How to overcome the challenge of high coefficients of variation (CV) in immunochromatography

Regarding chromatographic coefficient of variation (CV), a netizen responded, "I have worked on the quantitative production of colloidal gold! Indeed, precision is challenging to control. Achieving control within 15% is considered quite good; it's a tough problem!"


After reviewing literature from Clin Chem, the evaluation of ABBOTT and Biosite's NGAL products was conducted. Despite Biosite being a reputable company, the precision of their product's low and medium values is mostly around 15%. Some domestic companies claim to achieve precision within 10%, and some go as far as claiming 5%. I'm wondering, aside from ensuring the homogeneity of component drying and release, are there any other effective methods to improve the precision of chromatographic products? My thinking may be somewhat rigid, so let's discuss any good directions or strategies my colleagues might have.


Speaking specifically about colloidal gold, the colors of colloidal gold produced in different batches can inherently differ. When colloidal gold is retained at the T-line, it's not just what we see on the surface. The NC membrane has an actual thickness, and when we use a machine to transfer the protein onto the NC membrane, the solution diffuses not only laterally but also longitudinally on the NC membrane. During chromatography, a significant portion of colloidal gold may also be retained below the surface of the NC membrane. The machine reading is often just the surface grayscale.


Now, let's discuss machine-related issues. The principles of colloidal gold readers on the market today involve scanning or photographing, relying on grayscale peak or area readings. Machines inherently have differences; if there is light leakage in places where the card is stuck, the reading deviation will be even greater. Machines with slight defects can introduce errors of 5-10% for the same test strip.


At the same time, our samples, such as blood or urine, inherently exhibit significant color differences between individuals. In terms of NC membrane presentation, this translates to substantial differences in background color, posing a greater challenge to accurate readings. Additionally, some colloidal gold may remain on the NC membrane, leading to further errors.


Other factors influencing precision include:


1. **Antigen-antibody performance:**

   The quality of antibodies and antigens directly affects the accuracy of test results. Low purity, poor affinity, or instability of antibodies or antigens may result in higher CV values.


2. **Distribution of markers on the binding pad:**

   For immunochromatography, the uniform distribution of markers on the binding pad, critical for the precision of test results, is essential. Several methods can help ensure an even distribution:

   - Choosing the appropriate binding pad with characteristics suitable for experimental requirements and marker properties.

   - Controlling marker concentration in the solution to prevent oversaturation or insufficient coverage.

   - Optimizing methods such as point sampling, spraying, or brushing for marker application.

   - Controlling temperature and humidity to minimize uneven distribution caused by environmental changes.

   - Ensuring steady application of markers to prevent local concentration variations.


3. **Distribution of encapsulation fluid on the NC membrane:**

   For effective encapsulation, it's crucial to choose a suitable encapsulation fluid and adjust its concentration appropriately. Optimization of concentration can impact its binding efficiency on the NC membrane. Factors affecting distribution include:

   - Ensuring the NC membrane is clean, dust-free, and moderately dry before encapsulation.

   - Choosing the appropriate encapsulation fluid based on experimental requirements and NC membrane characteristics.

   - Adjusting the concentration of encapsulation fluid to achieve optimal binding.

   - Ensuring the NC membrane is clean and dry for improved binding efficiency.


4. **Uniform distribution of sample pad treatment fluid:**

   Adjusting the concentration of treatment fluid appropriately can impact its distribution on the sample pad. Optimization of concentration is essential for optimal distribution. Factors influencing distribution include:

   - Ensuring the sample pad is clean, dust-free, and moderately dry before treatment.

   - Choosing suitable treatment methods such as immersion, spraying, or brushing.

   - Optimizing these methods to improve the uniformity of treatment fluid distribution on the sample pad.

   - Considering the impact of environmental conditions on the treatment process.


By implementing these measures, it is possible to effectively improve precision in immunochromatography.