Quantitative differences among various proteins as blocking agents for ELISA microtiter plates

Journal oflmmunologicalMethods, 101 (1987) 43-50 Elsevier

43

J1M04391

Quantitative differences among various proteins as blocking agents for ELISA microtiter plates Robert F. Vogt, Jr., Donald L. Phillips, L. Omar Henderson, W a n d a Whitfield and Francis W. Spierto Division of Environmental Health Laboratory Sciences, Center for Environmental Health, Centers for Disease Control. U.S. Public Health Service, Atlanta, GA, U.S.A. Received 28 October 1986, revised received 2 March 1987, accepted 4 March 1987)

We tested instantlzed dry milk, casein, gelatins from pig and fish skin, serum albumin and several other proteins for their abilities to block non-specific binding (NSB) of a peroxidase-conjugated immunoglobulin to polystyrene microtiter plate wells. Each blocking protein was tested across a million-fold concentration range, both in simultaneous incubation with the peroxidase conjugate and as a pretreatment agent where excess protein was washed away before incubation with the conjugate. Overall, instantized milk and casein were the most effective proteins tested: they inhibited NSB by over 90% in both the simultaneous and pretreatment modes at far lower concentrations than most of eight other proteins. Enzymatically hydrolyzed porcine skin gelatin was the least effective protein tested: it did not reduce NSB by more than 90% even at its highest concentrations; its blocking ability fell rapidly upon dilution; and it was almost useless as a pretreatment agent. Fish skin gelatin showed much better blocking activity than hydrolyzed porcine gelatin, and it still had the practical advantage of remaining fluid even under refrigeration. Our results suggest that some proteins (such as casein) block NSB to plastic primarily through protein-plastic interactions, while others (such as porcine skin gelatin) block primarily through proteinprotein interactions. Although the optimal blocking agent for any particular ELISA system must be determined by empirical testing, these results should be helpful in selecting the best possible candidate proteins for further evaluation. Key words: ELISA; lmmunoassay; Blocking protein; Protein binding; Non-specific binding; Polystyrene

Introduction

E n z y m e - l i n k e d i m m u n o s o r b e n t assays (ELISAs) often include proteins (antigens or antiCorrespondence to: R.F. Vogt, Bldng. 17, Centers for Disease Control, Atlanta, G A 30333, U.S.A. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and H u m a n Services. Abbreviations: ELISA, enzyme-linked immunosorbent assay; NSB, non-specific binding; INH, inhibition (of NSB). See Table I for other abbreviations.

bodies) immobilized to a plastic surface by nonspecific binding (NSB). However, non-specific binding of other protein components during the subsequent steps of such assays is detrimental to their sensitivity and specificity. This undesirable non-specific binding may be minimized by saturating the plastic's remaining adsorptive surface with 'blocking' proteins, a collective term for various protein additives that have no active part in the immunochemical reactions of the assays. Blocking proteins have been chosen largely by convenience and empirical testing in specific

44

ELISA systems, since no assay providing a general categorization of blocking activity has been available. Many factors can influence NSB, including various protein-protein interactions that may be unique to a particular ELISA system. Avoiding these complications, we used a simple three-component system that measured direct protein-polystyrene binding and its inhibition by a variety of proteins. This system revealed large quantitative differences in blocking activity among many of the proteins tested. The pattern of results suggests a way of categorizing proteins based on different mechanisms by which they inhibit non-specific binding to the plastic surface.

Materials and methods The materials and reagents used in this study are summarized in Table I.

Preparation o/protein solutions Stock solutions of the blocking proteins tested

were prepared as described below. Working solutions were adjusted to comparable protein concentrations as determined by a Coomassie blue dye-binding assay (Bio-Rad, Rockville Centre, NY 11571) using bovine serum albumin as a standard. These protein concentrations should be considered as estimates within a two-fold range, since different proteins may vary that much in their dye-binding/concentration relationship (Pollard et al., 1978). Bovine serum albumin (BSA) and porcine thyroglobulin (PTG) were purified protein powders dissolved easily in phosphate-buffered saline (PBS). lnstantized dr), (bovine) milk (MILK) also dissolved easily in PBS. A 5% (total w/v) solution contained about 1.5% protein. Casein (CAS) solutions were prepared by stirring about 2 g of purified casein powder in 100 ml water while adding 0.1 M NaOH dropwise to maintain a pH of 6.8-7.2. After the pH had stabilized, the suspension was stirred overnight at room temperature, then filtered through standardgrade paper. This saturated casein solution (which

