Standardization of immunoassay-The difficulty of standardization of matrix effect immunoassay lies in protein heterogeneity, matrix effect, cross-reactivity, and the need to measure the concentration near the lower limit of the measurement method. A mixture of protein isoforms that differ in composite form.
Matrix effects in immunoassays are generally defined as non-specific factors that interfere with the reaction between antigen and antibody but are not related to the analyte itself. The matrix effect has a greater relationship with the assay mode and antibody selection, so it also affects different immunoassays in different ways. Matrix effects are usually caused by proteins, salts, phospholipids, complement, anti-immunoglobulin antibodies (rheumatoid factor and human anti-mouse antibodies), drugs, and substances that may contaminate the sample. These effects can be reduced by careful experimental design, such as the use of high-affinity antibodies or antibody fragments of a certain subtype, reducing the ratio of the sample to the total assay volume, adding immunoglobulin to the assay buffer, the ideal incubation temperature and comparison Long incubation time. The calibrator for immunoassay is usually prepared with a buffer solution similar to the matrix of the sample. The matrix can be human serum without analyte, animal serum without immunological cross-reactive antigen, or artificial buffer with protein content similar to the sample. Because the composition of serum is different, the difference in serum matrix of each batch may affect the calibration. Therefore, it is easier to use a pure protein solution as a matrix to maintain consistency. However, for hormones bound to serum proteins, other matrices than human serum cannot use.
The concentration of total protein and salt in serum and plasma is quite stable, but the salt concentration in urine changes greatly, and the increase in salt concentration plays an inhibitory role in the reaction between antigen and antibody. If the sample volume is less than 10% to 15% of the total reaction volume, the effect of protein is not obvious, but sometimes a larger sample volume is often required to increase the sensitivity of the assay. Therefore, the reference method is required to have a low lower limit of determination, and the sample volume to the total reaction volume should be small enough to exclude most of the matrix effects.
The immunoglobulin in the sample can affect the measurement result by reacting with the specific antibody used in the measurement. For example, patients with autoimmune diseases often have rheumatoid factor (RF) that can react with antibodies. Heterotropic natural antibodies against animal immunoglobulins are quite common, but generally their concentration and affinity are low. The presence of autoantibodies and heterophilic natural antibodies usually causes false positive reactions. These interferences can be reduced by adding sufficient amounts of immunoglobulins from the same line, or Fab or F'ab) 2 fragments of antibodies can be used in the assay instead of intact antibodies, but if the sample contains reagents This method is not effective for idiotype antibodies of antibodies. In this case, only the immunoglobulin is removed from the sample or measured using a method based on another antibody.
Various complement components can react with antibodies, thereby interfering with the reagent antibody's ability to bind antigen and react with secondary antibodies. It can cause false negatives in double-sandwich assays and false positives in competitive inhibition tests. Mouse IgG2, and IgG2, immunoglobulin subtypes are particularly sensitive to complement effects. It is possible to eliminate complement interference by heating the sample or adding a chelating agent to the reaction buffer, but heating will affect many analytes, and the chelating agent interferes with non-radioactive nuclide labels such as europium chelating agents and enzymes, so it tends to The use of antibodies or antibody fragments that are not sensitive to complement effects establishes assays.
It has been reported that phospholipids can interfere with the binding of antigens in the determination of steroids and cause false increases in results. Many other factors such as ingredients in serum separation gels, anticoagulants, detergents, drugs, non-esterified fatty acids, etc. are immune to certain immunity The measurement also interferes. Standardization of immunoassay-matrix effect
Hair Body Wave Human Hair,wholesale price brazilian,cuticle aligned bundles body wave
Xu Chang Zhuo YunQing crafts Co., LTD , https://www.wowqueenhair.com