A Micro-Immunochemical Procedure for the Measurement o f Total Protein in Cerebrospinal Fluid*

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1 A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e, Vol. 3, No. 4 Copyright 1973, Institute for Clinical Science A Micro-Immunochemical Procedure for the Measurement o f Total Protein in Cerebrospinal Fluid* M. GERALDINE HEINTGES, JOHN SAVORY, Ph.D. a n d LAWRENCE M. KILLINGSWORTH-j- Division of Clinical Chemistry, Departments of Medicine, Pathology, and Biochemistry. University of North Carolina, Chapel Hill, NC and Department of Pathology, University of Florida College of Medicine, Gainsville, FL ABSTRACT A procedure has been developed for the measurement of total protein in cerebrospinal fluid (C S F) based on light scattering of antigen-antibody complexes 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 correlations 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. 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 trichloroacetic 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 Pathology Training Grant NIMGH t NIH Academic Pathology Trainee, Department of Pathology, University of Florida College of Medicine. 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 reported but require preliminary column separations to remove interferring substances before measurement at 280 nm. Ultraviolet fluorometry12 and the micro Kjeldhal technique20 have also been used. An alternate approach to the measurement of proteins in aqueous solutions involves nephelometric measurements of antigen-antibody complexes formed in immunochemical reactions. Boyden et al in first showed that precipitin curves 2 6 5

2 6 6 H EIN TGES, SAVORY AND KILLIN GSW ORTH nm is used for the light scattering measurements. No secondary filter is required. The square micro flow cell of 0.

2 2 6 6 H EIN TGES, SAVORY AND KILLIN GSW ORTH nm is used for the light scattering measurements. No secondary filter is required. The square micro flow cell of 0.2 ml volume is used for the light scattering measurements. A recorder^ attached to the fluorometer is used in the present study but is not essential to the procedure. R e a g e n t s 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 methods for measuring albumin and total immunoglobulin in dilute solution and a similar procedure for measuring C'3 also has been described.2 A series of studies in our laboratory have led to the development of similar immunochemical methods9 17 using a fluorometer as a nephelometer for detecting antigen-antibody complexes. Continuous flow automation using these same principles has also been reported 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 A p p a r a t u s A fluorometer* equipped with a primary filterf to give a peak transmission at 360 Turner #111 from G. K. Turner Assoc., Palo Alto, CA Turner # Goat Anti-human Serum. Antiserum to human serum total proteins is obtained from a commercial source* and is diluted 50 fold with 0.9 percent saline before the determinations are carried out. Unused portions of the antiserum can be stored in the refrigerator at 5 and are stable at least two weeks. Saline Solution. Nine g of reagent grade sodium chloride are dissolved in one liter of distilled de-ionized water containing 0.5 ml per liter Triton X-lOO.f H u m a n S e r u m P r o t e in s Total Protein Standard (650 mg per dl). To obtain the concentration of total protein, replicate determinations were performed on a pooled human serum standardized against bovine serum albumin^ using a biuret procedure.7 Appropriate dilutions of the standard were made to obtain total protein concentrations of 130, 93, 59, 41, 21, 13 and 7 mg per dl. Serum Protein Fractions. The serum protein fractions are divided into two categories. Human Serum Albumin (693 mg. per d l). Human serum albumin obtained commercially was found to be electrophoretically homogenous demonstrating a single precipitin arc on immunoelectrophoresis. JHellma Cells #176F-QS from Hellma Cells, Inc., Jamaica, NY Technicon Instrument Corp., Tarrytown, NY *Meloy Laboratories, Falls Church, VA fharleco, Philadelphia, PA (Bovine Albumin, Fraction V, Technicon Corp, Tarrytown, NY Metrix, Chicago, IL

