1. Detection and quantification of proteins
MALIK ALQUB MD. PHD.

2. Performance of a test

3. True patients (gold standard)
Sensitivity of a test
Ability of a test to identify correctly affected individuals
proportion of people testing positive among affected individuals
True patients (gold standard) + －
True positive (TP)
Test
False negative (FN)
Sensitivity (Se) = TP / ( TP + FN )

4. Sensitivity of a PCR for congenital toxoplasmosis
Patients with toxoplasmosis
Rapid test
True positive 54
False negative 4 58
Sensitivity = 54 / 58 = 0.931= 93.1 %

5. + － Specificity of a test False positive (FP) Test True negative (TN)
Ability of test to identify correctly non-affected individuals
- proportion of people testing negative among non-affected individuals
Non-affected people + －
False positive (FP)
Test
True negative (TN)
Specificity (Sp) = TN / ( TN + FP )

6. Specificity of a PCR for congenital toxoplasmosis
Individuals without toxoplasmosis
Rapid test
False positive 11
True negative
114
125
Specificity= 114 / 125 =0.912 = 91.2 %

7. + ­ Performance of a test Disease Yes No TP FP Test FN TN TP Se =
TP + FN TN
Sp =
TN + FP

8. Distribution of quantitative test results among affected and non-affected people (ideal case)
Threshold for
positive result
Affected:
Number of people tested TN TP
Quantitative result of the test

9. Specific assay Specificity
A specific assay gives a response to the substance being assayed and to no other substance. Any other substance producing the same response as the substance being assayed is called an "interfering substance". Very few assays are 100% specific; virtually all respond to some interfering substance.

10. Specific assay Benedict's reagent
Benedict's reagent is used as a test for the presence of reducing sugars such as glucose, fructose, galactose, lactose and reducing disaccharides
Then the test is specific for reducing sugars.
But if we want to use it to detect monosaccharides then it would react with disaccharides. We can say the test is not specific for monosaccharide.

11. Detection of proteins

12. Proteins: Common properties
In spite of functional differences between the various proteins, they have certain common biophysical and biochemical properties. These include:
a basic composition of carbon, hydrogen, nitrogen and oxygen;
a backbone of covalent peptide bonds which join the amino acid units together; and
absorption maxima in the ultraviolet region.

13. The Biuret Method
The Biuret reagent is made of (NaOH) and copper (II) sulfate (CuSO4), together with potassium sodium tartrate (KNaC4H4O6).
A blue reagent which turns violet in the presence of proteins,
The Sodium hydroxide does not participate in the reaction at all, but is merely there to provide an alkaline medium so that the reaction can take place.

14. Principle: Biuret Method
Peptide bonds of proteins react with tartrate-complexed cupric ions in alkaline solutions to form a colored product.
In a positive test, a copper(II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution.
A violet color indicates the presence of proteins.
It is possible to use the Biuret reaction to determine the concentration of proteins because (for most proteins) peptide bonds occur with approximately the same frequency per gram of material.

15. The intensity of colaration depends on protein amout
High protein concentration the intesnsity of violet color would increase

16. Biuret Method The intensity of the color varies according to protein
concentration, and hence the absorption at 540 nm, is directly
proportional to the protein concentration, and can be determined
spectrophotometrically at 540 nm.
Using the Biuret Assay to determine protein concentration in an unknown sample requires, as a preliminary, construction of a standard curve made by assaying a series of protein solutions with known concentration. The protein bovine serum albumin (BSA) is frequently used for constructing standard curves because of its availability and low price.

17. Quantification of proteins
Standard amount of protein. We know exactly how much protein in each well.
Unknown concentration 1g 2g 3g 4g 5g

18. the intensity of color is proportionally dependent with absorbed light;
Or optic density
Detector

19. Standard curve
We draw the standard curve, concentration vs. Optic Density.
Then we measure the optic density of « unknown »
If optic density is known of the standard curve then we can find the concentration of the unknown
optic density 1g 2g 3g 4g 5g
concentration

20. BioRad DC Protein Assay