TABLE I REAGENTS USED FOR BLOCKING PROTEIN STUDY Reagent

Abbreviation

Source and/or formulation

Phosphate-buffered saline

PBS

Citrate-phosphate buffer

CPB

o-Phenylenediamine Hydrogen peroxide, 30% Goat anti-mouse antibody peroxidase conjugated Bovine serum albumin Porcine thyroglobulin High density lipoprotein (human) Whole goat serum Porcine skin gelatin Hydrolyzed gelatin Fish skin gelatin

OPD

0.01 M potassium phosphates, pH 7.4 0.15 M sodium chloride 0.05 M sodium citrate 0.05 M potassium phosphate (dibasic) pH adjusted to 5.0 Fisher Scientific Co. (cat. no. 0-4168) Sigma Chemical Co., St. Louis, MO (cat. no. H-1009) Atlantic Antibodies, Scarborough, ME (cat. no. 083-6: lot no. 009) Calbiochem-Behring, La Jolla, CA (cat. no. 12659) Sigma Chemical Co. (cat. no. T-1126) In-house preparation (see text) In-house preparation (single bleed from one animal) Sigma Chemical Co. (cat. no. G-2625) In-house preparation (see text) Hipure liquid gelatin, Norland Products, New Brunswick+ NJ (also available through Sigma Chemical Co., cat. no. G-7765) Fisher Scientific (cat. no. C-2021 Carnation non-fat dry milk Dynatech Laboratories, Alexandria, VA (cat. no. 011-010-2801 )

Casein Bovine milk (instantized) Microtiter plates (lmmulon 2)

H202 GAM-PO BSA PTG HDL WGS GEL HYGEL FSGEL

CAS MILK MTP

45

remained slightly turbid) contained about 1.5% protein. Whole goat serum (WGS) contained about 6% total protein and was diluted directly into PBS. High-density lipoprotein (HDL) was prepared from 1 U of human plasma by isophynic ultracentrifugation (Havel et al., 1955). The stock solution contained about 2% protein and was diluted directly into PBS. Porcine skin gelatin (GEL) was dissolved (5 g/100 ml) in warm (60 ° C) PBS. Hydrolyzed gelatin (HYGEL) was prepared by treating the 5% stock GEL solution with trypsin (Kato et al., 1980). Fish skin gelatin (FSGEL) was obtained as a viscous stock solution containing about 45% protein. It was diluted directly into PBS.

Microtiter plate ELISA Immulon 2 polystyrene microtiter plates (MTP) (Dynatech Laboratories, Alexandria, VA 22314) were used for all experiments. These plates contain 96 flat-bottom wells, each 6.3-6.4 mm in diameter, arranged in 12 columns and eight rows. The surface areas of plastic exposed to protein solutions was computed to be 125 mm2. All incubations were performed at 37°C in a total volume of 150 ~l/well. Each wash step consisted of 5 fill/empty cycles with deionized water. For the pretreatment study, MTP wells were first incubated with dilutions of the various blocking proteins for 1 h. After washing, the wells were incubated for 1 h with the suggested working dilution (1:2000) of the goat anti-mouse peroxidase conjugate (GAM-PO). For the simultaneous incubation study, MTP wells were incubated for 1 h with a solution containing both the 1 : 2000 dilution of GAM-PO and the blocking protein. Plastic-bound peroxidase activity was determined by incubating the washed plates for 30 min with 0.2% OPD dissolved in citrate buffer containing 0.015% hydrogen peroxide. Reactions were stopped by adding 50/tl of 10% sulfuric acid, and absorbances were read at 492 nm in an automated plate reader (Titertek Multiscan MC, Flow Laboratories).