3 M E A SU R E M E N T O F T O T A L P R O T E IN IN C E R E B R O SP IN A L F L U ID Concentrations of albumin 277, 231, 208, 187, 162, 139, 99 and 43 mg per dl were prepared from the stock albumin solution. Human Gamma Globulin (308 mg per dl). Human serum gamma globulin j shown by immunoelectrophoresis to contain mainly IgG with smaller amounts of IgA and IgM, was used in this study. The concentrations of gamma globulin prepared from the stock solution were 293, 270, 205, 154, 123, 116, 103, 98, 77 and 51 mg per dl. Procedure The diluted antiserum ( 1 :5 0 ) is transferred volumetrically in 2.5 ml aliquots to 13 X 100 mm screw-cap tubes and 25 microliters of the CSF sample or standard are added to each tube and the contents are gently mixed ten times. The tubes are allowed to stand for 25 minutes at room temperature for development of the antigenantibody complexes. The reaction mixtures are then introduced into the fluorometer (nephelometer) using a plastic syringe with a small tygon tubing extension. Diluted antiserum is first used to adjust the baseline to an arbitrary low setting on the fluorometer, and the light scattering from the immune complexes in the CSF samples and standards is measured. Rinsing with saline between readings of both the flow cell and syringe is essential to avoid sample interaction. Maximum recorder response should be set with the 130 mg per dl total protein standard to achieve optimal sensitivity. A blank correction for non-specific light scattering from CSF samples was found to be unnecessary. Concentration of total protein in the unknown samples is determined from the standard curve shown in figure 1. This standard curve is non-linear and, therefore, several points on the curve must be determined with each group of tests. Parke-Davis and Co., Detroit, MI F i g u r e 2. Light scattering from the antigenantibody complexes as a function of time. R esults T i m e D e l a y To determine the optimum time for development of the antigen-antibody complexes, a standard of known concentration was mixed with antiserum and was injected immediately into the fluorometer. Increasing light scattering from the antigenantibody aggregates, as they developed, could be followed. As shown in figure 2, a plateau was reached in approximately 15 minutes and remained stable for several hours. In the recommended procedure, 25 minutes was selected as the suitable delay period in development of the immune complexes. CSF TOTAL PROTEIN (mg/dl) F i g u r e 3. Precipitin curve for the total proteinanti total protein complexes.

4 2 6 8 H EIN TG ES, SAVORY AND KILLINGSW OBTH F i g u r e 4. Precipitin curves for albumin and gamma globulin. P r e c ip it i n C u r v e s Total Proteins. Conditions for measuring proteins by immunochemical reactions require the presence of antibody excess. Erroneous results are obtained from samples in antigen excess owing to solubilization of antigen-antibody complexes giving falsely low light scattering readings. It is necessary, therefore, to construct a precipitin curve to assure conditions of antibody excess with each new batch of antiserum. Experience with different batches from a single commercial source have shown, although precipitin curves vary slightly, this variation has not necessitated altering the reaction conditions. A typical precipitin curve constructed from dilutions of the standard is shown in figure 3. Screening of samples in antigen excess can be carried out by using 10 /xl of CSF in the reaction mixture and noting the light scattering response in comparison to the original test which requires 25 /J of CSF. Samples in antibody excess would exhibit a definite decrease in response whereas those in antigen excess most likely would demonstrate the opposite effect. The authors routinely screen for antigen excess on all samples. Albumin and Globulin. Since total protein in CSF is composed of many different fractions, it is necessary to ascertain that o> O cc CL < 1 O H liez) o F i g u r e 5. Total protein concentration of 50 CSF samples determined by both the immunochemical and TCA turbidimetric procedures. Solid line: theoretical relationship x = y; Dashed line: calculated regression line; Correlation coefficient, r, 0.995; and standard error of estimate, 4.5 mg per dl. C S F TOTAL PROTEIN (mg/dl) IM M U N O CHEM ICAL PRO CED U RE

$Although albumin constitutes the major fraction in CSF, pathologic samples particularly the IgG fraction of the gamma globulin, can demonstrate notable increases.$

