1.2.1The structure of antibodies

Antibodies are immunoglobulins that can specifically bind to antigens（immunoglobulin,Ig）.IgDivided into five categories, namelyIgGTheIgATheIgMTheIgDandIgERelated to immune testingIgmainlyIgGandIgM.IgMade up of two light chains（L）And two heavy chains（H）The composition of individual units.IgThe light chain is the same, there areMr（kappa）Andλ（Lambda）Two types. Five categoriesIgTheir heavy chain structures are different, which determines their antigenicity.IgGandIgMThe heavy chains are referred to asγ（gamma）Chain andM（mu）Chain.

The heavy chain and light chainNThe sequence of amino acid arrangement at the end varies among different antibodies, known as the variable region, which is used separatelyVHandVLexpress. The two constitute the antigen binding site of the antibody, which only matches the corresponding antigenic determinant clusterMatching and specific binding are the structural basis for antibody specific binding to antigens.

IgGCan be broken down into three segments by papain, of which two identical segments are called antigen binding fragments（Fab）. eachFabAll have the ability to bind antigens, but only oneEach antigen binding site is monovalent and does not undergo agglutination or precipitation upon binding to the antigen. Another section is calledFcSegment, no antibody activity, but possessingIgG*Antigenicity.

IgGCan be broken down into two fragments by gastric protease, oneFabTwins, calledF（ab'）2Can bind to two identical antigens; Another segment is similarFcSubsequently decomposedSmall molecule peptides with no biological activity.

IgMIt is a pentamer composed of five monomers, containing 10A heavy chain and10A light chain with10Due to the influence of spatial location, only five antigen binding valences are present.IgMThe molecular weight is approximately900000，IgGThe molecular weight is approximately150000.

After being infected by microorganisms, the body first producesIgMAntibodies are then producedIgGAntibodies. After a period of time,IgMThe amount of antibodies gradually decreases and disappears, andIgGAntibodies can exist for a long time and can last for several years after a diseaseFor a long time.

IgMAntibodies are generally protective antibodies with immunity. ThereforeIgMThe determination of antibodies has high clinical diagnostic value for certain infectious diseases such as hepatitis A. The image on the right shows hepatitis AIn the patient's serumIgGAntibodies andIgMThe time and level of antibody appearance.

1.3Antigen antibody reaction

1.3.1可逆性

The process of antigen antibody complex formation by antigen antibody binding is a dynamic equilibrium, and its reaction equation is:

Ag+AbAg·Ab

Affinity of antibodies（affinity）It is the inherent binding force between antigen and antibody, which can be expressed by an equilibrium constantKexpress:

K=[Ag·Ab]／[Ag][Ab]

Ag·AbThe degree of dissociation andKValue related. The antigen binding point of high affinity antibodies is very suitable for the spatial configuration of the antigen determinant cluster, and the two bind firmly and are not easily dissociated. dissociationThe antigen or antibody can maintain its original structure and activity, so affinity chromatography can be used to purify the antigen or antibody. In the serum, specificIgGAntibodies only account for the totalIgGmiddleA very small portion. The specific antibodies extracted by affinity chromatography are called affinity chromatography pure antibodies, which can achieve better results when applied in immunoassays.

1.3.2Zui appropriate proportion

The amount of antibody complex (precipitate) formed by adding incremental amounts of antigen to a constant amount of antibody is shown in the figure1-4The peak of the curve is the most suitable range for the ratio of antigen to antibody, which is called the equivalence band（zone ofequivalence）. Before and after the equivalence band are antibody excess band and antigen excess band, respectively. If there is an extreme excess of antigen or antibody, no precipitate will form, which can be detected in immunoassaysKnown as the band phenomenon（zone phenomenon）. Excessive antibody is called a prodrug（prezone）The excess of antigen is called the posterior band（postzone）. Using immunological methods to determine resistanceAt the beginning, there should be sufficient antibody levels in the reaction system, otherwise the measured amount will be lower than the actual content, and even false negatives may occur.

1.3.3specificity

The binding of antigen and antibody essentially only occurs between the antigenic determinant of the antigen and the antigen binding site of the antibody. Due to their complementary relationship in chemical structure and spatial configuration, the antigenAntibody reactions have high specificity. For example, the surface antigen in hepatitis B virus（HBsAg）TheeAntigen（HBeAg）And core antibodies（HBcAg）, originating from the same virus but only binding to its corresponding antibody, without binding to the other two antibodiesReaction. The specificity of antigen antibody reactions enables immunoassays to detect a specific substance in a very complex protein compound (such as serum) without the need to separate the test substance first.

