In the realm of pet diagnosis, three primary technologies are commonly employed: time-resolved immunofluorescence chromatography (TR-IF), colored latex microsphere immunochromatography (CLM), and colloidal gold immunochromatography (CGIA). Each method offers unique advantages and disadvantages, making it crucial to understand their distinctions to select the most appropriate test for your pet’s specific needs.
So, there are questions you may ask, como:
- What are the advantages and disadvantages of each of them?
- Which approach is ideal?
- Which product is best to choose for pet diagnosis?
We will explore the diagnosis technologies for dogs and cats to find out the answers.
What is Time-resolved immunofluorescence chromatography (TR-IF)
Driven by technological advancements, immunochromatographic assays (ICAs) in the field of rapid diagnostics have undergone a remarkable transformation, progressing from the first-generation colloidal gold and colored latex formats to the second-generation fluorescent microsphere technology. This evolution has enabled a significant leap from qualitative to quantitative analysis.
Time-resolved fluorescence immunochromatographic (TR-FIA) technology has taken this advancement a step further, dramatically enhancing the sensitivity, exactitud, and precision of rapid testing.
The third-generation time-resolved fluorescence immunochromatographic technology is a novel non-radioactive rapid immunoassay technique built upon traditional fluorescence analysis. It boasts the following unique features:
- Rapid Quantitative Analysis: Employing lanthanide element europium (Eu3+)-encapsulated fluorescent microspheres as markers, the technology determines results by analyzing the fluorescence intensity in the detection and control zones, enabling rapid quantitative analysis.
- Time-Resolved Performance: The fluorescence decay time of Eu3+ is exceptionally long, reaching 714ms, compared to less than 100ms for ordinary fluorescence. By utilizing instrument-specific settings, Eu3+’s specific fluorescence signal can be isolated.
- High Relative Specific Activity: The long decay time of Eu3+ allows for repeated excitation, resulting in a high relative specific activity of the fluorescent marker.
- Wavelength-Resolved Performance: Eu3+ exhibits a large Stokes shift of 255nm, effectively eliminating interference from non-specific fluorescence, and significantly enhancing assay specificity.
- Covalent Binding: During immunolabeling, Eu3+ is covalently linked to large molecules such as antigens and antibodies, forming a stable and irreversible bond.
- Ventajas: The technology offers a range of notable advantages, including ease of operation, high sensitivity, accurate quantification, excellent precision, and high throughput, making it suitable for therapeutic efficacy monitoring and epidemic surveillance.
- Aplicaciones: The technology is particularly well-suited for disease detection, customs quarantine, and agricultural applications, where rapid results and high assay accuracy are crucial.
What is Colored latex microsphere immunochromatography (CLM)
Immunochromatographic assays (ICAs) have gained widespread recognition in various fields, including clinical diagnostics, food and drug testing, pesticide detection, antigen-antibody interactions, and animal disease diagnosis. The development of ICAs is driven by three key goals: enhancing assay sensitivity, enabling quantitative analysis, and facilitating multiplexed detection.
Colored microsphere-based ICAs have emerged as a significant advancement in the realm of ICAs, offering enhanced sensitivity and improved visual clarity, making results easier to interpret. Here are the key features of colored microsphere-based ICAs:
Superior Sensitivity: Colored microspheres serve as a superior alternative to colloidal gold, offering enhanced sensitivity for a wider range of analytes.
Internal Dyeing Technology: The internal dyeing process ensures vibrant and durable colors, preventing dye leaching from the microsphere surface, and facilitating efficient coupling with antibodies or antigens.
Hydrophilic Surface and Abundant Functional Groups: The hydrophilic surface and high density of functional groups on colored microspheres enhance their protein binding capacity, leading to improved assay performance.
Scalable Production and Consistent Performance: Colored microspheres can be produced on a large scale with consistent performance, enabling the production of up to 100 liters per batch.
Uniform Microsphere Size: Colored microspheres exhibit exceptional uniformity in size, with a coefficient of variation (CV) below 5% for regular particle sizes, minimizing batch-to-batch variability.