Experimental design All blocking proteins were evaluated over a

range of 20 two-fold serial dilutions in both the pretreatment protocol and the simultaneous incubation protocol described above. Every dilution was tested in 8 replicate wells occupying one column of a microtiter plate (MTP). Two columns (16 wells) on each plate were not exposed to blocking protein, thus serving as reference wells for total NSB. The absorbance reading from each well on a given MTP was converted to its percentage (%ABS) of the mean absorbance of the 16 reference wells on that same MTP. The percent inhibition (%INH) was calculated as the complement of this percent absorbance (%INH = 100 %ABS).

Data reduction and analysis Means and standard errors (n = 8) of %INH were computed for every concentration tested, and the results were plotted on a linear scale for %INH and a logarithmic scale for concentration (Figs. 1 and 2). The concentration of each protein required for 50% inhibition of NSB was estimated by linear interpolation between the two concentrations surrounding the 50% value on the inhibition curve. The 16 data points from those two concentrations were grouped into eight pairs of matched readings from MTP wells with the same column position. The interpolated 50% INH concentration was derived for each pair of points, and the mean and standard error of those eight determinations were computed. Tests for significant differences in maximum %INH and the 50% INH concentration (Table II) were done using the two-tailed Wilcoxon rank sum procedure.

Results

All the proteins tested produced generally smooth, sigmoidal inhibition curves in both the pretreatment protocol (Fig. 1) and the simultaneous incubation protocol (Fig. 2). Table II lists for each protein the mean values and standard errors of the maximum %INH and the estimated 50% INH concentrations. All but one of the proteins tested were able to reduce NSB by more than 90% when used at high

46 INHIBITION O F NON-SPECIFIC B I N D I N G BY P R E - T R E A T M E N T O F MTP WITH V A R I O U S B L O C K I N G P R O T E I N S

-

100

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Protein w/v)

CAS

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(casein)

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CONCENTRAT,ON OF S O L U T I O N S (%

- -

-- ~ -. . . . . ...... - - - - ......

-20

(ug/MTPwell)

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GEL (gelatin) MILK (instantized bovine m i l k ) WGS (goat serum) HDL (lipoprotein) H Y G E L ( h y d r o l y z e d gelatin) BSA (albumin) PTG (thyroglobulin) FS G E L (fish gelatin)

q'" ~'" ~" %" %" %" %" ~" %" %" %" %. %" %" ~"

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[I

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Fig. 1. Pretreatment protocol: inhibition of non-specific binding by various blocking proteins. Experimental details are given in the text. Values represent the proportionate decrease of non-specific binding in microtiter plate wells exposed to blocking protein before incubation with a peroxidase-conjugated immunoglobulin, when compared to wells on the same microtiter plate that had not been exposed to blocking protein. Each point on the protein curve shows the mean _+ two standard errors of eight replicate determinations. The zero point (far right) shows the confidence interval ( _+two standard errors) for all of the total NSB determinations in untreated wells. c o n c e n t r a t i o n in b o t h the p r e t r e a t m e n t a n d the simultaneous incubation protocols. Although most o f t h e s e m a x i m u m % I N H v a l u e s w e r e q u i t e close, s o m e of the d i f f e r e n c e s a m o n g t h e m w e r e signifi-

c a n t ( T a b l e II). H y d r o l y z e d g e l a t i n ( H Y G E L ) was d i s t i n c t l y i n f e r i o r i n b o t h p r o t o c o l s , a n d it was e s p e c i a l l y p o o r as a p r e t r e a t m e n t agent. T h e c o m p l e t e s p e c t r u m of i n h i b i t i o n c u r v e s

47 INHIBITION OF NON-SPECIFIC BINDING BY SIMULTANEOUS INCUBATION OF MTP WITH VARIOUS BLOCKING PROTEINS 100 GEL

90 80

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HDL HYGEL BSA PTG FS GEL CAS

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FSGEL

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OF SOLUTIONS (% Protein w/v)

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Fig. 2. Simultaneous incubation protocol: inhibition of non-specific binding by various blocking proteins. Experimental details are given in the text. Values represent the proportionate decrease of non-specific binding in microtiter plate wells exposed to blocking protein during incubation with a peroxidase-conjugated immunoglobulim when compared to wells on the same microtiter plate that had not been exposed to blocking protein. Each point on the protein curve shows the mean _+two standard errors of eight replicate determinations. The zero point (far right) shows the confidence interval ( + two standard errors) for all the total NSB determinations in untreated wells.

