. We don't have the same problems with other tests. Thanks for your suggestions!