Journal o f Immunological Methods, 22 (1978) 165--174 @)Elsevier/North-Holland Biomedical Press

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THE PREPARATION OF LATEX PARTICLES WITH COVALENTLY B O U N D P O L Y A M I N E S , IgG A N D M E A S L E S A G G L U T I N I N S A N D T H E I R USE IN V I S U A L A G G L U T I N A T I O N T E S T S

G. QUASH, ANNE-MARIE ROCH, A. NIVELEAU, J. GRANGE, T. KEOLOUANGKHOT and J. HUPPERT Unitd de Virologic, U51 INSERM, Groupe de Recherches CNRS No. 33, 1 place Pr. Joseph Renaut, 69371 Lyon Cddex 2, France

(Received 3 January 1978, accepted 2 February 1978)

Carboxylated latex particles were substituted with side arms terminating in primary amine and hydrazine groups. The particles were coupled to aldehyde groups generated on glycoproteins which were treated with sodium periodate. Particles having the alipathic primary amine putrescine hapten as the sole substituent and particles linked to glycoproteins such as measles agglutinins and IgG were used to detect the presence of the corresponding antibodies or antigens in biological fluids by agglutination tests.

INTRODUCTION Linking o f p r o t e i n s t o insoluble s u p p o r t s is generally a c c o m p l i s h e d b y i n t e r a c t i o n o f t h e i r a m i n o , c a r b o x y l , t y r o s y l or s u l p h y d r y l residues with a p p r o p r i a t e spacer arms previously fixed t o t h e s u p p o r t . T h e greater the flexibility o f the spacer arm, the greater t h e possibility o f t h e insolubilised p r o t e i n r e t a i n i n g its biological activity a f t e r c o u p l i n g (Cuatrecasas and Anfinsen, 1 9 7 1 ) . F o r spacer arms t e r m i n a t i n g with a c a r b o x y l g r o u p (COOH), coupling with t h e a m i n o groups o f p r o t e i n s is achieved b y means o f a w a t e r soluble c a r b o d i i m i d e (Miller et al., 1 9 7 2 ) or b y W o o d w a r d ' s reagent K in a o n e or t w o step c o u p l i n g m e t h o d ( H o i - K a u - L e e et al., 1 9 7 6 ) . T h e c a r b o x y l g r o u p itself m a y also be p r e a c t i v a t e d with N-OH succinimide in n o n - a q u e o u s media, so t h a t c o u p l i n g with NH2 groups o f p r o t e i n s takes place d i r e c t l y O O w h e n the N-OH s u c c i n i m i d e c a r b o x y l i c acid ester (CH2--C--O--N (x_J)is m i x e d O with the p r o t e i n in a q u e o u s s o l u t i o n (Cuatrecasas and Parikh, 1 9 7 2 ) . With spacer arms t e r m i n a t i n g in a m i n o groups (NH2), and p r e a c t i v a t e d with a b i f u n c t i o n a l a l d e h y d e , e.g., g l u t a r a l d e h y d e (Avrameas and T e r n y n c k ,

