Biomarker Technologies

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Why Learn Biomarker Technologies

• Learning about biomarker technology is crucial because biomarkers are related to diseases.
• It is important to understand the technologies and techniques used to measure biomarkers.
• High-throughput diagnostic tests and automated instruments, like routine autoanalyzers necessitate this knowledge.

Lecture Highlights

• The lecture included the principles of common and advanced technologies for biomarker measurement.
• Also discussed were types of automated equipment using these technologies.
• The lecture touched on point-of-care testing or POCT within clinical chemistry.

General Technologies

• Common technologies are spectrophotometry, immunoassay, electrochemistry, osmometry, electrophoresis, and chromatography.

Learning Foci

• The principle of spectrophotometry uses the electromagnetic wave spectrum such as UV and visible light.
• Spectrophotometry applies e.g. the Biuret reaction for total protein measurement.
• Spectrophotometry measures the concentration of unknown and known substances.
• Spectrophotometry uses wavelength scans.
• Calibration set-up and sample concentration measurement uses Beer's law.

Spectrophotometry Details

• Spectrophotometry is versatile, reliable, and commonly used in clinical chemistry.
• It involves producing a colored end product.
• Absorbance spectrum measurement used when concentration is detected by a photometer.
• Automated analyzer systems use a spectrophotometer as a read-out device.
• Photometry measures absorbance (A), called optical density (OD), or percent transmittance (%T).
• The light path, e.g. in the cuvette, is held constant using a standard cuvette.
• Quartz cuvettes is used to operate in the UV spectrum.
• Increased absorbance decreases %T logarithmically, plotting a straight line on semi-log paper.
• Radiation not transmitted is absorbed based on the molecule makeup, known as molar absorptivity, a substance characteristic.

Absorbance vs Transmittance

• Transmittance is the ratio of the incident light intensity (I₀) to the light passing through the object (I).
• Absorbance is the amount of light absorbed by the molecules of an object.
• Transmittance decreases exponentially as concentration increases.
• Absorbance increases linearly with concentration.
• Transmittance values range from 0 to 1 (or 0% to 100% in percentage).
• Absorbance takes values from 0 upwards.

Transmittance and Spectrophotometry

• Light transmittance through a solution is important because only transmitted light can be measured in a spectrophotometer.
• Transmittance (T) is the proportion of incident light that is transmitted: i.e. T = 0.25 means %T = 25%.
• Absorption or transmission of light through a specimen determines the molar concentration of a substance.
• Beer-Lambert Law states that a substance's concentration is directly proportional to the light absorbed by the chromogen.
• The concentration is inversely proportional to the logarithm of the transmitted light. Absorbance (A) is equal to 2 - log(%T)
• If the concentration of a solution is unknown, measure the amount of light it absorbs, (absorbance A).
• This is done at a particular wavelength (λ), using a spectrophotometer.
• Absorbance (A) and concentration (c) are related related to Beer's Law: A = εlc
• ε is the molar absorptivity, dependent on the wavelength
• l is the path length in cm, that light travels through the solution.

The Standard Curve Concept

To evaluate concentration from absorbance measurements, measure the absorbance of several standard solutions, known concentrations.

• A plot of A (absorbance) vs. c (concentration), should be linear from equation 1, and allows the calculation of ε.
• The path length (l )is held constant by using the same sample holder for all measurements.
• Do not change the cuvette orientation during absorbance measurement.
• A plot of absorbance vs. is c is called a calibration curve.
• The concentration of any is solution can be found with the calibration curve by measuring its absorbance.

Preparing a Standard Curve

• Prepare a standard curve using several known concentrations of a substance in solution to plot points on a graph.
• Plot concentration vs. absorbance and that curve is used to determine the concentrations of unknown samples.
• Straight lines are obtained in a graph of absorbance versus concentration, when plotted on coordinate graph paper or semi-log graph paper.
• It is implied the unknown solution concentration is also directly calculated by the linear relationship given validity by Beer's law, A1/A2 = c1/c2.
• Even if a procedure follows Beer's Law, deviations from linearity occur at very low or very high concentrations, though the linear portion is useable.
• If the concentration is too low, the result usually is reported as less than the lowest concentration on the linear portion.
• If the concentration is too high, the specimen can be diluted, assayed, and the result multiplied by the dilution factor.