r e v e a l e d c o n s i d e r a b l e differences a m o n g m a n y of the p r o t e i n s tested. I n the p r e t r e a t m e n t p r o t o c o l , MILK, CAS and HDL retained maximal blocking a c t i v i t y across a 1 0 0 0 0 - f o l d c o n c e n t r a t i o n range,

while b l o c k i n g b y the o t h e r p r o t e i n s fell m u c h m o r e r a p i d l y o n d i l u t i o n . I n the s i m u l t a n e o u s i n c u b a t i o n protocol, M I L K a n d G E L r e t a i n e d m a x i m a l effectiveness over a 2000-fold d i l u t i o n . I n

48 TABLE II I N H I B I T I O N OF NON-SPECIFIC B I N D I N G BY VARIOUS B L O C K I N G PROTEINS: MAXIMUM I N H I B I T I O N VALUES A N D C O N C E N T R A T I O N R E Q U I R E D FOR 50% I N H I B I T I O N

Experimental details are given in the text. Under each experimental protocol (pretreatment and simultaneous incubation), two parameters are listed for each blocking protein: (1) the maximum x INH observed at any concentration: and (2) the concentration required for 50% inhibition, as estimated by interpolation from the points immediately surrounding the 50% value on the curve. The mean value and two standard errors (in parentheses, n = 8) are given for each entry. Proteins are listed within each category in order from the most effective to the least effective. Proteins that do not share a letter within the "Sig level" (significance level) column were significantly different for that parameter (two-tailed Wilcoxon rank-sum test, P < 0.05). Rank

Pretreatment protocol

Simultaneous incubation protocol

Maximum inhibition

Protein

%INH

Sig

Protein

Conc a

level 1

MILK

2

CAS

3

GEL

4

HDL

5

FGEL

6

BSA

7

WGS

8

PTG

9

HYGEL

94.0 (0.6) 93.9 (0.1) 93.7 (0.3) 92.7 (0.4) 90.8 (2.0) 90.4 (1.2) 90.4 (0.6) 90.0 (1.0) 55.5 (3.0)

Maximum inhibition

50%INH concentration Sig

Protein

%INH

level

a

CAS

a

MILK

a

HDL

b

PTG

bc

FGEL

c

GEL

c

WGS

c

BSA

d

HYGEL

560 (43) 620 (87) 710 (260) 3 300 (380) 5 400 (370) 11 000 (13 000) 15 000 (1 400) 96 000 (10000) > 10 7

50%INH concentration Sig

Protein

Conc a

level

a

FGEL

a

BSA

a

HDL

b

GEL

c

WGS

bc

CAS

d

MILK

e

PTG

f

HYGEL

95.7 (0.3) 95.3 (0.2) 94.7 (0.4) 94.6 (0.5) 94.5 (0.6) 94.4 (0.3) 94.4 (0.3) 93.3 (0.5) 86.9 (1.6)

Sig

level

a

MILK

ab

GEL

c

CAS

c

HDL

bc

PTG

c

WGS

c

FGEL

d

BSA

e

HYGEL

710 (160) 710 (62) 1 800 (100) 2 400 (230) 3 800 (430) 5 300 ( 1 200) 6 900 (1 200) 27 000 (8400) 4200 (6 900)

a a b c d e f g h

~' Concentrations are in ng/ml, equivalent to (10 7)%.

both protocols, BSA and HYGEL showed the quickest drop in blocking activity. These differences are reflected in the 50% INH concentration values (Table II). Comparison between curves generated by the two protocols for a given protein show that MILK, PTG, and FSGEL were comparable under both conditions, while GEL was much more effective in the simultaneous incubation, and CAS was somewhat more effective as a pretreatment agent.

