Hazırlanıyor
Biyokimya Test Prensipleri
Otto Bilimsel
Albumin g/dl
Colorimetric assay, endpoint method
• Sample and addition of R1
• Start of the reaction:
At a pH value of 4.1 albumin displays a sufficiently cationic character to be able to bind with bromocresol green (BCG), any anionic dyestuff, to form a blue‑green complex.
pH 4.1 albumin + BCG albumin BCG‑ complex
The color intensity of the blue‑green color is directly proportional to the albumin concentration and can be determined photometrical
Alkaline Phosphatase (ALP) U/L
Colorimetric assay in accordance with a standardized method.
ALP, Mg2
p ‑ Nitrophenylphosphate+ H2O Phosphate + p ‑ Nitrophenol
In the presence of magnesium and zinc ions, p‑nitrophenyl phosphate is hydrolyzed by phosphatases to form phosphate and p‑nitrophenol.
In this process AMP serves as transient phosphate acceptor. The release of coloured p‑nitrophenol is proportional to the ALP activity and can be measured photometrically.
Alpha Amylase
Colorimetric test with 2‑chloro‑4‑nitrophenyl‑a‑D‑maltotriose (CNP‑G3) as direct substrate. Colour is released directly as a result of a cleavage at the a‑glycone:
CNP ‑ G3 a‑amylase CNP + G3
(CNP = chloro‑nitrophenol; G = glucose)
The increase of absorption of chloro‑nitrophenol is directly proportional to the a‑ amylase concentration. The hydrolysis pattern in the formulation of the reagent show about less than 10 % CNP‑G2 and less than 1% CNP‑G4 as by products.
Amylase U/L
Colorimetric test with 2‑chloro‑4‑nitrophenyl‑a‑D‑maltotriose (CNP‑G3) as direct substrate. Colour is released directly as a result of a cleavage at the a‑glycone:
CNP ‑ G3 a‑amylase CNP + G3
(CNP = chloro‑nitrophenol; G = glucose)
AST U/L
UV test according to a standarrized method
Sample and addition of R1 (buffer)
Addition of R2 and start of reaction: AST
α‑ketoglutarate + L‑aspartate L‑ glutamate + oxaloasetate
AST is the enzyme which catalyzes this equilibrium reaction. The oxaloacetate in‑ crease is measured in a subsequent indicator reaction which is catalyzed by malate dehydrogenase.
MDH
oxalacetate + NADH + H+ L‑Malate + NAD+
In the second reaction, NADH is oxidized to NAD. The rate of decrease in NADH
(Measured photometrically) is directly proportional to the rate of formation of
oxaloasetate, and thus the AST activity.
ALT U/L
UV test according to the IFCC method.
ALT
L‑Alanin + 2‑Oxoglutarate ⎯⎯ L‑Glutamate + Pyruvat
LDH Range:20‑4000 U/L
Pyruvat + NADH + H+ ⎯⎯ D‑Lactate+ NAD+
The enzyme alanine aminotransferase (EC 2.6.1.2; L‑Alanine:2‑Oxoglutarate Aminotransferase,
ALT or A1aAT; Glutamate Pyruvate Transaminase, GPT) catalyzes the tran‑ saminase reaction between L‑Alanine and 2‑Oxoglutarate.
The pyruvate formed, is reduced to lactate in the presence of LDH. As the reactions proceed,
NADH is oxidized to NAD+. The disappearance of NADH per unit time is followed by measuring the decrease in absorbance at 340 nm.
Bilirubin Directmg/dl
Jendrassik‑Gróf method
In the presence of caffeine accelerator, total bilirubin couples with sulfanilic acid to form a red azobilirubin dye,
the color intensity which is proportional to the bilirubin concentration. Determination of direct bilirubin is performed without caffeine additive.
The addition of alkaline tartrate causes a transformation from the red azobilirubin dye to a blue dye and the absorbance maximum from 546nm to 578nm.
