principle of immunoenzymatic assay principle of clinical...

Principle of Immunoenzymatic Assay

Principle of Clinical Chemistry Assay

Principle of Specific Protein Assay

Principle of Immunoenzymatic Test

Immunoassay: Method Description

ECLECTICA™ uses a well-tested immunoreactionscheme based on immunoassays with magnetic particles. The separation method is so versatile that it allows to perform different assay schemes with the same separation reagent:

Direct Immunoenzymatic Assay (IEMA)

Competitive Immunoenzymatic Assay (EIA)

IEMA PHASE 1:IMMUNOLOGICAL REACTION

FITC-Labeled Antibody Analyte Enzyme-labeled Antibody Immunocomplex

Fisrt Incubation at +37°C

Anti-FITC Magnetic Particle

ReactionMixture

Bound enzyme-labeled antibody

(B) Excess FITC-labeled antibody

Free Excess Enzyme-Labeled

Antibody

BoundEnzyme-Labeled Antibody

IEMA PHASE 2MAGNETIC SOLID PHASE SEPARATION

Second Incubation at +37°C

Anticorpo marcato con l’enzima

Analyte in

sample

FITC-Labeled Analyte

Free Analyte and Free FITC-Labeled

Analyte

Enzyme-Labeled Antibody

Antibody-Bound Analyte (mostly FITC-Labeled)

EIAPHASE 1: IMMUNOLOGICAL REACTION

LOW SAMPLE

First Incubation at +37°C

Analyte in sample

FITC-Labeled Analyte

Enzyme-Labeled Antibody

Antibody-Bound Analyte (very little is FITC-

Labeled)

Free Analyte and Free FITC-Labeled Analyte

EIA : PHASE 1 IMMUNOLOGICAL REACTION

HIGH SAMPLE

First Incubation at +37°C

EIAPHASE 2 SEPARATION – LOW SAMPLE

Reaction Mixture


Complex with FITC-Labeled Analyte and

Antibody-Bound FITC-Labeled Analyte

Free Analyte and Free Antibody-Bound Analyte


EIAPHASE 2 SEPARATION – HIGH SAMPLE

ReactionMixture


Complex with FITC-Labeled Analyte and Antibody-Bound FITC-Labeled

Analyte

Free Analyte and Free Antibody-Bound Analyte


This phase is common for both methods.

The magnetic particles sediment and are separated out by a powerful magnet.

The liquid is aspirated

The complexes bound to the magnetic particles are then washed to eliminate the excess of reagent.

The number of wash cycles is 2

PHASE 3: WASHING AND SEPARATIONPHASE 3: WASHING AND SEPARATION

PHASE 4: ENZYMATIC REACTIONPHASE 4: ENZYMATIC REACTION


An enzymatic substrate, phenolphthalein monophosphate, is added to the reaction cells. This brings to the the resuspension of the magnetic particle complexes and kick-off of the enzymatic reaction.

The reaction cell is then incubated in the carousel.

The alkaline phosphatase enzyme in the magnetic particle complex removes the phosphate group from the substrate, producing phenolphthalein in direct proportion to the quantity of enzyme present.

Third incubation at +37°C

PHASE 5: COLOR DEVELOPMENTPHASE 5: COLOR DEVELOPMENT


At the end of enzymatic reaction a highly alkaline Stop Solution is added to stop the enzymatic reaction. It also develops the pink colour of the phenolphthalein.

The particles are attracted to the side of the reaction cell through the magnetic field, leaving a clear solution for the colour measurement step.

In IEMA assays, the more intense the colour, the higher the analyte concentration in the sample. In the EIA assays, the more intense is the colour, the lower is the amount of analyte in the sample.

PHENOLPHTHALEIN ABSORPTION SPECTRUMPHENOLPHTHALEIN ABSORPTION SPECTRUM

Wavelength (nm)

DATA MANAGEMENTDATA MANAGEMENTPhotometric reading is done at 546 nm, at which Phenolphtalein has maximum absorption, and at 492 nm where the absorption is arounda quarter of that at 546 nm.

The base measurement at 546 nm yields a high sensitivity, thus revealing minimal amounts of the target analyte. If the concentration of the analyte produces a strong colour, it cannot be measured directly at this wavelength, it will be measured at accurately at 492.

The data reduction system of the ECLECTICA™ is programmed to use the absorbance at 546 nm unless it exceeds a preset value (2.000 OD). If the preset value is exceeded, the absorbance at 492 nm is used, with a conversion factor for calculations (Overrange Factor)

The Overrange factor is set, instrument by instrument, by Adaltis Instruments Quality Control.

Principle of Clinical Chemistry Assay

Principle of the Assays: Clinical Chemistry Principle of the Assays: Clinical Chemistry

Clinical Chemistry determinations

Clinical Chemistry or Clinical Biochemistry are generic terms which commonly cover the majority of quantitative analyses of human fluids based on chemical or biochemical methods. It is one of the three major disciplines applied in medical laboratorywork, along with Haematology and Microbiology.

The biological fluids on which measurements are made are serum, plasma, urine, cerebrospinal fluid; in exceptional cases,pleural, pericardial and peritoneal or sino vial fluids may be used.

In Clinical Chemistry three major groups of analytes are measured:

Substrates: Glucose, Nitrogen, Cholesterol, etc.

