Difficulties in standardization of immunoassays are protein heterogeneity, matrix effects, cross-reactivity, and the need to measure the concentration close to the lower limit of the measurement method. The measured immunoreactivity is usually a protein that differs in glycosylation, degradation, and complex forms A mixture of the same type.

(1) Matrix effect

The matrix effect of immunoassays is usually defined as non-specific interference with the reaction between antigen and antibody but not with the analyte itself

factor. 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 the 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 stimuli that bind to serum proteins, other matrices than human serum Out of service.

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 antibody-reactive rheumatoid factor (RF) heterophilic natural antibodies against animal immunoglobulins that 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 Idiotype antibodies of reagent antibodies, this method is ineffective. 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 positives in double-sandwich assays and false positives in competitive inhibition tests. Mouse IgG2, IgG2, and immunoglobulin subunits are particularly sensitive to complement effects. The interference of complement can be eliminated 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 nuclear markers such as germanium 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 a false increase in results. Many other factors such as ingredients in serum separation gels, anticoagulant detergents, drugs, non-esterified fatty acids, etc. are used for certain immunoassays. There is also interference.

(2) Heterogeneity and cross-reactivity of antigens

Protein cords are obviously heterogeneous, in addition to biologically active forms in the blood circulation, there are prokinases, fragments and subunits. Thyroid-rich cord (PTH), ACTH and its precursors, large forms of prolactin and gastric juice, and splicing variants of growth hormone can all cause measurement problems. The two-point double-sandwich assay using monoclonal antibodies greatly improves the specificity of the assay, for example using anti-choriogonadotropin. The double-sandwich method established with monoclonal antibodies to the P subunit will not determine free subunits. The double-sandwich method established using two antibodies against the amino and carboxyl terminal portions of ACTH or PTH does not determine fragments that are not biologically active. In general, it is desirable to measure components with biological activity, but sometimes for specific clinical purposes, specific tests are required to determine degradation products, such as the core components of hCG. Therefore, in order to ensure the specificity of the assay, it is necessary not only to have a biologically active crystal standard crystal, but also a clinically relevant standard product of degradation products.

Some serum proteins have various genetic variants that differ in isoelectric point (eg. 1-antitrypsin) or in different degrees of polymerization (eg haptoglobin), although the ratio of these variants will affect their immunoassay Results, but this is not a prominent problem of serum protein immunoassay. Polyclonal antisera are commonly used in plasma protein assays. Polyclonal antibodies cannot detect small differences in protein structure. On the contrary, using monoclonal antibodies may not detect some variants, which is a common problem in serum protein immunoassay. The heterogeneity of glycoproteins mainly lies in the glycosylation, especially the difference in sialic acid content. This difference in turn causes differences in protein isoelectric point and electrophoretic mobility. Antibodies are generally insensitive to these differences, so sugar heterogeneity has little effect on immunoreactivity. However, the sugar chain on the protein can modify the protein's immunoreactivity by modifying or covering a peptide determinant. In contrast, the main epitope of mucin is composed of sugar components. Small differences in the design of immunoassay experiments can also cause large differences in polymorphic antigen measurement results. The determination of mucin antigens CA125, CA19-9 and CA15-3 can clearly prove this. For example, CA15-3 produced by different tissues contains different numbers of tandem repeats of 20 amino acids, and the monoclonal antibody used in the test to determine the antigen was prepared using tumor cells, so the antigen to be measured is not well-defined. molecule. Although most reagent manufacturers use the same standards and antibodies, the correlation between them is very poor, which is probably due to the difference in experimental design, so each method should have its own reference Range value.

(3) Experimental design

The experimental design of immunoassay mainly involves the use concentration of antibody or antigen, incubation temperature, incubation time, whether the measurement mode is competitive inhibition or double sandwich direct measurement, and whether the double sandwich measurement adopts one-step or two-step method, etc. The impact of the measurement is mainly the lower limit of the test, the simplicity of the specific nuclear, and so on. Generally speaking, a higher antibody or antigen reaction concentration, a higher incubation temperature (such as 37 ~ C instead of room temperature), and a longer incubation time can improve the lower limit of the immunoassay, but this is only a relative It is necessary to optimize rationally from the practical application of reagents. Due to its simplicity and convenience, the double-antibody sandwich enzyme immunoreagent has a better lower limit and specificity. It has now basically replaced the radioimmunoassay in the determination of most protein kinases and tumor markers. The double-antibody sandwich assay mode is performed in a two-step method (that is, clinical samples and enzyme-labeled antibodies are added in two steps), which can avoid many problems that may occur when using the one-step method, such as cross-reaction with labeled antibodies but with solid phase capture Substances with no cross-reaction of antibodies can be removed in the washing step after the first incubation; another example is the "hook" effect that often occurs in the one-step method (that is, the reaction color is very shallow when the antigen concentration is high, which is shown as false Positive) can be avoided by using the two-step method. However, because the two-step method requires a relatively long measurement time, the one-step method currently used in clinical trials is more. In addition, when the molecular size of the measured substance is inconsistent, the reaction of the large molecule is usually slower for the smaller molecule. For example, a short incubation time will also cause a measurement deviation. For example, when measuring the combined prostate-specific antigen (PSA) (Mr90Kd), if a shorter incubation time is used, the results will be lower compared to free PSA.

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