Customizable Design: Colored microspheres can be tailored to specific requirements, with adjustable particle size (ranging from 100 nm to 10 µm), surface functional group density, and a wide range of rainbow-inspired colors.
why colored microspheres are considered a superior alternative
| Característica | Colloidal Gold | Colored Microspheres |
|---|---|---|
| Visibility | Bien | Excelente |
| Color | Typically red | Variety of colors |
| Color Intensity | Purplish, somewhat dull | Vibrant, easy to observe |
| Multiplexed Detection | Difficult to achieve | Easily achievable |
| Sensitivity | Generally lower | Generally better |
| Estabilidad | Bien | Excelente |
| Ease of Preparation | Relatively simple | More complex |
| Preparation Reproducibility | Bien | Excelente |
| Escalabilidad | Easy to scale up production | Very easy to scale up production |
| Labeling Method | Relatively simple | More complex |
| Purification Method | Relatively simple | More complex |
| Production Cost | Relatively inexpensive | Slightly more expensive |
What is Colloidal gold immunochromatography (CGIA)
Immunochromatography is a rapid, simple, sensitive, intuitive, cost-effective, and field-deployable method.
This method possesses advantages not found in techniques such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence, and gas-liquid chromatography. It plays a crucial role in detection technology and serves as a powerful complement to traditional and large-scale instrument-based detection methods.
Its unique three-dimensional reaction mode endows it with rapid characteristics, with colloidal gold traditionally being used as the marker.
The characteristics of colloidal gold immunochromatographic technology include the use of colloidal gold as the marker. This technology was pioneered by Faulk and Taylor in the early 1970s and initially used in immunoelectron microscopy.
Colloidal gold, also known as gold sol, is a suspension of gold particles formed by the reduction of gold salts to elemental gold. Colloidal gold particles consist of a core of elemental gold (Au atoms) surrounded by a double ion layer. The inner layer of negatively charged ions (AuCl2-) is closely bound to the gold core, while the outer ion layer of H+ ions disperses in the solution between colloidal particles to maintain the colloidal gold in a suspended state.
Characteristics of colloidal gold immunochromatographic technology:
- Rápido: Each experiment takes 5 a 15 minutos, involving only simple steps of sample addition, reaction, and visual observation.
- Conveniente: The reagent composition is simple, requiring only a detection card without the need for microscopes or detection instruments.
- Suitable for field testing: Experiments can be conducted anywhere without the need for personnel to undergo specialized training.
- Reagents can be stored at room temperature: No special storage conditions such as refrigeration or dry ice are required.
Colloidal gold immunochromatographic technology, due to its reliance on electrostatic adsorption for labeling, is prone to detachment and instability. It suffers from shortcomings such as single-color output, inability for quantitative detection, and relatively low sensitivity. Consequently, it is gradually being replaced by colored latex microsphere immunochromatographic technology and immunofluorescence chromatographic technology.
The comparison of these diagnosis technologies
| Característica | Colloidal Gold Immunochromatography | Enzyme-Linked Immunosorbent Assay (ELISA) | Traditional Immunofluorescence Assay (IFA) | Time-Resolved Immunofluorescence Assay (TR-FIA) |
|---|---|---|---|---|
| Marker | Colloidal gold | Enzima | Fluorescein | Lanthanide-doped fluorescent microspheres |
| Qualitative or Quantitative | Cualitativo | Semi-quantitative | Cualitativo | Cuantitativo |
| Reagent Kit Components | 1-2 types | 7 components | 1-2 types | 1-2 types |
| Operation | 1-2 steps | 10-15 steps | 1-2 steps | 1-2 steps |
| Sensitivity | Lower | Better | Lower | Bien |
| Especificidad | Better | Better | Better | Bien |
| Detection Time | 15 minutos | 120 minutos | 10-15 minutos | 3-10 minutos |
| Ease of Detection | On-site testing | Centralized testing | On-site testing | On-site testing |
| Reagent Storage | Temperatura ambiente | 2-8°C refrigeration | Room temperature/2-8°C refrigeration | Temperatura ambiente |
| Epidemic Monitoring | √ | √ | √ | √ |
- Colloidal gold immunochromatography is the simplest and fastest method, but it has the lowest sensitivity and is only qualitative.
- ELISA is more sensitive than colloidal gold immunochromatography, but it is more complex and time-consuming.
- IFA is similar to ELISA in terms of sensitivity and complexity, but it requires a fluorescent microscope for detection.
- TR-FIA is the most sensitive and quantitative method, but it is also the most expensive and complex.
Resumen
you should consult with your veterinarian is essential to determine the most appropriate rapid diagnostic test for your pet’s needs. They can consider the specific signs and symptoms, the urgency of diagnosis, and the cost factors to recommend the best course of action.