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1969), coupling also takes place with NH2 groups on the protein molecule. A detailed mechanism for this reaction has been proposed (Monsan et al., 1975). NH2 groups on the spacer arm may also be coupled to COOH groups on protein by means o f an appropriate condensing agent, e.g., water soluble carbodiimide. For spacer arms having a free amino group at the para position of an H O aromatic ring e.g., p - a m i n o b e n z o a t e , CH2 N--C ( ~ NH2, diazotization of the NH2 groups allows effective coupling to take place with tyrosine residues on proteins. Sulphydryl groups at the end of the spacer arm may be coupled to SH groups on proteins by means of ammoniacal potassium ferricyanide (Epton and Thomas, 1971). Some proteins are liable to lose biological activity when coupled by their amino acid residues. In such cases, if these proteins also contain carboh y d r ate chains which do not c o n t r i b u t e to their reactivity, these carboh y d r a t e groups may be used to couple the proteins to insoluble supports. Methods for coupling glycoproteins such as immunoglobulin IgG, and certain viral agglutinins to latex particles and the use of these sensitized particles to d e tect corresponding antigens and antibodies are described here. MATERIAL AND METHODS Carboxylated p o l y s t y r e n e latex particles * ranging from 0.8 to 0.03 gm diameter were obtained as a 10% suspension in water from Rh6ne Poulenc, Aubervilliers, France and the Dow Chemical Co., Indianapolis, U.S.A. 1-Ethyl 3 , 3 - d i m e t h y l a m i n o p r o p y l carbodiimide HC1 (WSC), h e x a m e t h y l e n e diamine (HMD) were obtained from Merck (France). Spermine, spermidine and putrescine as their h y d r o c h l o r i d e salts were purchased from the Sigma Chemical Co., U.S.A. Sheep IgG anti-human fibrin degradation products (FDP) were a gift from Dr. I. Cayzer, Wellcome Research L a b o r a t o r y , Beckenham, U.K. Latex particles as obtained from the manufacturers were first systematically washed by repeated centrifugation and resuspension, or by filtration on Millipore filters of appropriate pore size, to eliminate non-dialysable carb o x y l a t e d p o l y m e r material.

Alipathic amine substitution (a) Hexamethylene diamine. To 12 mg of latex in suspension in 4 ml 0.14 M NaC1 containing 0.01 M borate--HC1 pH 8.1 (BBS) were added 80 t~moles HMD in solution in 1 ml of the same buffer. WSC was t hen added to a final c o n c e n t r a t i o n of 0.05 M. The mixture was gently shaken at 4°C overnight, after which the latex was spun down at 20,000 × g for 30 min in a * Estapor registered trade mark of Rh6ne-Poulenc, France.

167 Beckman J21 refrigerated centrifuge. The latex was resuspended in the same buffer and washed 4 times. (b) Polyamines. Identical procedures were used to couple spermine, spermidine and putrescine.

Aromatic hydrazine substitution To 10 mg of latex HMD in suspension in 10 ml of 0.1 M phosphate buffer pH 7 was added 50 t~moles of p-hydrazinobenzoic acid in solution in a 0.1 M borate buffer pH 8.1. The mixture was shaken at 20°C for 14 h in the presence of 0.05 M WSC. At the end of this period the treatment with WSC was renewed for a further period of 14 h, after which the substituted latex particles were washed by 3 successive cycles of centrifugation (20,000 × g for 30 min) and resuspension in 0.1 M borate buffer pH 8.1.

Alipathic hydrazine substitution Latex HMD (6.0 mg) suspended in 0.1 M sodium phosphate buffer pH 7 was shaken at 20°C for 1 h in the presence of glutaraldehyde at a final concentration of 1.25%. At the end of this period the suspension was dialysed against 0.1 M phosphate buffer pH 6.0 until the dialysis liquid gave a negative 2,4-dinitrophenylhydrazone test for aldehydes. To the latex suspension was then added 20 pmoles of adipic dihydrazide. After a contact time of 16 h at 20°C the excess adipic dihydrazide was removed by dialysis against 0.1 M sodium diphosphate buffer pH 7.0.

Preparation of latex labelled with [J4C]ethanolamine To 20 mg of carboxylated latex particles in suspension in BBS were added 50 gl [14C]ethanolamine (10 pCi/1.6 pmole) and 6.0 mg of WSC in a total volume of 4.0 ml. After shaking at 4°C for 2 h a further 20 pmoles of WSC were added and shaking continued for an additional 16 h at 4°C. The mixture was then dialysed against BBS until the dialysis liquid no longer showed the presence of radioactivity. The particles so obtained had a specific activity of about l 0 s cpm/mg. The radioactive particles were then substituted first with HMD as previously described and then with an alipathic hydrazine.

