As of 2021, reported detection methods for lactoferrin include enzyme-linked immunoassay, high-performance capillary electrophoresis, biosensor assay, high-performance liquid chromatography, and high-performance liquid chromatography-mass spectrometry.
Enzyme-Linked Immunoassay
Enzyme-linked immunoassay is a detection method based on specific antigen-antibody reactions, with advantages such as high sensitivity and strong specificity. In a microplate, anti-lactoferrin antibody (primary antibody) is coated. After adding the target lactoferrin, the lactoferrin binds to the primary antibody, then lactoferrin antibody (secondary antibody) is added to form a sandwich structure. Finally, a chromogenic agent is added, and the absorbance at a specific wavelength is measured using a microplate reader. The absorbance is proportional to the lactoferrin content, and a standard curve can be plotted to quantitatively detect the lactoferrin concentration in a sample. Although enzyme-linked immunoassay has high sensitivity, it usually requires multiple incubations, washes, and cleanings, making the operation cumbersome. Additionally, the preparation of antibodies and anti-antibodies is difficult and expensive, making it unsuitable for large-scale sample analysis.
High-Performance Capillary Electrophoresis
The smaller the diffusion coefficient or the larger the molecular weight of the sample molecules, the higher the separation efficiency of the capillary column. High-performance capillary electrophoresis mostly uses quartz capillaries as separation channels, using high-voltage direct current electric fields as the driving force to achieve liquid-phase separation. It has the advantages of simple operation and high sensitivity, but the problem of poor reproducibility due to adsorption of the target lactoferrin on the capillary wall needs to be solved. Moreover, the detection results are greatly affected by the complex matrix of the sample.
Surface Plasmon Resonance Technology
Incident light at the critical angle onto the interface of two metal media with different refractive indices can cause free electron resonance in the metal. The electrons absorb light energy, greatly reducing the reflected light. The incident angle at which the reflected light completely disappears within a certain angle is called the SPR angle. The SPR angle changes with the refractive index of the medium surface, and the change in refractive index is proportional to the mass of molecules bound to the medium surface. Therefore, specific signals of interactions between biomolecules can be obtained by detecting动态 changes in the SPR angle during biological reactions, achieving detection purposes. The biosensor immunoassay based on surface plasmon resonance technology can automatically detect low levels of lactoferrin in real time without labeling. However, the experimental temperature and sample composition affect the measurement results, and the equipment is relatively expensive.
High-Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) is a technique that uses different chromatographic columns to separate components based on differences in partition coefficients and adsorption strengths, which are then recognized by detectors. Among these, reversed-phase high-performance chromatography, a liquid chromatography system composed of a non-polar stationary phase and a polar mobile phase, is the most widely used HPLC method. Its advantages are high sensitivity, accurate and reliable results, and good reproducibility. However, its disadvantage is that it requires high sample purity, often requiring complex pretreatment processes before injection.
High-Performance Liquid Chromatography-Mass Spectrometry
Mass spectrometers accurately identify analytes by measuring the mass-to-charge ratio of specific ions. They are typically coupled with high-performance liquid chromatography or gas chromatography to achieve precise quantification. Due to the large molecular weight of proteins, in protein analysis, they are often hydrolyzed into peptide mixtures using various methods such as trypsin, and then detected by measuring specific peptide segments. Some reports indicate that researchers have combined proteomics technology, isotope labeling technology, and high-performance liquid chromatography-mass spectrometry to detect lactoferrin in dairy products. Liquid chromatography-mass spectrometry is sensitive, reliable, with high reproducibility and recovery rates. However, it requires breaking down large protein molecules into small amino acid molecules for qualitative and quantitative analysis using specific amino acid fragments. At this point, the protein has already been denatured, which is不利 for determining whether lactoferrin in the test sample is active. Furthermore, the equipment is expensive and requires higher professional skills from operators.
Article source: Baidu Baike: https://baike.baidu.com/item/%E4%B9%B3%E9%93%81%E8%9B%8B%E7%99%BD/3407131?fr=aladdin