Clin. Biochem. 11 (2) 54-56

(1978)

Silica Gel Radioimmunoassay for Myelin Basic Protein H A N S E N M. HSIUNG, J. WU and T . A. McPHERSON Department of Medicine, Cross Cancer Institute and University of Alberta, Edmonton, Alberta (Accepted August 29, 1977)

CLBIA, 11 (2) 54-56 Clin. Biochem.

(1978)

Hsiung, Hansen M., Wu, J. and McPherson, T. A.

Department of Medicine, Cross Cancer Institute and U~ziversity of Alberta, Edmonton, Alberta. SILICA GEL RADIOIMMUNOASSAY FOR MYELIN BASIC P R O T E I N 1. A radioimmunoassay which relies upon the capacity of silica gel to adsorb free h u m a n basic protein of myelin (BP) is described. 2. Reagents involved in the assay include unlabelled BP, ~25I-BP, and rabbit anti-BP. The assay is performed in excess normal rabbit serum, and uses 3-4~ silica gel for adsorption of free ~ I - B P . 3. The assay is simple, reproducible and not particularly time-consuming. It is capable of detecting BP at a level of 1-10 or more nanograms in samples containing varying amounts of serum. The assay is thus highly suitable as a routine test in clinical laboratories interested in detecting BP in cerebrospinal fluid in patients with suspected or proven demyelination.

1 M) was reacted with ~ I (0.5-1 mCi; carrier free, New England Nuclear, Boston) and excess chloramine-T in phosphate buffer (25 ~tl, 10 m g / m l ) . The reaction was quenched by excess sodium metabisulfite in phosphate buffer (25 ~l, 20 m g / m l ) and the labelled protein was isolated by passage through a coarse G-25 Sephadex column eluted in a 0.2 M trisacetate buffer, pH 7.4, containing 1 m g / m l histone (type II, calf thymus, Sigma Chemical Co., St. Louis, Missouri) or a solution of 25 m g / m l of bovine serum albumin (Connaught Lab., Willowdale, Ont.) in 0.1 M acetic acid. The specific activity of labelled BP was calculated to be approximately 175 ~,Ci/~.g Iodinated BP was stored in the histone-containing buffer or in the acetic acid/bovine serum albumin solution. Radioactive BP was prepared at least every four weeks. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis confirmed co-migration of ~SI-BP with unlabelled BP.

Radioimmunoassay A specific anti-BP serum raised in rabbits was used

QUANTITATION OF HUMAN MYELIN BASIC PROTEIN ( B P ) in c e r e b r o s p i n a l fluid ( C S F ) m a y be useful for the d i a g n o s i s and m o n i t o r i n g of d e m y e l i n a t i o n"'~'. Several methods to detect BP, or a n t i - B P , have been described and used in the s t u d y of e x p e r i m e n t a l allergic e n c e p h a l o m y e l i t i s ( E A E ) as well as in h u m a n d e m y e l i n a t i o n ''~8'. The e a r l i e r assays were u n s a t i s f a c t o r y for use in detection a n d m o n i t o r i n g of h u m a n d e m y e l i n a t i o n because they were e i t h e r too i n s e n s i tive or too t i m e - c o n s u m i n g . Recently developed assays have shown improved s e n s i t i v i t y , and, in general, detection of BP in the low n a n o g r a m r a n g e is now possible. F o r use as a r o u t i n e test in a clinical l a b o r a t o r y a r a d i o i m m u n o a s s a y for B P in C S F should be technically simple, h i g h l y reproducible, a n d n o t p a r t i c u l a r l y t i m e - c o n s u m i n g . I n a d d i t i o n , the a s s a y should be s e n s i t i v e a n d capable of d e t e c t i n g B P w i t h h i g h consistency in samples c o n t a i n i n g v a r y i n g q u a n t i t i e s of protein. We describe here a r a d i o i m m u n o a s s a y for BP which fulfills these c r i t e r i a .