Oxidation of glycoproteins (a) Immunoglobulin G. Immunoglobulin G (IgG) was prepared by either (NH4)2SO4 precipitation or the caprylic acid technique (Steinbuch and Audran, 1969) and purified by chromatography on DEAE-cellulose. To 60 mg of horse IgG anti-polyamines in solution in 2 ml of 0.1 M phosphate buffer pH 6 were added to 0.2 ml of 0.15 M NaIO4 in aqueous solution. The mixture was left for 3 h at 4°C and protected from light. The reaction was then terminated by the addition of 0.015 M glycerol (final concentration). After a contact time of 30 min the contents of the tubes were dialysed at 4°C against 1 litre of 0.1 M phosphate buffer pH 6 with several changes of

168 buffer until the dialysis liquid no longer showed the presence of aldehyde products. An identical procedure was used for the preparation of oxidised IgGs from sheep, rabbit and goat with specificities for human IgG, human IgM, human fibrin degradation products (FDP) and rabbit IgG. (b) Viral agglutinins. Measles virus suspensions were a gift from Dr. F. Wild. The suspensions were concentrated with a m m o n i u m sulphate (50% saturation) and then either used directly or partially purified by adsorption on DEAE-cellulose equilibrated with 0.05 M NaC1, 0.05 M Tris--HC1 pH 7.6, followed by elution with 0.3 M NaC1, 0.05 M Tris--HC1 pH 7.6. The biological activity of the preparations as measured by the haemagglutination assay was 160,000 HA U/mg protein. To 5 ml of a measles virus preparation containing 16 mg of protein in 0.1 M sodium phosphate buffer pH 6 was added 0.5 ml of 0.15 M NaIO4 dissolved in water. The mixture was left at 4°C in the dark for 3 h. At the end of this time 0.6 ml of 0.15 M NazSO3 was added to arrest the action of the NaIO4 on the viral carbohydrate residues. After 30 min contact the mixture was dialysed against 0.1 M phosphate buffer pH 6 for 18 h at 4°C with several changes to eliminate low molecular weight oxidation products.

Coupling of oxidised IgGs to HMD substitu ted latex particles To 20 mg of HMD substituted latex particles were added 40 mg of oxidised goat IgG anti-polyamine in a final volume of 5 ml of 0.01 M borate-HC1 pH 8.1 (BBS). The suspension was gently rotated at 4°C for 16 h, and the Schiff bases formed were stabilised by addition of 5 mg NaBH4 to the suspension, the pH having been adjusted to pH 8.8 with 0.1 M borate buffer. After 5 h at 4°C the mixture was centrifuged and the pellet of sensitized particles washed 4 times with BBS pH 8.1 to eliminate uncoupled material. The first two washings were pooled and saved for protein determination; the pellet was suspended in BBS pH 8.1 and sonicated on a Bronson sonifier at setting 2 for about 3 sec with a microprobe to ensure thorough suspension. Coupling o f oxidised IgG to hydrazine substituted latex particles To 10 mg of hydrazine substituted latex were added 10 mg of oxidised IgG anti-human fibrin degradation products in a final volume of 5 ml 0.1 M phosphate buffer pH 6.0. The mixture was left at 4°C for 16 h with gentle shaking. At the end of this period the contents of the tubes were spun down at 20,000 × g for 30 min, the supernatants collected and the pelleted particles washed by 3 repeated cycles of resuspension and centrifugation. The first two supernatants were pooled and saved for protein estimations. The particles were finally suspended in 5 ml of 0.14 M NaC1, 0.1 M glycine-NaOH buffer pH 8.1 and sonicated as described above. Coupling o f oxidised viral agglu tinins to hydrazine su bstitu ted particles An identical procedure was used for coupling oxidised measles virus to

169 non-radioactive spheres using the same weight of protein to When radioactive particles were used, their specific activity increased by using labelled viral agglutinins which had been Vero cells growing in the presence of 14C-labelled amino acids. measles agglutinin coupled particles had a specific activity of latex.