These are known molecules which on addition of the reagents turn a measurable color. Their concentration is represented by the ratio between the color developed by the sample and the color obtained by the reference standard.

Enzymes: GOT (AST), GPT (ALT), CPK, etc.

These are molecules which act on the reagent and vary its extinction value. The delta extinction, measured over a standard period of time, is proportional to the concentration.

It is usually read in UV at 340/365 λ because the ΔE is obtained from the action of the enzyme on a NAD+-NADH system.

Electrolytes:

The most common method used for these measurements is spectrophotometry.


Types of reactionThe chemical reactions can be classified into three

main groups:

End - PointKineticFixed-Time


End-Point reactionsEnd-Point reactions are the classical reactions used to measure the

concentration of substrates. They can be enzymatic reactions in equilibrium due to the complete exhaustion of the analyte (e.g. formation of a complex of the analyte with a specific relating substance).

Calculation: Reading in the visible spectrum after termination of the reaction with production of stable colour.

The reaction may involve one or two reagents.

Example:Concentration of calcium in the sample (mg/dL) =

OD SAMPLE= --------------- x Standard concentration (mg/dL)

OD STANDARDwhere:OD SAMPLE = optical density of sampleOD STANDARD = optical density of standardStandard concentration = known concentration of reference standard


End Point reaction – single reagentThe above graph shows a typical End Point reaction: after a certain period of time (depending on the concentration of the enzyme, the temperature, the type of reagent, etc.) the reactionreaches equilibrium and the colour remains stable for a variable period of time (minutes to hours).

Principle of the tests: Clinical Chemistry Principle of the tests: Clinical Chemistry AssaysAssays

There are two types of End Point reaction: single reagent (see previous slide) or two (or three or more) reagents

End–Point reaction – two reagents

Principle of the Assays: Clinical Principle of the Assays: Clinical ChemistryChemistry

Kinetic reactionsThis type of reaction is generally used to measure the catalytic activity of

enzymes.Calculation:Reading in UV at fixed, well-defined intervals of time. The resulting ΔE is

multiplied by the specific factor for the analyte in question.

17680min/200,0122,6

1000200,2min/1000min/××Δ=

××××

=××

××Δ AAVcde

VTA

Example:

U/L = AST activity in international units per litre.

ΔA/min = Diminution in absorbance per minute (mean of three readings).

VT =Total reaction volume

1000 = Conversion into litres of volumes

E = Micromolar extinction coefficient of NADH+H+ (6,22 cm2/µmole at 340 nm)

d = optical range (1 cm.)

Vc = Volume of sample in final reaction mixture.


• Kinetic reactions can also involve one or two reagents

Kinetic reaction – Single Reagent Kinetic reaction – Two Reagents


Fixed-TimeThis type of reaction is generally used in enzymatic and chemical

reactions and measures the variation of absorbance over a set time.

The variation of absorbance over a set time is not generally linear.

Calculation: After reading in the visible spectrum after a set time for both the sample and the standard.

Example:Concentration Creatinin in sample (mg/dL) =

ΔA/min sample--------------- x Standard concentration of creatinin

ΔA/min standard

Principle of the Assays: Clinical ChemistryPrinciple of the Assays: Clinical Chemistry

Principle of Specific Protein Assay

Light scatteringLight scatteringWhen a light beam strikes particles, some light is reflected (scattered),

some is absorbed and some is transmitted.

Depending on the size of struck particles, the scattering may occur:

1) in all the directions ((particle ØØ lower than light λ λ ) ) or

2) mostly forward ((particle ØØ comparable to light λ λ ).).

Principle of the Assays: Specific Protein Principle of the Assays: Specific Protein

Light scatteringLight scatteringAntigen-Antibody complexes have size

comparable to light λ, thus a struck light beam is scattered mostly forward:

NephelometryNephelometry and and TurbidimetryTurbidimetry

Both these analytical methods are based on the phenomenon of light scattering, but they differ in the measured signal:

Nephelometry measures the intensity of the scattered light whereasTurbidimetry measures the light of the transmitted light, which is decreased due to the scattering phenomenon.

TurbidimetricTurbidimetric detection of detection of SpecificSpecificProteinsProteins

Eclectica uses a turbidimetric detection system for Specific Proteins.

Indeed, the advantage of turbidimetry is that such a methodcan be easily adapted to clinical chemistry instrumentsequipped with a photometer. This perfectly applies toEclectica.

The absorbance of the reaction solution (in the flow cell) ismeasured by ECL photometer and fitted on a calibrationcurve

Specific Proteins: Specific Proteins: summary of method descriptionsummary of method description

SP Calibration SP Calibration

The calibration process uses the Samples Carousel.

There are 2 groups depending of the assays.

– 1st Group: IgA, IgG, IgM, C1r, Ceruloplasmin, AntiTrombin 3, Alfa-Glycoprotein, Albumin,C3, C4, AAT, Aptoglobin, Transferrin, Kappa and Lambda chains ,Prealbumin, Microalbumin, Fibrinogen, Fibronectin, CRP, Apo A/B

These assays use :1 vial for the Standard Calibration (only 1 tube)2 tubes for the dilution of the standard (the other dilution are made using

needle)

– 2nd Group: ASO, b2-microglobulin, Lp(a)– These assays use a standard set containing 4 vials (4 tubes)

principle of immunoenzymatic assay principle of clinical...

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