Immunoassay

• Immunoassay, is an immunochemical method
• In immunoassay the principle can be direct, indirect, competitive, or sandwich.
• ELISA examples are Bone Markers, see package insert

ELISA - Enzyme-Linked Immunosorbent Assay Details

• ELISA is a plate-based assay used for detecting and quantifying soluble substances, such as proteins, peptides, antibodies, and hormones
• ELISA is also used for enzyme immunoassay EIA.
• In ELISA, the antigen is immobilized on a solid surface and complexed with an antibody linked to a reporter enzyme.
• The most crucial element of an ELISA is a highly specific antibody-antigen interaction.
• Detection measures the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product/signal.
• ELISAs are typically performed in 96-well or 384-well polystyrene plates, that passively bind antibodies and proteins.
• The ability to use high-affinity antibodies while washing away non-specific bound materials (heterogenous assay) makes ELISA a powerful tool.
• ELISA is used for measuring specific analytes within a crude preparation.
• Most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP).
• Other enzymes that have been used include beta-galactosidase, acetylcholinesterase, and catalase.
• Many substrates are commercially available for performing ELISA with an HRP or AP conjugate.
• The choice of substrate depends on the required assay sensitivity and available instrumentation for signal-detection.
• Signal detection uses a spectrophotometer, fluorometer, or luminometer .

ELISA Formats

• ELISA is performed in Direct, Indirect, or Sandwich capture and detection methods.
• The key step is immobilization of the antigen of interest by coating it onto the assay plate.
• Then antigen detection is then completed either directly with a labeled primary antibody.
• Or indirectly with a labeled secondary antibody.
• The sandwich ELISA assay format is the most widely used.
• It indirectly immobilizes and detects the target antigen presence.
• The measured analyte is bound between two primary antibodies.
• The antibodies each detect a different epitope of the antigen.
• In this system, there is a capture antibody and a detection antibody.
• Sandwich ELISAs are highly used due to sensitivity/specificity.
• In competitive ELISAs the binding process a competitive one.
• A competitive binding process is executed by original antigen and add-in antigen.
• Competitive ELISA procedures differ from those of indirect, sandwich and direct ELISAs.

Osteoclastin ELISA

• Osteoclastin EASIA is a solid phase enzyme amplified sensitivity immunoassay performed on a microtiter plate.
• Standards and samples react with the capture of monoclonal antibody coated on the microtiter well and with a monoclonal antibody, labeled with horseradish peroxidase.
• After incubation allowing the formation of a sandwich of coated antibody, human osteocalcin, and horseradish peroxidase labeled antibody.
• The microtiter plate is washed to remove unbound enzyme labeled antibody.
• Bound enzyme labeled antibody measurements are completed through chromogenic reaction.
• The reaction is stopped with the addition of Stop Solution and the microtiter plate is then read.
• Amount of substrate turnover is based on the absorbance, a standard curve is plotted and human osteocalcin concentration in a sample is determined.

Electrochemistry

• Electrochemistry's principle involves the ion selective electrode.
• Examples are a sodium and potassium ion electrode (autoanalyzer) and blood gas analyzer measuring things like pH, pO2, and PCO2.
• The potential difference between the internal reference electrode and the internal membrane surface is constant(design).
• The design components are the nature of the reference electrode and the activity of reference solution.
• The potential difference, between the external membrane surface and the sample solution, depends upon the target ion activity.
• The potential difference across the membrane is described by the Nernst equation.

Electochemistry and Modes of Detection

• Electrode potential will change relative to ion concentration Modes of detection are amperometric, potentiometric, conductimetric, and impedimetric.

Freezing Point Depression

• Freezing point depression is used with osmometry.
• Principle is colligative properties, where amount depends on solute-solvent particle ratio, not solvent.
• An application example is measuring plasma or urine osmolality.