5 M E A SU R E M E N T O F T O T A L P R O T E IN IN C E R E B R O SP IN A L F L U ID the region of antibody excess exists in all instances. Although albumin constitutes the major fraction in CSF, pathologic samples particularly the IgG fraction of the gamma globulin, can demonstrate notable increases. It is therefore important to establish a albumin and gamma globulin. In figure 4 condition of antibody excess for both are shown two curves constructed for these fractions; it is seen that antibody excess is present up to 130 mg per dl for albumin and 103 mg per dl for gamma globulin. The light scattering response observed here for the respective precipitin curves is not indicative of absolute sensitivity derived from the immune complexes but is simply a reflection of the change in sensitivity setting on the fluorometer. The total protein standard curve used in the measurement of CSF samples employs a 130 mg per dl standard for the highest concentration which certainly is well within the range of antibody excess that exists up to a concentration of 390 mg per dl. COBBELATION CSF samples from 50 hospital patients were analyzed by both the turbidimetric trichloracetic acid method of Henry et al6 and the present immunochemical technique. The results of this correlation are shown in figure 5. P b e c is io n In table 1 is shown the precision of the method as determined by performing 50 duplicate measurements of CSF samples encompassing a broad range of concentrations. D iscussion The present procedure described for the measurement of total protein in CSF is a microtechnique using only 25 to 35 jul of sample. Other methods for performing this same determination require 0.5 to 1.0 ml. TABLE I P r e c i s i o n o f CSF T o t a l P r o t e i n M e t h o d N um ber o f D uplicate Determinations M ean S.D. Range mg/ à i Coefficient o f V ariations percent ± The real advantage, therefore, of the immunochemical technique is that larger quantities of unused CSF are available for additional studies such as the measurements of immunoglobulins and other specific proteins which can have importance in evaluating diseases of the central nervous system. There are, however, inherent problems using the immunochemical approach for measuring mixtures of several proteins such as proposed in the present procedure. The precipitin curve is a composite of several curves and different proteins will obviously provide varying light scattering responses when bound to their specific antibodies. However, this problem of proteins responding dissimilarly is not unique to immunochemical techniques and a similar problem exists with both turbidimetric and ultraviolet absorbance approaches. For example, different proteins in CSF produce varying degrees of turbidity with TCA and also have differing molar absorptivities at 280 nm. In the present procedure, errors due to the presence of several antigen-antibody reactions occurring in the reaction mixture are reduced by using a standard containing all of the CSF protein fractions in amounts similar to those encountered in CSF. Pooled human serum is used as this standard and dilutions are made to achieve protein levels in the range frequently encountered in CSF. Relative proportions of CSF proteins are changed in a few disease states but not enough to cause significant errors.

2 7 0 H EIN TG ES, SAVORY AND KILLINGS W ORTH Sum m ary A method is proposed for measuring total protein in CSF using a nephelometric immunochemical technique.