Discussion The choice of blocking agent(s) to inhibit NSB can be critical to the sensitivity and specificity of ELISA-type systems (Spinola and Cannon, 1985;

Bjercke et al., 1986; Sarma et al., 1986). However, no evaluation of potential blocking proteins has presented a systematic approach to categorizing the many possible candidates. Our results show that proteins do differ considerably in their ability to block NSB, and that amphipathic proteins such as casein and lipoprotein are especially effective. These results may be explained by differences in the proportion of soluble protein that binds to the plastic surface, differences in the effectiveness of surface 'coverage' provided by the bound protein, and additional blocking activity due to proteinprotein interactions at or near the plastic surface. These points are best illustrated by comparing two proteins that exhibited the opposite extremes in blocking effectiveness, CAS and HYGEL. CAS, the most effective purified protein tested,

49

maintained a consistent 92%-94% inhibition of NSB when solutions containing as little as 400 ng total protein were used to pretreat plastic MTP wells. This amount of protein, even if entirely bound to the plastic, is just in the range of minimal saturation for the 125 mm2 of plastic surface exposed during the incubations: 3-4 n g / m m 2 (Pesce et al., 1977; Morrisey and Han, 1978; Cantarero et al., 1980; Fair and Jamieson, 1980; Hosaka et al., 1983). The rapid loss of blocking activity seen immediately below this concentration is commensurate with protein levels falling below the theoretical range of saturability. Therefore, the blocking activity of CAS may be attributed entirely to a monomolecular film that prevents almost all adsorption of the GAM-PO to the plastic. CAS thus serves as a prototype for high-avidity blocking protein with very effective surface coverage. HYGEL, the least effective blocking agent tested, could not reduce NSB in the pretreatment protocol by more than 50%, even at concentrations that would insure complete saturation of the plastic surface. In contrast, HYGEL was able to reduce NSB by almost 90% in the simultaneous incubation protocol. This pattern suggests that excess HYGEL in solution interacts with the protein molecules adsorbed to the plastic surface to provide a more effective barrier against NSB. HYGEL thus serves as a prototype for low-avidity, poor-coverage blocking protein that depends largely on protein-protein interaction to inhibit NSB. Such agents would be expected to do poorly in the pretreatment protocol, since the weak protein-protein interactions would not prevent disruption of the multi-layered molecular barrier when the plates were washed. Non-fat dry milk, which has been used as a blocking agent for both MTP ELISAs and nitrocellulose membrane Western blots (Johnson et al., 1984), was as effective as pure casein in the pretreatment protocol, and somewhat more effective in simultaneous incubation. The stronger blocking shown by MILK over CAS in simultaneous incubation may relate to the instantization process, in which dry milk solids are rendered readily miscible with water. Instantized milk probably provides a more dispersible form of casein that binds more quickly to the plastic.

Since non-fat dry milk is easily dissolved and inexpensive, it offers certain advantages over purified casein. However, it must be used with care in ELISA systems because of two potential problems. First, it is a complex mixture containing substances that may interfere with certain assays; for instance, histones that interfere with anti-DNA determinations (Waga et al., 1986). Second, MILK can 'mask' other solid-phase proteins, causing the complete loss of immunoreactivity of antigens bound to nitrocellulose (Spinola and Cannon, 1985). Perhaps related to this, we have noticed a reduction in hapten-specific antibody titer when MILK was used to block microtiter plates sensitized with hapten-protein conjugates (see Spierto et al., 1987). Fish skin gelatin (FSGEL), an excellent blocker for nitrocellulose Western blots (Saravis, 1984), appears to be the best of the gelatin preparations tested. It does not solidify (even at high concentrations under refrigeration), it is readily available without need for further processing, and it shows considerably better blocking ability than HYGEL. In summary, our results would recommend MILK, CAS, and FSGEL as promising candidates for evaluation as blocking proteins. Of course, a typical ELISA includes several factors not addressed by this study that can influence NSB (e.g., detergents and other protein components). The final result of the interaction among all such factors is never completely predictable, and empirical testing remains essential in the development of any immunoassay. However, in concordance with our findings, CAS has been shown to give much better results than several other blocking agents in specific ELISAs for certain proteins (Kenna et al., 1985) and haptens (Bjercke et al., 1986). The results presented here may therefore reflect properties of blocking proteins that are of general importance in solid-phase immunoassays.