in all studies. The silica gel technique for detecting binding of anti-BP to 12~I-BP was used. A dilution of anti-BP capable of binding approximately 35-50% of added ~2~IBP, which was on the linear aspect of an antibody dilution curve, was used in the silica gel radioimmunoassay. The radioimmunoassay was performed by adding increasing amounts of unlabelled BP to ~=I-BP (0.1 ng, 20-50 × 103 cpm) and 75 ~1 of the appropriate antiserum dilution. The unlabelled BP and ~ I - B P was contained in 30% normal rabbit serum (Grand Island Biologicals, Grand Island, New York) in a buffer consisting of 0.2 M trisacetate (pH 7.4), 1% Triton X-100 (alkylphenoxypolyethoxythanol, Rohm and Haas, Philadelphia, Pa.), and 0.1% Trasylol (Calbiochem, LaJolla, Calif.). Incubation was allowed to continue for 30 rain at room temperature. Following this, an equal volume of 6-8% silica gel (silica gel G, Macherey-Nagel and Co., D-5160 Doren, Germany) suspension was added so that the final concentration of silica gel in the solution was 3-4%. The incubation mixture was vortexed three times over a 30 rain period. The suspension was then centrifuged at 2,000 rpm for 19-20 rain at room temperature. The hound and free ~ I - B P was calculated after t r a n s f e r r i n g the s u p e r n a t a n t from the original tube to a second tube. Results were expressed as percentage of ~5I-BP bound minus the amount of ~-~I-BP bound in the control sample, which contained no anti-BP. The control sample containing no anti-BP regularly showed 15-20% nonspecific binding of ~=I-BP.

MATERIALS AND METHODS

EXPERIMENTAL RESULTS AND DISCUSSION

Human Myelin Basic Protein (BP) BP was isolated from human brain as described by Deibler et al ~ . Iodination of BP was by the chloramine-T procedure c~°~ as used by McPherson, Gilpin and Seland c1~. In brief, 4 ~g BP in 100 ~I phosphate buffer (pH 7.2, Correspondence: T. A. McPherson, M.D., Department of Medicine, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, Canada T6G lZ2

Binding of BP to Silical Gel Radioactive a n d unlabelled B P a d s o r b s r e a d i l y to glass or plastic s u r f a c e s ' U " . I n a d d i t i o n , B P f o r m s m i c r o - a g g r e g a t e s w h e n kept a t n e u t r a l p H ~'2''3~. T h e presence of a n excess of n o r m a l s e r u m i n the B P c o n t a i n i n g s o l u t i o n effectively p r e v e n t s the adsorpt i o n of B P to glass or plastic '~', a n d also g r e a t l y reduces the t e n d e n c y for B P to f o r m m i c r o - a g g r e g a t e s

SILICA GEL RADIOIMMUNOASSAY FOR MYELIN BASIC PROTEIN

55

50' 40

Fig. 1 - - Typical antibody dilution curve obtained w i t h ~ I - B P (0-1 ng) and rabbit anti-BP.

I

25

:01 IXl,

.,_

,

I

I

I

125 625 1/Antiserum Dilution

3125

30Fig. ~ - - Typical standard curve obtained w i t h I ~ I - B P (0.1 rig), 1/155 dilution of rabbit a n t i - B P and unlabelled B P .

I

2

5

I0 20 BP (ng)

50

in buffer at neutral pH. These observations were confirmed in the present study, where it was shown that up to 40% of '~I-BP was adsorbed to glass when a serum-free BP-containing buffer was vortexed, and more than 30% of '~I-BP was precipitated when a serum-free neutral buffer containing I~I-BP was centrifuged at 2,000 rpm for 10 min. The presence of more than 18% normal rabbit serum in the BPcontaining solution largely prevented this adsorption, and micro-aggregation. The presence of an excess of normal rabbit serum was also important with respect to binding of '~I-BP to silica gel. Thus, in excess of 18% normal rabbit serum was needed in the radioimmunoassay to clearly detect binding of ~ I - B P to anti-BP. Approximately 15-20% of added "~I-BP was detected as bound by