But this specificity is not the same. If two compounds have partially identical structures, cross reactivity may occur in antigen antibody reactions. For example: chorionic gonadotropin（hCG）Luteinizing hormone（LH）All byalphaandBComposed of two subunits, their structural differences lie inBSubunits, and both of themalphaSubunits are of the same kind. usehCGimmunityThe anti serum obtained from animals contains antibodiesα-hCGAnd resistanceβ-hCGTwo types of antibodies, antiα-hCGAntibodies will be associated withLHin
alphaEnzyme site undergoes cross reactivity. In clinical testing, if using antibioticshCGAntiserum as a pregnancy diagnostic reagent for detecting urinehCG, can only be used forhCGHigh concentration experiments, otherwiseTrace amounts of physiological excretion into urine by womenLHThere will be a cross reaction with it. Therefore, in the diagnosis of early pregnancy, the sensitivity should reach50mIu/mlhCG）In reality, it is necessaryApplication only applies tohCGSpecific resistanceβ-hCGTo avoid cross reactivity with other hormones.

1.3.4sensitivity

The sensitivity of chemical colorimetric method in determining the content of a substance in serum ismg/mlThe sensitivity of the enzyme reaction assay method is approximately5~10μg/mlThe sensitivity of gel diffusion method and turbidity method in immunoassay is similar to that of enzyme reaction method. Exemption from MarkingThe sensitivity of epidemic detection can be increased by thousands of times, reachingng/mlLevel. For example, using radioimmunoassay or enzyme immunoassay to determineHBsAgIts sensitivity can reach0.1ng /ml.

1.4Application of Immunoassay in Clinical Laboratory Testing

Due to various antigen components, including small molecule haptens, they can be used to prepare specific anti serum or monoclonal antibodies, which can be used as reagents to detect the corresponding antigens in the specimenTherefore, the application range of immunoassay is extremely wide, and it can be used in clinical testing to determine:

1）Various proteins in body fluids, including proteins with extremely low levels such as alpha fetoprotein.

2）Hormones, including small molecular weight steroid hormones.

3）Antibiotics and drugs.

4）Pathogen antigens,HBsAgTheHBeAgWait.

5）In addition, purified antigens can also be used to detect antibodies in specimens, such as antibodies-HBsWait.

5Immunoassay with labeled markers

As mentioned above, immunoassay is a highly sensitive method that directly measures the precipitate or turbidity formed after antigen antibody reaction, with a sensitivity of up to5~10μg/mlHowever, in clinical testing, the content of certain test substances in the specimen is much lower than this level, so methods to increase sensitivity need to be sought. The labeled immunoassay isLabel the antigen or antibody in the detection reagent with a trace detectable substance, and increase sensitivity by measuring the label. In radioimmunoassay and enzyme immunoassay, the markers are used separatelyFor radioactive isotopes and enzymes, the sensitivity can be increased by hundreds to thousands of times compared to directly measuring sediment by measuring radioactivity and enzyme activity to calculate the amount of the analyte. In labeled immunoassays,Generally, excessive labeling reagents are added to ensure reaction with the analyte *. To label antibodies（Ab^※）Detecting antigens（Ag）For example, the reaction equation is as follows:

Ag+ Ab^※ AgAb^※+Ab^※

In the reaction products, there areAgcombinedAb^※And free and wanderingAb^※ If the marker is determined without separating the two, the result obtained will be the sum of the two. Therefore, the separation of free and bound markers is an important step in label immunoassay. collectibleSolid phase carrier is one of the various methods used. If the antigen or antibody is encapsulated on a solid-phase carrier and then directly reacted with the labeled antigen or antibody, the bound label is fixed on the carrierOn the body, while free markers remain in the solution. This can be washed to remove free substancesAb^※In addition, the determination of binding markers can be carried out on the solid phase.

6Enzyme immunoassay

Enzyme immunoassay（enzymeimmunoassay）Can be classified as homogeneous（homogenous）And non-homogeneous（heterogenous）Two types. In homogeneousEIAZhongkeDirect determination of markers without the need for separation of free and bound markers. For example, under certain conditions, the enzymes in the enzyme labeled antigen antibody complex formed after antigen antibody reaction are lostThe enzyme activity measured directly reflects the free enzyme marker due to its ability to act on substrates. homogeneousEIALess commonly used in clinical testing. heterogeneousEIANeed to undergo dissociation firstSeparation of bound markers. As mentioned earlier, solid-phase carriers can be used as a separation method. This solid-phase enzyme immunoassay method1971At the beginning of its establishment, it was called enzyme-linked immunosorbent assay (ELISA) adsorbentMeasurement（enzymelinked immunosorbent assay）, abbreviated asELISAIn China, there is a translation for enzyme-linked immunosorbent assay, although its meaning is not precise, it has been widely used.