This article describes a detailed protocol to estimate the concentration of protein in cerebrospinal fluid (CSF) by the Pyrogallol dye binding method which was originally developed by Watanbe et.al. The reference to the original article is N Watanabe, S Kamei, A Ohkubo, M Yamanaka, S Ohsawa, K Makino and K Tokuda (1986) “Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a Hitachi 726 automated analyzer” Clinical Chemistry August 1986 vol. 32 no. 8 1551-1554.
This protocol has been adapted to quantitatively estimate the concentration of protein in cerebrospinal fluid (CSF). Although this is the standard operating protocol (SOP) set by the World Heath Organization (WHO), this article aims to explain every single step of the protocol in very simple, lucid and understandable terms to researchers in a step-by-step manner.
The protein molecules present in the sample human cerebrospinal fluid (CSF) solution will quantitatively bind with pyrogallol red molybdate reagent dye at pH 2.2 to form a violet colored complex. The intensity of this colored complex is measured at 600 nm in a spectrophotometer. The color intensity of the CSF solution is directly proportional to the concentration of protein in the CSF. In simplistic terms, the higher the concentration of protein, more intense or darker will be the color of the CSF solution, lower the concentration of protein, less intense or lighter will be the color of the sample human CSF solution. The intensity of the color of the sample CSF solution is compared with the color developed by a set of protein standards to which pyrogallol red molybdate reagent has also been added and estimated at 600 nm in the spectrophotometer.
The normal concentration or reference range of total protein in human cerebrospinal fluid (CSF) is 15-45 mg/dl.
The protein normally present in human CSF is about 2/3rds of albumin. However, in disease states affecting the central nervous system such as in spinal tumor, bacterial and fungal meningitis, multiple sclerosis, paralysis, sub-arachnoid hemorrhage, cerebral infarction and traumatic tap, a mixture of albumin and globulins are observed, as well as, a marked increase in the total protein concentration which can be as high as 400 mg/dl. In inflammatory lesions, an increase in protein concentration is also accompanied by an increase in the number of white blood cells which are normally very low in the CSF. The increase in total protein concentration in the CSF is due to increased permeability of the brain capillary endothelial cells that form the blood brain barrier.
1) Test tubes 2) Test-tube rack 3) Labels for test tubes 4) Reagents as follows
Glycine pyrogallol red dye reagent :-
1) Glycine buffer – Dissolve 7.5 g glycine and 32 mg ammonium molybdate in 350 ml distilled water (D/W). Adjust the pH to 2.2 with 1N HCl, and make up the final volume to 500ml with D/W.
2) Pyrogallol red dye – Dissolve 26.8 mg pyrogallol red in 1 ml methanol in a test tube. Mix well until dye dissolves. Add 0.1ml 0.1 N NaOH.
– Mix glycine buffer & pyrogallol red dye solution with constant stirring. Add 50 mg EDTA, 5-10 drops of 10% triton X-100, and make up the volume to 1000ml with D/W.
3) Protein Standards:
Protein stock solution (2mg/ml) Bovine serum albumin (BSA) containing 0.1% sodium azide. (* Sodium azide is a hazardous material, hence it should not be swallowed, and contact with skin and mucous membranes should be avoided).
Prepare working standards (1ml) from the protein stock solution as follows:-
Working stds. S1 S2 S3 S4
Conc. (mg/dl) 20 40 80 120
Stock soln. (ml) 0.1 0.2 0.4 0.6
Normal saline (ml) 0.9 0.8 0.6 0.4
– Prepare 6 test-tubes (1 for blank, 4 for standard protein working solution as S1, S2, S3, S4 respectively) and 1 for test CSF sample solution as follows:-
Blank Standards Test
S1 S2 S3 S4
Reagent dye (ml) 1.5 1.5 1.5 1.5 1.5 1.5
D/W (ml) 0.025 – – – – –
Stds (ml) – 0.025 0.025 0.025 0.025 –
Test sample CSF(ml) – – – – – 0.025
– After addition into respective tubes, mix well. Leave test-tubes at 25 – 35oC for 15 minutes. Read absorbance of standard protein solutions and CSF sample solution at 600nm in a spectrophotometer using reagent blank set to 0. Plot a graph of concentrations of standard protein solutions on the X axis and O.D. (optical density or absorbance) on Y axis. Derive concentration of protein in sample CSF solution from the graph. Basically, the graph is a straight line passing through 0. First record the O.D of the CSF sample solution, then see what concentration it corresponds to on the x-axis ON the straight line formed when the concentrations of standards are plotted.
For more information about how the graph should look like, please see:-
– If protein concentration of sample CSF solution is within the range of 15-45 mg / dl – NORMAL
– If protein concentration exceeds above range, it could be indicative of neurological diseases as indicated above and must be confirmed by serological, hematological and microbiological tests.
If the protein concentration in the CSF solution EXCEEDS 120mg/dl, the CSF solution should be diluted with 0.9% saline in either 1:2 or 1:3 and reassayed. The value obtained should be multiplied by the appropriate dilution factor. Since CSF is an ultrafiltrate of plasma, the presence of blood and pus in the CSF will increase CSF protein. Hence, it is very important to note the appearance of the CSF both before and after centrifugation.