RANIBIZUMAB

PROTEIN ASSAY

QUANTITATIVE METHODS

Copper Ion Based Assays (Lowry & BCA) 

In copper ion based assay protein solution mixed with alkaline solution of copper salt. In  alkaline conditions, cupric ions (Cu2+) react with peptide bonds and reduce to cuprous ions (Cu+). If the alkaline copper is in excess over the amount of peptide bonds, some of the cupric ions (Cu2+) will remain unbound to the peptide bonds and are available for detection. Protein assays based on copper ions can be divided into two groups, assays that detect reduced cuprous ions (Cu+) and assays that detect the unbound cupric (Cu2+) ions. The cuprous ions (Cu+) are detected either with bicinchoninic acid (BCA) or Folin Reagent (phosphomolybdic / phosphotungstic acid) as in the protein assays based on Lowry method. 

Lowry Method
As discussed above its two step reaction in first step Cu+2 reduced under alkaline condition to Cu+1 which form complex with peptide bonds. In second step Cuprous ions (Cu+) peptide bond complex  reduces the Folin Reagent and produces a blue color that can be read at 650-750 nm. The amount of color produced is proportional to the amount of peptide bonds, i.e. size as well as the amount of protein/peptide.This assay is useful to detect protein from 10 to 1000 μg /ml. 
Folin method sensitive to pH changes and pH should maintain 10 to 10.5.

Step 1 : Protein + cu+2   = OH- + protein-Cu+1complex 

Step 2 : protein-Cu+1complex + Folin ciocalteu = OH- and reduced Folin reagent

Other Method

This assay is based on the detection of unbound cupric ions (Cu+2), the protein solution is mixed with an amount of alkaline copper that is in excess over the amount of peptide bond. The unchelated cupric ions are detected with a color-producing reagent that reacts with cupric ions. The amount of color produced is inversely proportional to the amount of peptide bond. 

Bicinchoninic Acid (BCA)

The first step here is to complex the protein with copper ions. In the second step, this protein-bound copper chelates BCA to give an intense purple color with an absorbance at 562nm. The purple color's intensity is linear with the amount of protein. This assay is useful to detect protein from 0.5 to 1000 μg /ml. The BCA assay is a great option if your protein samples contain > 5% detergents.

Step 1 : Protein + cu+2   = OH- + Cu+1 

Step 2 : 2BCA + Cu+1 = BCA-Cu+1 complex

Bradford

This is colorimetric assay in which coomassie brilliant blue 250 G dye is used. This dye has three forms cationic (red color, absorbance 470 nm), Neutral (green color, absorbance 650 nm), Anionic (blue color, absorbance 595nm). Normally Dye is available in cationic form (red color), it interact with protein in acidic solution by non-covalent interaction like van der waals bond with carboxylic group and ionic interaction with amine group of basic amino acid and gives blue color. The basic amino acid arginine, lysine and histidine play a role in the formation of dye-protein complexes color. The intensity of blue color directly proportional to amount of protein. The absorption can then be compared to a standard curve. It can generally detect protein larger than 3KD. This assay is useful to detect protein from 20 to 2000 μg /ml.

This assay is susceptible to detergent like SDS Triton -100 while less affected by the presence of sodium, potassium or carbohydrate like sucrose. 

Ultraviolet Absorption

Amino acids containing aromatic side chains (i.e., tyrosine, tryptophan and phenylalanine) exhibit strong UV-light absorption. This assay is useful to detect protein from 0.1 to 100 μg /ml.
The main limitations of this method is, it measure the protein by measuring the absorption of tyrosine and tryptophan at 280 nm. As all protein have different amount of aromatic amino acid. Other factor affect to this method are presence of Alcohols, certain buffer ions, and nucleic acids all absorb at 280 nm, hence this method is not suitable if any of above molecules are present. 
Peptide bond absorbed at 205nm. nucleoside absorbed at 260nm. ratio of 260/280 shows the purity of sample if it is 2.0 pure RNA, 1.8 pure DNA and if it is <1.8 then pure protein.

Biuret Assay
Biuret reagent is the condensation product of two molecules of urea. It contain hydrated copper sulphate, potassium hydroxide, sodium potassium tartrate. Biuret test is used for peptide bond detection and useful for the compound having two or three peptide bonds eg. tripeptide gives positive result. Cu(ii) ion in alkaline condition formed complex with unshared electron pairs of peptide nitrogen and gives violet colour which measure at 540nm using spectrophotometer. Absorption is directly proportional to presence of peptide bonds and thus by comparing with standard, amount of protein in sample can be determined.