HCl
Sulfanilic acid+NaNO2 diazotized Sulfanic acid
HCl
Bilirubin + diazotized Sulfanic acid azobilirubin
Bilirubin Total mg/dl
Jendrassik‑Gróf method
In the presence of caffeine accelerator, total bilirubin couples with sulfanilic acid to form a red azobilirubin dye,
the color intensity which is proportional to the bilirubin concentration. Determination of direct bilirubin is performed without caffeine additive.
The addition of alkaline tartrate causes a transformation from the red azobilirubin dye to a blue dye and the absorbance maximum from 546nm to 578nm.
HCl
Sulfanilic acid+NaNO2 diazotized Sulfanic acid
HCl
Bilirubin + diazotized Sulfanic acid azobilirubin
Calciummg/dl
Calcium in the sample reacts with arsenazo III forming a coloured complex that
can be measured by spectrophotometry.
Cholinesterase U/L
Cholinesterase (CHE) catalyzes the hydrolysis of butyrylthiocholine to thiocholine and butyric acid.
The catalytic concentration is determined from the rate of decrease of hexacyanoferrate (III), measured at 405 nm, by means of the following reactions
CHE
Butyrylthiocholine + H2O Thiocholine + Butyric acid
2 Thiocholine + 2 OH‑ + 2 Hexacyanoferrate (III) Dithiobis(choline) + 2 Hexacyanoferrate (II) + H2O
The atypical isoenzyme of cholinesterase (AA) can be estimated through the “dibucaine number” indicating the inhibition of enzyme activity in the presence of dibucaine and expressed in per cent
Clolesterol Totalmg/dl
Cholesterol ester + H2O Cholesterol + fatty acids
Cholesterol esters are ceaved by the action of choesterol esterase to yield free
choesterol and fatty acids Cholesterol oxidase Cholesterol + O2 Cholesten‑3‑on + H2O2
Peroxidase
2H2O2 + Phenol + 4‑Aminoantipyrine Quinoneimine dye + 4 H2O
Cholesterol is converted by oxygen with the aid of cholesterol oxidase to A4‑ Cholestenone and hydrogen peroxide.
Hydrogen peroxide created forms a red dyestuff by reacting with 4‑aminoantipyrine and phenol under the catalytic action of peroxidase.
The color intensity is directly proportional to the concentration of cholesterol and can be determined photometrically.
Creatine Kinase Range: 5‑2500U/l
UV Test
• Sample and addition of R1
• Addition of R2 and start of reaction: CK Creatine phosphate + ADP Creatine + ATP HK ATP + glucose Glucose‑6‑phosphate + ADP G6PDH Glucose‑6‑P + NADP Gluconate‑6‑P + NADPH+H+
Equimolar quantities of NADPH and creatine are formed at the same rate. The photometrically measured rate of formation of NADPH is proportional to the CK activity.
CK‑MB U/l
Immunological UV assay
• Sample and addition of R1 (buffer/enzymes/coenzyme/antibody)
• Addition of R2 (buffer/substrate) and start of reaction.
Human CK‑MB is composed of two subunits, CK‑M and CK‑B which both have an active site.
With the aid of a polyclonal antibody to CK‑M, the catalytic activity of CKM subunits in the sample is inhibited to 99.6% without affecting the CK‑B subunits.
The remaining CK‑B activity, corresponding to half the CK‑MB activity, is determined by the CK‑MB method analogous to total CK.
As the CK‑BB isoenzyme only rarely appears in serum and the catalytic activity of the CK‑M and CK‑B subunits hardly differ, the catalytic activity of the
CK‑MB isoenzyme can be calculated from the measured CK‑B activity by multiplying the result by
CRP (C‑Reactive Protein) mg/L
Immunoturbidimetric assay
Anti‑CRP antibodies react with antigen in the sample to form an ntigen/antibody complex. Following agglutination, this is measured turbidimetrically.
Addition of PEG allows the reaction to progress rapidly to the end point,increases sensitivity, and reduces the risk of samples containing excess antigen producing false negative results.