Acknowledgements The authors wish to thank Dr. Hans Feindt (Becton-Dickinson Corporation) for suggesting the evaluation of instantized milk and fish gelatin. We also thank Drs. Roger Clemens (The Carnation

50

Company), John Langone (Baylor University), Richard Norland (Norland Products), Alan Scott (Johns Hopkins University), Shinobu Waga (Scripps Clinic and Research Foundation), James Woodford (Web of Research) and J.C. Wang (Pandex Corporation) for their help in this study. References Bjercke, R.J., Cook, G., Rychlik, N. et al. (1986) Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays. J. Immunol. Methods 90, 203. Cantarero, L.A., Butler, J.E. and Osborne, J.W. (1980) The adsorptive characteristics of proteins for polystyrene and their significance in solid-phase immunoassays. Anal. Biochem. 105, 375, Fair, B.D. and Jamieson, A.M. (1980) Studies of protein adsorption on polystyrene latex surfaces. J. Colloid Interface Sci. 77, 525. Havel, R.J., Eder, H.A. and Bragdon, J.H. (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34, 1345. Hosaka, S., Murao, U., Masuko, S. and Miura, K. (1983) Preparation of microspheres of poly(glycidyl methacrylate) and its derivatives as carriers for immobilized proteins. Immunol. Commun. 12, 509. Johnson, D.A., Gautsch, J.W., Richard Sportsman J. and Elder, J.H. (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Tech. 1, 3. Kato, K., Umeda, Y., Suzuki, F. and Kosaka, A. (1980) Improved reaction buffers for solid-phase immunoassay without interference by serum factors. Clin. Chim. Acta 120, 261. Kenna, J.G., Major, G.N. and Williams, R.S. (1985) Methods for reducing non-specific binding in enzyme-linked immunosorbent assays. J. Immunol. Methods 72, 251

Morrisey, B.W. (1977) The adsorption and conformation of plasma proteins: a physical approach. Ann. N.Y. Acad. Sci. 283, 50. Morrisey, B.W. and Han, C.C. (1978) The conformation of gamma-globulin adsorbed on polystyrene latices determined by quasielastic light scattering. J. Colloid Interface Sci. 65, 423. Norland, R. (1986) Fish Gelatin: Technical Aspects and Applications. Special Report (Norland Products, New Brunswick, N J). Pesce, A.J., Ford, D.J., Gaizutis, M. and Pollak, V.E. (1977) Binding of protein to polystyrene in solid-phase immunoassays. Biochim. Biophys. Acta 492, 399. Pollard, H.B., Menard, R., Brandt, H.A. et al. (1978) Application of Bradford's protein assay to adrenal gland subcellular fractions. Analyt. Biochem. 86, 761. Saravis, C.A. (1984) Improved blocking of nonspecific antibody binding sites on nitrocellulose membranes. Electrophoresis 5, 54. Sarma, J.K., Hoffman, S.R. and Houghten, R.A. (1986) Enzyme linked immunosorbent assay (ELISA) for beta-endorphin and its antibodies. Life Sci. 38, 1723. Spierto, F.W., Vogt, R.F., Whitfield, W. et al. (1987) Development and evaluation of a microtiter plate enzyme immunoassay for antibodies to 3,3'-dichlorobenzidine. J. Anal. Toxicol. 11, 31. Spinola, S.M. and Cannon, J.G. (1985) Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods 81, 161. Vroman, g., Adams, A.L., Klings, M. et al. (1977) Reactions of formed elements of blood with plasma proteins at interfaces. Ann. N.Y. Acad. Sci. 283, 65. Waga, S., Tan, E.M. and Rubin, R.L (1986) Histones in biological fluids: effect on anti-histone antibody specificities. Arthritis Rheum. 29, s72. Wedege, E. and Svenneby, G. (1986) Effects of blocking agents bovine serum albumin and Tween 20 in different buffers on immunoblotting of brain proteins and marker proteins. J. Immunol. Methods 88, 233. Whitfield, W. and Spierto, F.W. (1986) Modified ELISA for the measurement of urinary albumin. Clin. Chem. 32, 561.