Immunoassays in tdm

Most TDM and drugs of abuse (DAU) testing are now performed by immunoassay methods on automated systems. Currently, there are over 25 immunoassays commercially available and are routinely used in TDM/DAU laboratory analysis. Most immunoassay methods use specimens without any pretreatment and are run on fully automated, continuous, random access systems. The assays require very small amounts of sample (mostly <100 ^L), reagents are stored in the analyzer, and most analyzers have stored calibration curves on the system. In immunoassays, the analyte is detected by its complexation with a specific binding molecule, which in most cases is an analyte-specific antibody (or a pair of specific antibodies). This reaction is further utilized in various formats and labels, giving a whole series of immunoassay technologies, systems, and options (Table 1).

With respect to assay design, there are two formats of immunoassays: competition and immunometric (commonly referred as "sandwich"). Competition immunoassays work best for analytes with small molecular weight, requiring a single analyte-specific antibody. In contrast, sandwich immunoassays are mostly used for analytes with larger molecular weight, such as proteins or peptides, and use two different specific antibodies. Since most TDM immunoassays involve analytes of small molecular size, these assays employ the competition format. In this format, the analyte molecules in the specimen compete with analyte (or its analogues), labeled with a suitable tag provided in the reagent, for a limited number of binding sites provided by, for example, an analyte-specific antibody (also provided in the reagent). Thus, in these types of assays, the higher the analyte concentration in the sample, the less of label can bind to the antibody to form the conjugate. If the bound label provides the signal, which in turn is used to calculate the analyte concentration in the sample, the analyte concentration in the specimen is inversely proportional to the signal produced. If the free label provides the

Chapter 3 / Assay of Therapeutic Drugs

69

Table 1

Various Types of Commercial Immunoassay Kits

Immunoassay

types

Analyte Assay format Example

Assay Signal

Competition

Small Homogeneous FPIA (TDx® from

Fluorescence

molecular Abbott, Abbott Park,

polarization

weight IL, USA): TDM,

(<1000 D) DAU, other (e.g.,

T4, T3)

EMIT®

Colorimetry

(Dade-Behring,

(enzyme

Newark, DE, USA):

modulation)

TDM, DAU, other

(e.g., T4, T3)

CEDIA®

Colorimetry

(Microgenics,

(enzyme

Fremont, CA, USA):

modulation)

TDM, DAU, other

(e.g., T4, T3)

Competition

Both small Heterogeneous ADVIA Chemistry:

Turbidimetry, latex

and sandwich

and large Siemens, Tarrytown,

micro-particle

NY, USA TDM,

assisted

DAU, other (HbAlc,

plasma proteins, etc.)

Centaur® (Siemens):

Chemiluminescence

TDM, others (thyroid,

(acridinium ester

steroid, etc.)

label)

ACCESS® (Beckman,

Enzyme

Fullerton, CA, USA):

immunoassay

TDM, others (thyroid,

(using

steroid, etc.)

chemiluminescent

substrate)

Elecsys® (Roche,

Electro-

Indianapolis, IN,

chemiluminescence

USA): TDM, others

(thyroid, steroid, etc.)

AxSym® (Abbott):

Enzyme

TDM, others (thyroid,

immunoassay

steroid, etc.)

(using fluorescence

substrate)

signal, then signal produced is proportional to the analyte concentration. The signal is mostly optical—absorbance, fluorescence, or chemiluminescence.

There are several variations in this basic format. The assays can be homogeneous or heterogeneous. In the former, the bound label has different properties than the free label. For example, in fluorescent polarization immunoassay (FPIA), the free label has different Brownian motion than when the relatively small molecular weight (a few hundreds to thousand Daltons) label is complexed with a large antibody (140,000 D). This results in difference in the fluorescence polarization properties of the label, which is utilized to quantify the bound label (1). In another type of homogeneous immunoassay, an enzyme is used as the label, the activity of which is modulated differently in the free versus the complexed conditions with the antibody. This forms the basis of the enzyme-multiplied immunoassay technique (EMIT®) or cloned enzyme donor immunoassay (CEDIA®) technologies (2,3). In the EMIT method, the label enzyme glucose 6-phosphate-dehydrogenase is active in the free form but inactive in the antigen-antibody complex. The active enzyme during reaction with the substrate also reduces the cofactor NAD to NADH, and the absorbance is monitored at 340 nm. Because antibody-bound enzyme is inactivated or reacts slowly with the substrate, the unbound or free portion is responsible for the signal. Therefore, at equilibrium, the amount of unbound enzyme-labeled analyte will be directly proportional to the drug concentration.