Creatininemg/L
Immunoturbidimetric assay
Anti‑CRP antibodies react with antigen in the sample to form an ntigen/antibody complex. Following agglutination, this is measured turbidimetrically.
Addition of PEG allows the reaction to progress rapidly to the end point,increases sensitivity, and reduces the risk of samples containing excess antigen
producing false negative results.
Ferritinug/L
Serum ferritin causes agglutination of latex particles coated with anti‑human ferritin antibodies.
The agglutination of the latex particles is proportional to the ferritin concentration and can be measured by turbidimetry.
GGT (Gamma GT)U/l
Enzymatic colorimetric assay
• Sample and addition of R1 (Buffer/Glycylglycine)
• Addition of R2 (substrate) and start of reaction Gamma‑glutamyltransferase transfers the g‑glutamyl group of L‑g‑glutamyl‑3‑
carboxy‑4‑nitroanilide to glycylglycine. The amount of 5‑amino‑2‑nitrobenzo‑nate liberated is proportional to the GGT activity and can be determined photo‑metrically.
Glucose mg/dl
Enzymatic colorimetric test on basis of Trinder – Reaction:
Glucose oxidase Glucose + O2 Gluconic acid + H2O2
Peroxidase
2H2O2 + Phenol + 4–Aminoantipyrine Red Quinoneimine + 4H2O
(Otto Scientific)
HbA1c %
The principle of the test is latex agglutination method that measures the ratio of hemoglobin A1C that occupy in total hemoglobin in the whole blood.
The sample (hemolysis sample) is added to the sensitized latex particles, and the surface of the latex adsorbs total hemoglobin in the sample.
Anti‑human HbA1c Mouse monoclonal antibody complex agglutination by anti‑mouse IgG antibody. At this time, the amount of agglutination caused
depends on the amount of HbA1c that adsorbs the surface of the latex, this this agglutination is measured as a turbidity.
The concentration of HbA1c (%) in the sample is determined by referring to the calibration curve obtained by the same test of diluted standard solutions.
HDL Cholesterol mg/dl
Enzymatic colorimetric test
• Sample and addition of R1
• Addition of R2 and start of reaction
In the first step LDL, VLDL and Chylomicrons are eliminated and transformed to
non reactive compounds and specific condition for the reaction. By the second
reagent only the HDL‑Cholesterol is subject to color reaction
Cholesterol Esterase
Cholesterol ester + H2O Cholesterol + fatty acid
Cholesterol Oxidase
Cholesterol + O2 Cholesten‑3‑on + H2O2
Peroxidase
H2O2 + phenol + 4‑aminoantipyrine quinoneimine dye+4 H2O
LDL Cholesterolmg/dl
İlk adımda, HDL, VLDL ve Şilomikronlar elimine edilir ve reaksiyon için özel koşulda reaktif olmayan bileşiklere dönüştürülür. İkinci reaktif sadece LDL‑kolesterol renk reaksiyonudur.
Kolesterol esteraz
Kolesterol ester + H2O kolesterol + yağ asidi
Kolesterol Oksidaz
Kolesterol + O2 kolesten‑3‑on + H2O2
peroksidaz
H2O2 + fenol + 4‑ aminoantipirin kinon boyası +4 H2O
lgA mg/dL
Immunoglobulins A (IgA) selectively react with an anti‑IgA antibody and form an immunocomplex.
The produced turbidity is proportional to the concentration of IgA in the sample, and can be measured at the wavelenght of 600 nm
lgE IU/mL
Anti‑human IgE antibodies when mixed with samples containing IgE, form insoluble complexes.
These complexes cause an absorbance change, dependent upon the IgE concentration of the patient sample,
that can be quantified by comparison from a calibrator of know IgE concentration.
lgG mg/dL
Immunoglobulins G (IgG) selectively react with an antiIgG antibody and form an immunocomplex.