I00

the silica gel assay in the absence of anti-BP even when 18% or more normal rabbit serum was used in the assay. It was thought that this represented either nonspecific binding of '~I-BP or incubation damage, and accordingly this amount of binding was subtracted from the percentage binding in the control sample when results were expressed. Early results indicate that this nonspecific binding of m I - B P to normal rabbit serum probably results from binding to cz and fl globulins rather than albumin or y globulins. When less than 18% normal rabbit serum is used in the radioimmunoassay, a proportion of I"'I-BP which dissociates from anti-BP is adsorbed to silica gel, and precipitated with centrifugation - - thus introducing error in the assay. This dissociation of IUI-BP-

56

HSIUNG, WU AND McPHERSON

•anti-BP, and subsequent adsorption of ~'~I-BP to silica gel, does not appear to occur when greater than 18% normal rabbit serum is used in the assay. The binding of BP to silica gel was shown to be charge-related. SDS (a strong anionic detergent) is known to change the charge properties of protein molecules. Thus, treatment of BP with SDS should change BP from a highly positively charged molecule to a more negatively charged one. I f binding of BP to silica gel is largely charge-related, then treatment with SDS should prevent adsorption of BP to silica gel. This, in fact, was the result of such an experiment, and, in addition, was shown to be the case for binding of histone to silica gel as well. The optimal amount of silica gel for the assay was shown to be 4%. However, a wide range of silica gel concentration was satisfactory in the assay, and errors in silica gel would therefore have little effect on results from the assay. The silica gel assay was equally effective in detecting ~'~I-BP-anti-BP binding at pHs of 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. Errors, therefore, in pH of the solution in which the assay is performed would also have little effect on the assay.

Silica Gel Radioimmunoassay An antibody dilution curve and a BP-standard inhibition curve are shown in Figures i and 2 respectively. It can be seen that a 1/125 antiserum dilution, of this particular antiserum, was satisfactory for establishing a BP-standard inhibition curve. The range of detection of free BP using the silica gel radio-immunoassay was from 1 to 10 ng. Preliminary results indicate that the assay, which is normally done using a 1 ml volume before addition of silica gel, can be scaled up to a volume, before addition of silica gel, of 2-3 ml without a major loss in sensitivity. This indicates that test samples suspected of containing BP, such as CSF, can either be lyophilized and reconstituted so as to allow the

assay to be performed at a 1 ml volume, or could be tested directly using a volume of 2-3 ml of CSF. This latter possibility greatly extends the practicability of the assay for clinical laboratory use. ACKNOWLEDGEMENTS We wish to acknowledge the technical assistance of Ms. S. McDonald, and Dr. Jack Miller, Chairman, Department of Radiology, Universtiy of Alberta, for providing us with some CSF samples used in this study. This work was supported by the MS Society of Canada. R*:*'~NCES 1. McPherson, T. A., Gilpin, A. and Seland, T. P. Can. Med. Assn. J. 107: 856-859 (1972). 2. Cohen, S. R., Herndon, R. M. and McKhann, G. M. New England J. Med. 295:1455-1457 (1976). 3. Kibler, R. R. and Barnes, A. E. J. Exp. Med. 116: 807 (1962). 4. Caspary, E. A. and Field, E. J. Ann. N.Y. Acad. Sci. 122:182 (1965). 5. McPherson, T. A. and Carnegie, P. R. J. Lab. Clin. Med. 7 2 : 8 2 4 (1968). 6. Day, E. E. and Pitts, O. M. Immunoehemistry 11: 651-659 (1974). 7. Schmid, G., Thomas, G., Hempel, K. and Gruninger, W. Eur. J. Neurol. 12:173-185 (1974). 8. Cohen, S. R., McKhann, G. M. and Guarnieri, M. J. Neurochem. 25:371 (1975). 9. Deibler, G. E., Martenson, R. E. and Kies, M. W. Prepr. Biochem. 2:139-165 (1972). 10. Hunter, W. M. and Greenwood, F. C. Nature 194: 495 (1962). 11. Lamoureux, G., Carnegie, P. R., McPherson, T. A. and Johnston D. Clin. Exp. Immunol. 2 : 6 0 1 (1967). 12. McPherson, T. A. Studies on the production of expermiental allergic encephalomyelitis using purified encephalitogenic proteins and polypeptides. Ph.D. Thesis University of Melbourne, Australia (1968). 13. Jones, A. J. S. and Rumsby, M. G. J. Neurochsm. 25: 562-572 (1975).