RESIDENT CHEMICAL PATHOLOGY

TWO CONTROL SYSTEMS

• Two systems control all physiologic processes:

• The nervous system exerts point-to-point control through nerves, similar to sending messages by conventional telephone. Nervous control is electrical in nature and fast.

• The endocrine system broadcasts its hormonal messages to essentially all cells by secretion into blood and extra cellular fluid.

• Like a radio broadcast, it requires a receiver to get the message - in the case of endocrine messages, cells must bear a receptor for the hormone being broadcast in order to respond.

STRUCTURAL GROUPS OF HORMONES

• Most commonly, hormones are categorized into four structural groups, with members of each group having many properties in common:

• Peptides and proteins

• Steroids

• Amino acid derivatives

• Fatty acid derivatives - Eicosanoids

Peptides and Proteins Steroids

Amino Acid Derivatives

DEFINITION OF IMMUNOASSAY

ASSAY literally means:

“ test” “analyze” “measure” “run test on”

IMMUNOASSAY:

“Any binding assay in which binding protien is an antibody.”

(Clinical Chemistry Lawrence A.Kaplan.Amadeo J.Pesce)

Immunoassay is divided in to two basics types

• Competitive (Ags + Fixed No. of Ab)

• Non-competitive

(Fixed Ab+ Ag + Anti Ab)

Competitive immunoassay

• Use labeled antigens• Carry out in the presence of excess

antigens.• Competes for the binding sites on

antibody.• RIA(Radioimmunoassay) is a prototype of

competitive heterogeneous immunoassay.• EMI (Enzyme multiplied immunoassay) is

a example of homogenous immunoassay.

Two subtypes of competitive immunoassay

A-Isotopic( radio isotopes labels are use)

B-Non isotopic immunoassay (other labels are used)

It is further classified according to types of the labels used:

1)Enzymes immunoassay (EIA)

2)Fluorescence immunoassay (FIA) or

Fluorescence polarization immunoassay

( FPIA)

3)Chemiluminescence immunoassay (CIA)

CRITERION FOR RELIABILITY OF

COMPETATIVE IMMUNOASSAY

1-Accuracy________ Nearness to true

2- Precision _______ Repeatability

3-Sensitivity_______ Minimum detectable limit

4-Specificity______Ability to exclusively

discriminate a single entity

The RIA is the conventional prototype of a competitive-binding assay:

There are three fundamental components to the RIA –

1. radioactive ("hot") hormone

2. unlabeled ("cold") hormone (standard or sample)

3. antibody

-- Radioisotopes of tritium (b emitter) and iodine (high specific activity g emitter) are incorporated into steroid and protein (Tyr or His residues) hormones, respectively; this must be done without significant damage to the immunoreactivity of the hormone.

Principle of RIA

• Tracer and standard or unknown sample compete for a limited number of binding sites on the antibody.

• Amounts of (excess) tracer and antibody (sufficient to bind 40-60% of radio labeled antigen) for each reaction are held constant, while quantities of standard hormone are increased step-wise.

• Reactions are allowed to proceed to equilibrium, and free (unbound) hormone is segregated from antibody-hormone complexes.

• Emission of energy from the bound complex is monitored by radiation detection equipment.

Interpretation of RIA

• As content of standard is increased from 0 (i.e.100% of antibody is bound by tracer), the amount of antibody-bound tracer declines reciprocally –

• A standard curve is constructed from these data. Reaction tubes containing sample in place of standard are assayed simultaneously.

• Estimates of mass of hormone within a sample are interpolated from the standard curve .

Standard curve

Demerits of RIA

• Waste disposal (costly and inconvenient)• Potential health hazards(exposure to radiation)• Obtaining government license( bureaucratic

procedure)• Regulation of safety and disposal• Expensive instruments for counting radioactivity• Short shelf life of labeled reagents

paticularly(Iodine-125) because of radioactive decay.

Enzyme immunoassay (EIA)

In EIA Antigen is labeled with a stable enzyme e.g.