The produced turbidity is proportional to the concentration of IgG in the sample, and can be measured at the wavelenght of 600 nm
lgM mg/dL
Immunoglobulins M (IgM) selectively react with an antiIgM antibody and form an immunocomplex.
The produced turbidity is proportional to the concentration of IgM in the sample, and can be measured at the wavelenght of 340 nm.
Lipase U/l
Enzymatic colorimetric assay;
• Sample and addition of R1 (buffer/colipase/cholate)
• Addition of R2 (emulsion/chromogenic substrate/cholate) and start of reaction:
lipase
1,2‑O‑dilauryl‑rac‑glycero‑3‑ 1,2‑O‑dilauryl‑rac‑glycerol +
glutaric acid‑(6‑methylresorufin)ester glutaric acid‑(6‑methylresorufin)ester
spontaneous
glutaric acid‑ glutaric acid + methylresorufin
(6‑methylresorufin) ester decomposition
The chromogenic lipase substrate 1,2‑O‑dilauryl‑rac‑glycero‑3‑glutaric acid‑(6‑
methylresorufin) ester is cleaved by the catalytic action of alkaline lipase solution to form 1,2‑O‑dilauryl‑rac‑glycerol and an unstable intermediate,
glutaric acid‑(6‑ methylresorufin) ester. This decomposes spontaneously in alkaline solution to form glutaric acid and methylresorufin.
The colour intensity of the red dye formed is directly proportional to the lipase activity and can be determined photometrically.
MagnesiummEq/l
Colorimetric endpoint method
Sample and addition Reagent start of reaction:
In alkaline solution, magnesium forms a purple complex with xylidyl blue, a
diazonium salt. The magnesium concentration is measured photometrically via the decrease in the xylidyl blue absorbance.
(Otto Scientific)
Phosphorusmg/dl
Inorganic phosphate forms an ammonium phosphomolybdate complex having the formula(NH4)3[PO4(MoO3)12] with ammonium molybdate in the presence of sulfuric acid.
The complex is determined photometrically in the ultraviolet region (340 nm).
RF (Rheumatoid Factor) IU/ml
Particle enhanced immunoturbidimetric assay.
Sample and addition of R1 (buffer)
Addition of R2 (latex‑bound IgG/buffer) and start of reaction.
Latex‑bound heat‑inactivated IgG (antigen) reacts with the RF‑antibodies in the sample to form antigen/antibody complexes. Following agglutination is measured turbidimetrically.
Total Protein g/dl
Colorimetric assay, Sample and addition of Reagent start of the reaction:
Divalent copper reacts in alkaline solution with protein peptide bonds to form the characteristic purple‑colored biuret complex.
Sodium potassium tartrate prevents the precipitation of copper hydroxide and potassium iodide prevents auto reduction of copper. alkaline protein + Cu2+ solution Cu‑protein complex
The color intensity is directly proportional to the protein concentration which can be determined photometrically.
Triglyceridesmg/dl
Triglycerides in the sample originates, by means of the coupled reactions described below, acoloured complex that can be measured by spectrophotometry.
Triglycerides + H2O lipase Glycerol + Fatty acids
Glycerol + ATP glycerol kinase Glycerol – 3 – P + ADP
Glycerol – 3 –P + O2 G‑3‑P‑oxidase Dihidroxyacetone – P +H2O2
2 H2O2 + 4 – Aminoantipyrine + 4 – Chlorophenol G‑3‑P‑oxidas Quinoneimine + 4 H2O
UIBC μg/dl
Photometric test using chromagen ferrozine.
Total Iron‑Binding Capacity(TIBC): A known amount of ferrous ions are added to serum at an alkaline pH.
The ferrous ions bind with transferrin at unsaturated iron‑binding sites. The additional unbound ferrous ions are measured using the ferrozine reaction.
The difference between the amount of ferrous ions ions added and the unbound ions measured is the unsaturated iron‑binding capacity (UIBC). The TIBC is equal to the serum iron concentration plus the UIBC.