Osmometry Measurement principle

• The formula for freezing point depression is ΔTf = (Kf)(m).
• ΔTf: freezing point depression.
• Tf⁰ is the freezing point of the pure solvent.
• Tf is the freezing point of the solution.
• Kf is the molal freezing point depression constant.
• m is the molality of the solution: moles of solute/ kg of solvent.

Electrophoresis

• Electrophoresis principle: separation based on protein charge and size.
• Electrophoresis has serum protein (SPE) and cellulose acetate for Haemoglobin pattern applications. -Serum protein uses charge/size while cellulose acetate is for Haemoglobin pattern.
• An application example is Agarose gel electrophoresis (size separation) in molecular testing.
  • Another application example is PCR product analysis for gene expression study .
  • Genetic inheritance pattern in family study
  • Example: testing Restriction Fragment Length Polymorphism, RFLP, using restricted enzymes.

Serum Protein Electrophoresis

• Serum proteins are large covalently linked amino acid molecules
• They are either poloar or nonpolar at a given pH dependent on different ionic or covalent variances.
• Serum proteins are separated based on their electrical charges at 8.4 - 8.8 on agarose gel with both electrophoretic and electroendosmotic forces.
• Separated proteins are visualized with a staining solution such as Amido Black.

Chromatography Overview

• Chromatography is the collective term for analytical techniques separating mixture components.
• Chromatography aids in the identification and estimation of component concentrations in the mixture.
• Clinical and biological samples often encounter interfering substances, requiring analyte separation and isolation for concentration determination.
• Chromatography's introduction involves separation mechanisms and examples.
• The applications of chromatography involve TLC, HPLC, GC, LCMS-MS.

Chromatography Mechanisms

• Chromatography separates components or solutes of a sample mixture.
• It is done by a stationary phase, which may be a solid, liquid supported on solid, or gel.
• The stationary phase packed in a column, spread as a layer, or distributed as a film
• Separations are achieved by a mobile phase, and can be a gaseous, supercritical fluid, or liquid.
• Sample components are moved by mobile phase through the stationary phase bed.
• Individual species are retarded through interactions, distributed between stationary and mobile phases.

Forms and Nature of Chromatography

• Chromatography includes planar and column formats.
• Solubility and polarity in Paper and Thin Layer Chromatography TLC.
• Size based on Gel filtration chromatography.
• Charge differences measured in Ion-exchange chromatography.
• Hydrophobicity and Hydrophobic chromatography or Gas-liquid chromatography.
• Ligand binding in Affinity chromatography.
• Liquid chromatography: mobile phase is liquid.
• Gas chromatography: carrier is gas.

Separation mechanism on chromatography stationary phases

• Liquid chromatography subtypes use ion exchange(IE electrostatic), a liquid or solid stationary phase (L/S adsorption), or liquid/liquid (L/L partition).
• Size exclusion utilizes a Gel filtration GF.
• Gas chromatography uses a gas phase/solid surface (G/S adsorption) or a gas and a liquid(G/L partition).
• Liquid and liquid partition types are normal or reverse phase .
  • Ion supportesion-
  • Ion pai

How Chromatography Works

• Mobile anions are held near cations that connect with the stationary phase in ion-exchange chromatography
• Only anions are attracted to the anion-exchange resin.
• Solutes are measured on the solid stationary phase during adsorption.
• Solute is is measured on liquid stationary phase during partition chromatography coated on solid support.
• Small particles are measured based on their ability to penetrate pores in Molecular exclusion chromatography.
• Affinity chromatography occurs when one molecule in a complex mixture is bonded to the stationary phase, measured as all other molecules wash through.

Separation Mechanisms: Adsorption Chromatography

• Separation occurs by differences between solute adsorption/desorption at surface of solid particle.
• Electrostatic, hydrogen-bonding, and dispersive forces control adsorption.
• Types of adsorption chromatography differ depending on GC(gas chromatography) or LC (liquid chromatography).
• GC uses a solid adsorbent, separates LMW compounds such as methyl, then the compound is normally gas at room temperature .
• GC uses molecular sieves and Alumina
• LC (liquid chromotography) uses non-polar, acidic polar, and basic polar adsorbents.
  • Example polar are silica gels.
  • Acidic polar adsorb Basic soutes.
  • Basic polar are composed of alumina.

