Immunoprecipitation Flashcards

Affinity. Affinity refers to the strength of a single antibody-antigen interaction. Avidity is the strength of interactions between many different antibodies in a serum against a particular antigen( ie, the sum of many affinities.)

Specificity. Specificity is defined as a negative result in the absence of the disease. The non-rheumatoid arthritis specimens would be expected to test negative if the assay has hogh specificity. Percision is the ability of the assay to repeatedly yeald the same results on a single specimen. both bias and sensitivity calculations would include specimens from rheumatoid arthritis specimens. Although those specimens would be included in the evaluation. they are not listed in the question.

Prozone Phenomenon, Although performance error and low specificity should be considered. if a test system fails to yeald the expected reaction, excessive antibody preventing a precipitation reacton is usually the cause. Prozone occurs when antibody molecues saturate the antigen sites, preventing cross linking of the antibody-antigen complexes by other antibody molecules.Because the antigen and antibody do not react at equlivance a visable product is not formed. , leading to a false negative resut.

In the RIF test system, for example, one measuring hemopexin concentration, the gel would contain the antihemopexin. a standardized volume of serum containing the antigen is added to each well. Antigen diffuses from the well into the gel and formes a precipitation ring by reaction with antibody. At equivalence, the area of the ring is proportional to antigen concentration.

Crossed lines indicate non-identity. between wells 1 and 2. The antibody from well 1 recognises a different antigenic determinant than the antibody from well 2.

RIA is extreamly sensitive, but because of strict regulations for handling radioactive materials and saftey concerns it is used only when an alternative method in unavailable. The sensitivity of EIA methods producing fluorescent and chemilluminesant products can be equlivilant to that of RIA. These detection systems hsve greatly dimniished the number of analytes that must be measured by RIA.

The ELISA test measures antibody using immobilized reagent antigen. the antigen is fixed to the walls of a tube or bottom of a micro-titer well. sreum is added and incubated anf the antibody binds if present. After washing the antigen antibody complexs are dtcted by addein and enzyme labeled anti-immunoglobulin. Unbound enzyme label is removed by washing, and the bound enzyme is dected by addind chromogenic substrate. The enzyme catalizes the conversion of substrate to colored product.

If unbound enzyme conjugate antimunogloublin is not washed away , it will catalize conversion of substrate to colored product , yealding a falsley elevated result.

If the color reaction is not stopped within the time limits specified by the procedure the enzyme will continue to act on the substrateproducing a falslry elevated test result.

Usually when a test sample reads at a value above the highest standard in an ELISA test , it is diluted and measured again. In those instances where no where no additional clinical value can be obtained by dilution , the result may be reported as greater than the highest standard. ( citing the upper reporyable limit of the assay.).

Serum and urine protein electrophoresis should be performed initially to detect the presence of an abnormal immunoglobulin which demonstrates restricted eletrophoretic mobility. A patient producing only monoclonal light chains may not show any abnormal serum finding because the light chains may be excreted in the urine . A positive finding for either serum or urine should be followed by IFE on the positive specimen. This is required to confirm the presence of monoclonal immunoglobulin, to identify the light anf heavy chain type and to determine whether free monoclonal light light chains are being produced.

A narrow dark band formed in both the lane containing anti gamma and anti kappa indicates the presence of a monoclonal IgG kappa immunoglobulin. A diffuse dark band would indicate a poly clonal increse in IgG that often accompanies chronic inflammatory disorders such as systemic lupus erythematosus.(SLE)

Rate. Rate nephelometry is used to measure formation of small immune complexes as they are formed under conditions of antibody excess . the rate of increase in photodetector oputput is measured within seconds or minutes. and increases with increasing antigen concentration. Antigen concentration is determined by compairing the rate for the sample to that for standards. using an algorithm that compensates for linearity. In endpoint nepheolmetry, reactions are read after equlivance. Immune complexes are of maximal size but may have a tendancy to settle out of solution, thereby deccreasing the amount of scatter.

In an IFA for example, and antinuclear antibody (ANA) tesst, the fluorescence pattern must be coorelated correctly with the specificity of the antibodies. Both pathological and nonpathological antibodies can occur, and antibodies may be detected at significant titer in a patient whose disease is inactive. Failure to correctly identify subcellular structures may result in misinterpretation of the antibody specificity, or a false- positive caused by nonspecific fluoresence.

An unexpected pattern may indicate the presemce of more than one antibody. Diluting the sample may help to clearly show the antibody specificitues; if they are found in different titers. If the patteren is still atypical. a new sample should be collected and the test repeated.