6 2 7 0 H EIN TG ES, SAVORY AND KILLINGS W ORTH Sum m ary A method is proposed for measuring total protein in CSF using a nephelometric immunochemical technique. Only 25 to 35 A of sample are required and measurements can be made 25 minutes after mixing sample and antiserum. The method is precise and correlates well with a conventional turbidimetric procedure. References 1. A l p e r, C. A.: Automated nephelometric determination of serum haptoglobin, C'3, and aiantitrypsin. Advan. Automat. Anal. ( Technicon International Congress) 1: , A l p e r, C. A., P r o p p, R. P., K l e m p e r e r, M. R., a n d R o s e n, F. S.: Inherited deficiency of the third component of human complement (C'3). J. Clin. Invest. 48: , B o n i t a t i, J., E l l i o t t, W. B., a n d M i l e s, P. G.: Interference by carbohydrate and other substances in the estimation of protein with the Folin-Ciocalteu reagent. Anal. Biochem. 31: , B o y d e n, A., B o t t o n E., a n d G e m e r o y, D.: Precipitin testing with special reference to the measurement of turbidity. J. Immunol. 57: , E c k m a n, I., R o b b i n s, J. B., V a n d e n H a m e r, C. J. A., L e n t z, J., a n d S c h e i n b e r g, I. H. : Automation of a quantitative immunochemical micronanalysis of human serum transferrin: A model system. Clin. Chem. 16: , H e n r y, R. J., S o b e l, C. a n d S e g a l o v e, M.: Turbidimetric determination of proteins with sulfosalicylic acid and trichloroacetic acids. Proc. Soc. Exp. Biol. Med. 92: , D E l a H u e r g a, J., S m e t t e r s, G. W., a n d S h e r r i c k, J. C.: Colorimetric determination of serum proteins: The biuret reaction. Serum Proteins and the Dysproteinemias. Sunderman, F. W. and Sunderman, F. W., Jr., eds. Philadelphia, J. B. Lippincott Co., pp , K i l l i n g s w o r t h, L. M. a n d S a v o r y, J.: Automated immunochemical procedures for measurement of immunoglobulins IgG, IgA, and IgM in human serum. Clin. Chem. 17: , K i l l i n g s w o r t h, L. M. a n d S a v o r y, J.: Manual nephelometric methods for immunochemical determination of immunoglobulins IgG, IgA, and IgM in human serum. Clin. Chem. 18: , K i l l i n g s w o r t h, L. M., S a v o r y, J., a n d T e a g u e, P. O.: Automated immunoprecipitin technique for analysis of the third component complement (C'3) in human serum. Clin. Chem. J 7: , M e u l e m a n s, O.: Determination of total protein in spinal fluid with sulfosalicylic acid and trichloroacetic acid. Clin. Chim. Acta 5: , O M a l l e y, W. E. a n d O D o h e r t y, D. S.: Estimation of protein content of spinal fluid by direct ultraviolet fluorometry. Trans. Amer. Neurol. Assoc. 94: , P e n n o c k, C. A., P a s s a n t, L. P., a n d B o l t o n, F. G.: Estimation of cerebrospinal fluid protein. J. Clin. Path. 21: , R i c e, E. W. a n d L o f t i s, J. W.: Critique of the determination of proteins in cerebrospinal fluid: Evaluation of the biuret method of Goa and the TCA-turbidimetric method of Meulemans. Clin. Chem. S , R i t c h i e, R. F.: A simple, direct, and sensitive technique for measurement of specific protein in dilute solution. J. Lab. Clin. Med. 70: , S a v o r y, J., H e i n t g e s, M. G., K i l l i n g s w o r t h, L. M., a n d P o t t e r, J. M., Manual and automated determination of immunoglobulins in unconcentrated cerebrospinal fluid. Clin. Chem. 18:37-42, S a v o r y, J. a n d K i l l i n g s w o r t h, L. M.: Determinations of alpha-l-antitrypsin by a nephelometric procedure. Ann. Clin. Lab. Sci. 3:43-47, S c h u l t z e, H. E. a n d S c h w i c k, G.: Quantitative immunologische bestimmung von plasmaproteinen. Clin. Chim. Acta 4:15-25, T o m b s, M. P., S o u t e r, F., a n d M a c L a g a n, N. F.: The spectrophotometric determination of protein at 210 mu. Biochem. J. 73: , T o u r t e l l o t t e, W. W., P a r k e r, J. A., A l v i n, R. E., a n d D e J o n g, R. N.: Determination of total protein in cerebrospinal fluid by an ultramicro-kjeldahl nitrogen procedure. Anal. Chem. 30: , W a d d e l l, W. J.: A simple ultraviolet spectrophotometric method for the determination of protein. J. Lab. Clin. Med. 4S , W e g f a h r t, P. F., F i s h, M. B., A l d a n a, F. B., a n d A r o n s o n, S. B.: Automated immunoprecipitin determination of albumin in serum. Advan. Automat. Anal. (Technicon International Congress) 1: , Z o n d a g, H. A. a n d v a n B o e t z e l a e r, G. L.: Determination of protein in cerebrospinal fluid; sources of error in the Lowry method. Clin. Chim. Acta 5: , 1960.

PROTEIN TURBIDITY PRODUCED BY TRICHLORACETIC ACID AND SULFOSALICYL1C ACID AT VARYING TEMPERATURES AND VARYING RATIOS OF ALBUMIN AND GLOBULIN

THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 44, 1V0. 6 Copyright 1965 by The Williams & Wilkins Co. Primed in U.S.A. PROTEIN TURBIDITY PRODUCED BY TRICHLORACETIC ACID AND SULFOSALICYL1C ACID AT VARYING