1. G6PD (generates NADH),

2. alkaline phosphatase( splits into a colored compound and phosphate),

3. β-galactosidase (splits off a fluorescent product from the substrate)

(NON –ISOTOPIC ASSAY)

FLUORESCENCE IMMUNOASSAY (FIA and FPIA)

• In this labeled antigen has a capacity to fluorescence

• If the fluorescent emission can be measured directly this is known as FIA.

• If the fluorescent emission can be measured by a change in polarization of the exciting light this is known as FPIA.

Chemiluminescence's Immunoassay

• It is the name given to light emission produced during a chemical reaction.

• Isoluminol and acridinium esters are the most important example of chemiluminescent labels.

Chemiluminescence's Immunoassay

• Oxidation of isoluminal by hydrogen peroxide in the presence of a catalyst produces a relatively long lived light emission at 425mm .

• oxidation of an acridium esters by alkaline hydrogen peroxide in the presence of a detergent produces a flash of light at a 429mm.

• Acridium esters are high specific activity detection limits of labels is 800 zeptomoles.

NON-COMPETITIVE IMMUNOASSAY

• Use labeled antibody.

• Carry out in the presence of excess antibody.

• Competes between binding sites between

Capture solid phase AB and labeled AB in a solution .

-- Non-competitive assay also called immunometric (sandwich) assay.

Immunometric immunoassay

Antibody-excess immunoassays include the 1. Immunoradiometric assay (IRMA) 2. Enzyme-linked immunosorbent assay (ELISA).• In the IRMA cold ligand is "sandwiched"

between an antibody coated to a solid phase and a second radiolabeled antibody raised against a different hormonal epitope (this works best with macromolecular hormones).

• In a sandwich ELISA, hormone is bound to an antibody attached to a solid phase, and then an antibody-enzyme conjugate and substrate are added as shown in the (Figure).

Merits of immunometric assay

• Sensitivity of these assay is not mandated by competition, and therefore, reactions can be carried out expeditiously over a wide range of detection.

• These methods engender a direct relationship between radioactivity/enzymatic activity measured in the final complex and concentration of standard or analyte (in contrast to the inverse correlation between bound radioactivity and standard or sample concentrations in an RIA).

ELISA

• In this the antibody is adsorbed on a solid surface.g.wall of a test tube,microtitre well or a plastic tube.

• The antigen in the patient serumand a fixed amount of a enzyme-labeled antigen are added and allowed to compete for limited number of antibody binding sites,

• Enzymatic activity of the bound labeled antigen is determined by incubation with substrate.

ELISA

HORMONES AND THEIR METHODOLOGIES IN AKU LABORATORY

HORMONES DONE BY RIA :

• HYDROXYCORTICOSTEROIDS

• HYDROXYPROGESTERONE

• 17-KETOSTEROIDS

• ALDOSTERONE

• ANDROSTENEDIONE

• CALCITONIN

HORMONES DONE BY CLIA• ALPHA FETO PROTIEN• TESTESTERONE• PROGESTERONE• DHEA-SO4• TOTAL AND FREE T3,T4,TSH• GROWTH HORMONE• PROLACTIN• ß-HCG

• HORMONES BY FPIA• SERUM UNCONJUGATED ESTRIOL• URINE ESTRIOL

HORMONES BY(ELISA) MEIA:

• ESTRADIOL

• FSH

• LH

• INSULIN

• CORTISOL

HORMONES BY FPIA

NEWER METHODOLOGIESOther analytical systems that exploit the same basic

principle as the RIA include 1. Protein-binding assay2. Radioreceptor assay (RRA) 3. Scintillation proximity assay (SPA)Protein-binding and radio receptor assays are

radioligand assays that utilize an endogenous plasma protein (eg., for steroid hormones) or cellular receptor, respectively - instead of an antibody. Protein-binding assays lack the specificity of an immunoassay. The radioreceptor assay has an advantage over the RIA in that it only detects bioactive hormone (i.e.antibodies can interact with sites on the hormone molecule not involved in receptor binding).