In the CEDIA method, two genetically engineered inactive fragments of the enzyme beta-galactosidase are coupled to the antigen and the antibody reagents. When they combine, the active enzyme is produced and the substrate, a chromogenic galactoside derivative, produces the assay signal. In a third commonly used format of homogeneous immunoassay (turbidimetric immunoassay or TIA), analytes (antigen) or its analogs are coupled to colloidal particles, for example, of latex (4). As antibodies are bivalent, the latex particles agglutinate in presence of the antibody. However, in presence of free analytes in the specimen, there is less agglutination. In a spectrophotometer, the resulting turbidity can be monitored as end-point or as rate.

In heterogeneous immunoassays, on the contrary, the bound label is physically separated from the unbound labels, and its signal is measured. The separation is often done magnetically, where the reagent analyte (or its analog) is provided as coupled to paramagnetic particles (PMP), and the antibody is labeled. Conversely, the antibody may be also provided as conjugated to the PMP, and the reagent analyte may carry the label. After separation and wash, the bound label is reacted with other reagents to generate the signal. This is the mechanism in many chemiluminescent immunoassays (CLIA), where the label may be a small molecule which generates chemiluminescent signal (5). The label also may be an enzyme [enzyme-linked immunosorbent assay (ELISA)], which generates chemiluminescent, fluorometric, or colorimetric signal. In older immunoassay formats, the labels used to be radioactive [radio-immunoassay (RIA)]. But because of safety and waste disposal issues, RIA is rarely used today. Another type of heterogeneous immunoassay uses polystyrene particles. If these particles are micro-sizes, that type of assay is called microparticle-enhanced immunoassay (6). Microparticle enzyme immunoassay (MEIA) is also used for analysis of drugs in biological matrix.

The main reagent in the immunoassay is the binding molecule which is most commonly an analyte-specific antibody or its fragment. Several types of antibodies or their fragments are now used in immunoassays. There are polyclonal antibodies, which are raised in an animal when the analyte (as antigen) along with an adjuvant is injected into the animal. For small molecular weight analyte, it is most commonly injected as a conjugate of a large protein. Appearance of analyte-specific antibodies in the sera of animal is monitored, and when sufficient concentration of the antibody is reached, blood is collected from the animal. The serum can be used directly as the analyte-specific binder in an immunoassay but common practice is to purify the antibody before use. As there are many clones of the antibodies specific for the analyte, these antibodies are called polyclonal. In newer technologies, however, a mast cell of the animal can be selected as producing the optimum antibody and then can be fused to an immortal cell. The resulting tumor cell grows uncontrollably producing only the single clone of the desired antibody. Such antibodies, termed as monoclonal antibody, may be grown in live animals or cell-culture. There are several benefits of the monoclonal antibodies over polyclonal ones: (a) the characteristics of polyclonal antibodies are dependent on the animal producing the antibodies, and when the source individual animal is changed, the resultant antibody may be quite different; (b) as polyclonal antibodies constitute many antibody clones, these antibodies are less specific compared with monoclonal antibodies for the analyte. Sometimes, instead of using the whole antibody, fragments of the antibody, generated by digestion of the antibody with peptidases, for example, Fab, Fab' (or their dimeric complexes) are used as a reagent.

The other major component of the immunoassay reagents is the labeled antigen (or its analog). There are many different kinds of labels used in commercially available immunoassays, generating different kinds of signals. As described earlier, an enzyme may be also used as the label.

Even though the immunoassay methods are now widely used, there are few limitations of this technique. Antibody specificity is the major concern of an immunoassay. Many endogenous metabolites of the analyte (drug) may have very similar structural recognition motif as the analyte itself. There maybe other molecules unrelated to the analyte but producing comparable recognition motif as the analyte. These molecules are generally called cross-reactants. When present in the sample, these molecules may produce false results (both positive and negative interference) in the relevant immunoassay (7-9). Other components in a specimen, such as bilirubin, hemoglobin, or lipid, may interfere in the immunoassay by interfering with the assay signal, thus producing incorrect results. These effects have been described in Chapter 5. A third type of immunoassay interference involves endogenous human antibodies in the specimen, which may interfere with components of the assay reagent such as the assay antibodies, or the antigen-labels. Such interference includes the interference from heterophilic antibodies or various human antianimal antibodies and is described in Chapter 12.

Dealing With Asthma Naturally

Dealing With Asthma Naturally

Do You Suffer From ASTHMA Chronic asthma is a paralyzing, suffocating and socially isolating condition that can cause anxiety that can trigger even more attacks. Before you know it you are caught in a vicious cycle Put an end to the dependence on inhalers, buying expensive prescription drugs and avoidance of allergenic situations and animals. Get control of your life again and Deal With Asthma Naturally

Get My Free Ebook


Post a comment