Urea (BUN)mg/dl
Urea is hydrolysed in presence of urease to produce ammonia and CO2. The ammonia produced combines with 2 – oxoglutarate and NADH in presence of GLDH to yield glutamate and NAD.
urease
Urea+H2O+2H+ 2 NH4+ + 2+ CO2
GLDH
2 NH4+ + 2‑Oxoglutarate + 2 NADH H2O + 2 NAD+ + Gl utamate The decrease in absorbance due to consumption of NADH is measured kinetically.
Uric Asitmg/dl
Colorimetric endpoint assay
Uricase
Uric acid + 2 H2O + O2 Allantoin + CO 2 + H 2O 2
Uricase cleaves uric acid to form allantoin and hydrogen peroxide.
POD
2H2O2 + 4H+ + phenole + 4‑aminoantipyrine quinonimine dye + 4H2O
The increase in absorbance is measured.
HbA1c
The principle of the test is latex agglutination method that measures the ratio of hemoglobin A1C that occupy in total hemoglobin in the whole blood.
The sample (hemolysis sample) is added to the sensitized latex particles, and the surface of the latex adsorbs total hemoglobin in the sample.
Anti‑human HbA1c Mouse monoclonal antibody complex agglutination by anti‑mouse IgG antibody. At this time, the amount of agglutination caused depends
on the amount of HbA1c that adsorbs the surface of the latex, this this agglutination is measured as a turbidity.
The concentration of HbA1c (%) in the sample is determined by referring to the calibration curve obtained by the same test of diluted standard solutions.
Iron (FE) (μg/dl)
Colorimetric assay pH < 2,0 Transferrin‑Fe‑complex apotransferrin + Fe3+ ascorbate Fe3+ Fe2+ FerroZine® + Fe2+ colored complex Under acidic conditions,
iron is liberated from transferrin. Lipemic samples are clarified by the detergent. Ascorbate reduces the released Fe3+ ions to Fe'2+ ions which react with
FerroZine to form a colored complex. The color intensity is directly proportional to the iron concentration and can be measured photometrically.
Potassium
Ion Selective Electrode (ISE)
Sodium
Ion Selective Electrode (ISE)
Potassium (K)
Potassium Assay Kit is an assay kit is an Assay Kit by Colorimetric Method designed for the detection and quantification of Potassium concentrations.
Sodium (Na)
Sodium Assay Kit (Colorimetric) provides a convenient two‑step method to detect sodium ions (Na ) present in serum, urine and saliva. In this assay,
sodium ions present in the sample are used by the enzyme β‑galactosidase to produce an intermediate product, which reacts with a developer to generate a color signal that can be detect at OD = 405 nm.
ICP‑MS
An ICP‑MS instrument uses a plasma (ICP) to ionize the elements in a sample and then measures the ions using a mass spectrometer (MS)
Perkin Elmer NexION 2000‑c
NEFA
Enzymatic endpoint method
Non‑esterified fatty acids and coenzyme A react in the presence of acyl coenzym A synthetase (ACS) to acylated coenzyme A.
Subsequent oxidation of the acylated coenzyme A by acyl‑CoA oxidase releases H2O2. H2O2 is converted to a colored product by the use of Trinder substances in the presence of peroxidase (POD).
ACS
NEFA + Coenzyme A + ATP ◄─────► Acyl‑CoA + AMP + PPi
ACOD
Acyl‑CoA + O2 ◄─────► 2,3‑Trans‑enoyl‑CoA + H2O2
POD
2 H2O2 + Trinder ◄─────► Dye + 4 H2O
At 546 nm the intensity of the red dye is directly proportional to the concentration of free fatty acids in the sample.
BHBA
Enzymatic determination with β‑hydroxybutyrate‑dehydrogenase
β‑Hydroxybutyrate in presence of NAD+ is converted to acetoacetate and NADH + H+ by β‑hydroxybutyrate‑dehydrogenase. The absorbance at 340 nm is proportional to the β‑hydroxybutyrate concentration in the sample.
β‑Hydroxybutyrate‑dehydrogenase