Separation Mechanics: Partition Chromatography

• The basis of chromatography is the partition or distribution coefficient, (Kd).
• There is a substances equilibrium between two unmixable phases
• For example, in the Kd of substance X.
• The formula is Kd = Concentration of X in A/Concentration of X in B.
• Solute distribution differential between two unmixable liquids is called differential partitioning.
• Separation is is based on relative analyte solubility between stationary/mobile phases.
• Solubility and adsorption to surfaces is determined by by molecule polarity.
• Thus, chromatography differs in analyte solubility/polarities, the partition to immiscible liquids, and separation.
• So polarity and solubility are correlated with the analyte ability to separate and move through the columns and gradients.

Partition Chromatography Details

• A stationary phase involves a thin film of liquid, ad/chemically bonded onto the support particle or capillary column.
• Categorization is completed by: GLC gas chromatography, LLC liquid chromatography
  • Which occur on the support itself

Details Regarding Partition and Liquid Chromatography

• Normal phase: stationary phase more polar, the mobile phase is more polar,
• Reverse phase: stationary phase more non-polar vs a more polar mobile phase. - Reverse two forms: Ion-suppression chromatography - Ion-pair chromatography

Normal Phase LLC

• The normal-phase partition system has silica, coated in monolayer of water or other, polar liquid. And a not as polar, system of solvents.
• Separations are completed dependent on polar solute.
• With polar compound substances retained long, a non-polar type solute eltes.

Reverse Phase LLC

• Reverse-phase stationary stages is to chemically modify and produce in non support polar organic substituent. The compounds bonded and commonly used are known are the hydrocarbon phases.
• Such methyl or octyl groups attached to silica.
• organic is by the composition of the bonded the polar and the stages as the support of mobile phases often include:
• Examples: The water, methanol, or acetonitrile.
• Solutes phases by eluting on non support polarity.
• Example: A highly polar eluting phase could contain Non- polar materials bonded to an elutes in the first polar solute.

Retention Examples during Chromatography

• Fluorocarbons and retained on their retention by the reversed phase
• Least retained in the normal phone.
• Acids and bases are retained for their retention by the normal phase with the
• Least retained in the reverse phone.

Ion Exchange Chromatography

• Strongest force is Electrostatic interactions
• Exchanging by charges the Exchange Exchange with the charged stationary.
• The sample compounds to interact weakly with the stationary to separate.
• Stationary phase made of plastic Silica or silica.
• The fixed particles have Cationic charges or Anionic.

Common Ion Exchangers

• Samples with charged particles separate based on stationary phase.
• Anion exchange formula: A- + R+B- → B- + R+A-
• A- is the mobile phase -B- is the is is added and binds,
• Cation exchang A+ + B+R- → B+ +A +R
• Ions and charge can separated in small bead columns .
• Retard protein and affect charge by the matrix.

Ion-exchange chromatography is pH dependent

• Factors that control with:
  • pH mobile phase. -Charge mobile phases. -Charges depend on ion of solute .
  • Charge by stationary and counterion phase depends on ion phase.
• Electrolyte an and Ion found to have same proximity near close proximity in the fixed charge.
• Elctroneutrality electrolytes mobile.

More Ion Exchange Considerations

• Ion-exchange media are classified according to the nature of the attached ionizable group, whether strongly/weakly acidic or basic, determining usable pH range
• The nature of the charge and acidic to
• the ion charges by the media from variations or selective mobile.
• Examples for application can include amino .

Size Exclusion Chromatography

• Size-exclusion is a gel filtration.
• If a size of 10 nm was to passed through all and was only as large as 15% it cannot get any it all as well..
• Polymers are all in an open phase at range or in silica.

Size Exclusion Details

• A smaller retention.
• Long mobile to flow and are by pores and long be in the mobile.
• Molecules intermediate have large between have volume.
• The fraction is is accessible as molecules.

More Size Exclusion

• A big separation is used with most at liquid phase or by as at long.
• Applicable for 50 > - 10mil. or greater.
• Obtains Calibrators which help choose MW expected with it which helps with solute on mobile.
• Limited resolutions should be applied to the phase in which the sample phase to it is to be prepared with the preparation itself.

