Chemistry and Immunochemistry Methodologies:
- Chemiluminescence (Immunoassays)
- Sometimes referred to as "Chemiflex"
- Reagents are sets including diluent, conjugate, and reagent containing antibody-coated paramagnetic microparticles
- EXAMPLE: TSH (thyroid-stimulating hormone)
- Sample anti-B TSH antibody-coated paramagnetic microparticles are combined with TSH assay diluent
- Next, TSH in the sample (antigen) binds to anti-TSH antibody found in the reagent (coated microparticles)
- The sample is washed
- A dye referred to as anti-A TSH acridium labeled conjugate is added
- Pre-trigger and trigger solutions are added to the mixture
- A chemical reaction occurs and it is referred to as a chemiluminescent reaction
- The chemiluminescent reaction is measured by a special photometer as relative light units (RLU's)
- There is a direct relationship between the amount of TSH in the sample and the RLU's detected by the optics (photometer)
- This is an antibody-antigen complex that forms and is read by a photometer and measured in relative light units (RLU's)
- Enzymatic
- Enzymes are catalysts that kick off and accelerate chemical reactions
- They speed up in the presence of heat
- Most are proteins
- Require optimal temperature, pH, and are very specific
- Many of these 3D or 4D molecules are permanently denatured in the presence of excessive heat
- "Lock-and-key" fit
- End in "ase"
- Some require coenzymes to help get the reaction started (NAD+ or vitamins)
- They can be measured by any of the following in the chemistry lab:
- Measurement of enzymes
- Measurement of substrate (substance upon which the enzyme acts)
- Measurement of products (end products form when enzyme acts on substrate)
- Measurement of inhibitors (substances that decrease enzymatic activity)
- Therapeutic drugs
- Toxic drugs and alcohol and poisons (lead, ammonium, acetaminophen, salicylate, street drugs, ETOH, antifreeze, etc...)
- Measurement of activators (substances that increase enzymatic activity)
- Measurement of the reaction rate
- Immunoturbidimetry
- Useful in the diagnostic field of clinical chemistry
- Used to detect serum proteins
- Based on the antibody-antigen complex, which can be detected by a photometer
- Turbidity or turbidimetry is the measurement of the loss of intensity of transmitted light, because it is scattered by particles within a solution.
- Light passes through a special filter and creates light of a known wavelength. The wavelength is passed through a cuvette or reaction vessel containing a solution.
- A photoelectric cell in the analyzer absorbs the light and it passes through the cuvette. This absorbed light gives a measurement.
- CNP-triose/CNPG 3
- Urease
- This is a measurement of the waste product of nitrogen metabolism, urea, which further hydrolyzes into CO2 and ammonia.
- Urease is the enzyme that kicks off the urea cycle
- Used to measure BUN (blood urea nitrogen)
- UV
- Arsenazo III
- This method is used to detect Calcium levels
- Useful in detecting hypercalcemia (may be caused by hypoparathyroidism, hypervitamin D, Multiple Myeloma, bone diseases and cancers, long-term lithium therapy)
- Useful in detecting hypocalcemia (may be caused by hypoparathyroidism, low vitamin D, steatorrhea, nephrosis, nephritis, pancreatitis)
- Arsenazo dye reacts with calcium in the sample in acidic solution
- It forms a purple-blue complex, which is measured by spectrophotometry/colorimetry at a range of 600-660 nm and the amount of this complex is directly proportional to the amount of calcium in the sample
- Arsenazo is a calcium-sensitive dye and is a metallochromic indicator
- NOTE: arsenic present in arsenazo dye is a poison and a health hazard
- This method is used to detect Calcium levels
- ALK-picrate-kinase
- Benzethonium chloride
- EIA
- Hexokinase
- Lactate reduced to pyruvate
- Used to measure glucose
- Diazotization: This is the conversion of a primary aromatic amine into its diazonium salt, which is an intermediate that is able to go through a coupling reaction and form an azo dye such as methyl red or pigment-red 170.
- Example: Direct Bilirubin
- The bilirubin reacts with a diazonium sulfanilic acid or a diazonium salt in the presence of sulfanilic acid and it forms a colored compound, which is then detected via spectrophotometry/colorimetry.
- The bilirubin-diazonium compound is referred to as azobilirubin.
- Example: Direct Bilirubin
- Diazonium ion
- Diazonium is a salt
- Diazo breaks down near or under UV light
- An azo compound is utilized as a dye
- This dye can be used as a pH indicator
- Diazonium is a salt
- Bromcresol purple
- pH indicator
- Used to measure albumin
- Yellow when acidic; purple when neutral to basic (alkaline)
- PNPP, AMP Buffer
- Direct measure, Polymer-polyanion
- Chol. oxidase, esterase, peroxidase
- Ferene
- G-Glutamyl-carboxyl-nitroanilide
- Biuret
- The biuret method uses biuret reagent, consisting of sodium hydroxide (NaOH), hydrated copper (ii) sulfate, and potassium sodium tartrate. The potassium sodium tartrate both chelates and stabilizes the copper (cupric) ions. Copper (ii) is reduced to copper (I). Cupric ions react with nitrogen atoms present in peptide bonds holding proteins together. This causes the hydrogen atoms present in the bonds to become displaced in alkaline conditions. The chelation with nitrogen atoms forms the characteristic purple color when proteins are present. This is detected by spectrophotometry/colorimetry read at 540 nm.
- This detects the concentration of protein in the sample, and the amount of protein present in the sample is directly proportional to the color complex formed, according to the Beer-Lambert Law.
- Sometimes referred to as the Modified Lowry Test
- NOTE: Ammonia interferes with this test (Try not to put total protein reagent and ammonia reagent next to each other on the analyzer rack or wheel)
- Colorimetric
- Used to measure lithium
- Similar to spectrophotometry
- Used to detect the concentration of colored compounds in solution
- Colorimeter measures the absorbance of a specific wavelength of light
- Uses the Beer-Lambert Law to calculate the intensity of light before and after it passes through a sample in order to determine the concentration
- Different solutions must be made and a water calibrator is often used
- Colorimetric assays use reagents that react to form a color change when the analyte is present
- Indirect measure
- Used to measure K+ and Cl- and Na+ (the electrolytes)
- Molybdate
- Used in the colorimetric determination of phosphate quantity in association with the dye malachite green
- A type of colorimetric reaction
- Used in the colorimetric determination of phosphate quantity in association with the dye malachite green
- Arsenazo Dye
- Used to measure MG
- Oxidation
- Used to measure Lac M
- Modified Rate Jaffe'
- Used to measure creatinine
- CE/CO/Peroxidase
- Used to measure cholesterol
- BCG
- Used to measure albumin
Chemiluminescence/Chemiflex:
Spectrophotometry:
Photometry is a method that depends on the measurement of light by a photodetector. Light is either:
- absorbed by a (solute) that has been dissolved in a solution (this is called "absorbance")
- refracted by particles that are suspended in a solution (scattered; referred to as "turbidimetry"
- emitted from a substance that absorbs light at one particular wavelength and emits light at another wavelength (referred as "fluorescence")
- depends upon the properties of the substance that is being measured
- 400 nm (violet light) to
- 700 nm (red light)
- 200 nm to
- 400 nm