latex (1/1). was further prepared on The labelled 106 d p m / m g

RESULTS

Determination o f the quantity of glycoprotein linked per mg latex particles An aliquot (0.3 ml) of the two combined supematants was taken for protein determinations by the Lowry technique. If the protein concentration was too low, 5 ml of the supernatant were first precipitated in the cold with 5% TCA (final concentration). The precipitate was washed with 5% TCA and finally dissolved in 0.3 ml of I N NaOH. Aliquots of this solution were used for protein determinations. From this value and the total volume of the supernatant, the a m o u n t of protein remaining in the supernatant was calculated and the a m o u n t of protein linked per mg latex thus determined. For anti-IgG and anti-FDP a value of 600--720 pg protein/mg latex (0.8 pm) was obtained and for measles agglutinins this value was between 130--322 pg protein/mg latex (0.3 pm). Use o f antigen linked latex particles to detect antibodies Measles antigen. (i) Macroscopic agglutination on glass plates (circles of 3 cm diameter). Measles agglutinin latex spheres were suspended at 2--3 mg latex/ml in 0.14 M NaC1, 0.01 M glycine--NaOH pH 8.1. Two-fold dilutions of a rabbit anti-measles serum were made in this same buffer but containing 0.2% bovine serum albumin. In each circle on the glass plate were placed 2 drops of the glycine--BSA buffer, 1 drop of the appropriate serum dilution and 1 drop of measles latex at 2--3 mg latex/ml. The contents were mixed with a wooden stirrer and the plates gently rotated by hand for 3 min and agglutination assessed visually. Serum from non-immunized rabbits served as negative controls. A typical result is shown in Fig. 1. (ii) Agglutination in microtitre plates was performed by mixing 50 gl of the glycine--BSA buffer pH 8.1, 50 pl of 2-fold dilutions at the measles rabbit antiserum (diluted in the same buffer) and 50 pl of measles latex spheres at 300 pg latex/ml. The mixture was incubated 18 h at 4°C. Where agglutination was present the latex particles failed to form pellets in the wells (Fig. 2). The reaction could also be read by inclining the plates; in the negatives the particles flowed in a straight line, whereas the positives either did not flow, or did so as a continuous sheet. Polyamine antigens. In an a t t e m p t to develop a test for polyamines in

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f

Fig. 1. A g g l u t i n a t i o n o n a glass slide of latex measles s u s p e n d e d at 2.6 m g l a t e x / m l in 0.14 M NaC1, 0.01 M glycine-NaOH pH 8.1. 1: measles virus specific a n t i s e r u m ; 2: n o r m a l rabbit serum.

serum and urine, latex-putrescine (put) was prepared as described under Methods (HMD substitution) using [14C]putrescine. Putrescine linked particles, which contained 0.88 nmole put/rag latex, were suspended at 4 mg latex/ml in 0.14 M NaC1, 0.2% BSA, 0.1 M Tris--HC1 pH 8.1. T hey were used t o d e t e c t antipolyamine antibodies in the sera of animals which had been previously injected with lysozyme-spermine. As judged by the slide agglutination test a rabbit serum was positive up to 1/5 and a goat serum up to 1/24. Both sera were diluted in the BSA, NaC1, Tris buffer described above.

A [3 C

Fig. 2. A g g l u t i n a t i o n o f l a t e x measles particles s u s p e n d e d at 300 p g l a t e x / m l in 0.2% BSA, 0.14 M NaCl, 0.01 M Tris--HCl pH 8.1, b y 2-fold d i l u t i o n s of measles virus-specific a n t i s e r u m in m i c r o t i t r e plates. A a n d B are t w o positive sera, C is a negative serum.

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The preparation of latex particles with covalently bound polyamines, IgG and measles agglutinins and their use in visual agglutination tests.

Journal o f Immunological Methods, 22 (1978) 165--174 @)Elsevier/North-Holland Biomedical Press 165 THE PREPARATION OF LATEX PARTICLES WITH COVALENT...
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