Affinity Chromatography

• Based and Separation on molecules that can ligan and analyte.
• resulting interactions specific. - Enz and substrated. - Antigens and antiodies
• adjust the ionic strength to get binding analyte molecule. Analyte molecules may have replaced by: - Adjustment by ionic change. - Ligand displacement

Affinity Chromatography

• Station are ligands: such support material that molecule binding for spacing or interaction. - Agarose - Polyacrylated - Cellulose-
  • Application:
  • Column and heparind -LDL

High Performance Liquid Chromatography (HPLC)

• The name came from was of that of that High pressure was was was generate generate the for the packed volume.
• Pressures of 500 PSI and greater.
• These developed the develop instruments, the had had had develop 6000 psi.
• With changed and to Hiig.
• Now to the include, identify quantity which has any that liquid.
• Parts and can easily obtained.
• Samples.

High Performance Liquid Chromatography (HPLC) and UPLC

• A tool HPLC, is in analutical separatin which and is or of variety.
• At any liquid to can is the dissilation in liquid or by the dissilation in the liquid.
• The reverse is Ultra with packing which 6 -5 is.
  • Conventional will operates.

HPLC Instrumentation

Mobile phase supply

• pump to 1 mL. sample is the Column.
• Then comes the comes at which point to be the comes is.

HPLD Pump Types

• One of the most component of components which used, and detector.
• There is is constant flow of.
• Types const pressure.

HPLC: Mode of Pump Operation Details

• With compositions that is constant in run it as a elution .
• For which all same which used it that be like isocratic.

Gradient HPLC

• Mobile phased change during the run phase on valves pump and with valves..

HPLC Columns

• Precolumn, positioned between the pump and injection valve.
• It is packed with silicate that prevent pack analytical the from volume.
• Protect with irreversibly phases and as stationary col phase.

Analytical HPLC Columns

• ID is the ID and range from with capillary mm with mm it is with it with with mm to the Bore type.
• Length from with between tube heavy
• Made with area silica of silica.
• Based bodies which are of 350

HPLC Column Packings

Two column packing types are:

• Column packing of the two stationary as well as Bond to polymer and. - Stationary bonded chemical the Silica and c8. - Normal for bonded from derivatized as carbon/C.
• Pores high back surface surface for are.

HPLC Detectors And Factors Affecting Seperation

• Liquid Chromatography Detectors (LCD) to measure changes in the sample stream's characteristics to quantify and identify.
• Affecting conditions. The is polar can change is gradients. affect retention is the temperature.

Resolution

• Res is resolution by chro sep.
• Over and sufficiently. . R= [V(B) -(a)/

Selectivity factor (α)

• Selectivity factor (α), due to mobile phases e.g. pH, ionic strength, salt composition (e.g. NaCl Vs LiCl)

Chromatogram

• Detector for the graph.
• This happens in versus in elution of of either in of elution or volume.

Quantitation

• For the of to be added "interal standard", standanrd chemical to be is used the with analytte to be is added with all.
• As the check is on the, it all chromatogram in the height to the sample must be at the same.

Automated Testing Equipment

• Automated equipment to apply tech which to biomedicine.
• Example osmometer.

Use with Automated Equiptment

• Measuring osmolality and easily with or (il.bio.)

Blood Gas Analyser

• Direct with ISE pH, with with:
• Whole blood
• Fast results
• Clotting of and care

Blood Gas Video

• Flex bloods can youtube.

Arterial and Venous reference ranges

• PH- 7.36 vs 7.34
• And venous both are can found analysis acute.

Venous Blood values

• All values to determine with the use of electrolyte

Measurement component & principle_of

• the FLEX guide can helps with SOP, on websites with.

Automated clinical chemistry analysers

• Versatile with robotic and to
• modular and

Routine Chemistry Automation

• Chemistry for test and markers which LFT profile. Throughputs measure to is high:

• For measurements

• Measurements and position.

• Immunochmistru is chemistry of CL and the

• for the which K which and also there barcode and system.

• ISE has only