A micro-immunochemical procedure for the measurement of total protein in cerebrospinal fluid.

A procedure has been developed for the measurement of total protein in cerebrospinal fluid (C SF) based on light scattering of antigen-antibody com­ plexes formed with anti-whole human serum. Twenty-five microliters of CSF are required. Precision of the method is 6.3 percent (R.S.D) and close cor­ relations are obtained with the trichloroacetic acid turbidimetric procedure. The essential condition of antibody excess has been shown to exist up to 390 mg per dl of total protein. photometric measurement at 330 nm14 as a means of improving sensitivity. Folin and Ciocalteu reagent in conjuction with biuret3’23 has been adopted but is subject to some interferences. Ultraviolet spectrophotometric methods13*19’21 have been re­ ported but require preliminary column separations to remove interferring sub­ stances before measurement at 280 nm. Ultraviolet fluorometry12 and the micro Kjeldhal technique20 have also been used. An alternate approach to the measure­ ment of proteins in aqueous solutions in­ volves nephelometric measurements of antigen-antibody complexes formed in im­ munochemical reactions. Boyden et al in 19474 first showed that precipitin curves 2 6 5 In trod u ction Several methods for the measurement of total protein in CSF have been proposed and, at the present time, the relatively simple turbidimetric procedures are most commonly employed using either tri­ chloroacetic acid6 or sulfosalicylic acid11 as a means of producing turbidity. These methods are technically straightforward and are adaptable to emergency use but suffer from a lack of sensitivity. The biuret reaction has been proposed with spectro* This work was partially supported by Pathol­ ogy Training Grant NIMGH 01996-02 t NIH Academic Pathology Trainee, Department of Pathology, University of Florida College of Medicine. 2 6 6 H E IN T G E S, SAVORY AND K ILLIN G SW O R T H F i g u r e 1. Standard curve for the measurement of total protein in CSF. could be characterized by measuring the turbidity developed in antigen-antibody mixtures. Schultze and Schwick18 described the turbidimetrie measurement of several plasma proteins. Ritchie15 developed meth­ ods for measuring albumin and total immu­ noglobulin in dilute solution and a similar procedure for measuring C'3 also has been described.2 A series of studies in our labora­ tory have led to the development of similar immunochemical methods9’17 using a fluorometer as a nephelometer for detecting anti­ gen-antibody complexes. Continuous flow automation using these same principles has also been reported.1’5’8’10’16’22 These nephelometric immunochemical procedures are sensitive, rapid and involve only simple technical manipulations. The present study was undertaken to evaluate a technique for the measurement of total protein in CSF with emphasis being placed on the development of a micro procedure possessing both accuracy and precision. M aterials and M ethods