Immune adherence: A quantitative and kinetic analysis

Journal of Immunological Methods, 23 (1978) 99--108

99

© Elsevier/North-Holland Biomedical Press

IMMUNE ADHERENCE: A QUANTITATIVE AND KINETIC ANALYSIS

TERUAKI SEKINE

Department of Virology, National Cancer Center Research Institute, Tsukiji 5-Chome, Chuo-Ku, Tokyo, Japan (Received 31 Ja~auary 1978, accepted 10 March 1978)

Quantitative and kinetic analysis of the immune-adherence reaction (IA) between C3b fragments and IA receptors as an agglutination reaction is difficult. Analysis is possible, however, by use of radio-iodinated bovine serum albumin as antigen at low concentrations {less than 200 ng/ml) and optimal concentration of antibody to avoid precipitation of antigen--antibody complexes with human erythrocytes without participation of complement. Antigen and antibody are reacted at 37°C, complement is added, the mixture incubated and human erythrocytes added; after further incubation, ice-cold EDTA containing buffer is added and the erythrocytes centrifuged and assayed for radioactivity. Control cells reacted with heated guinea pig serum retained less than 5% of the added radioactivity. The method facilitates measurement of IA reactivity and permits more detailed analysis of the mechanism underlying the reaction.

INTRODUCTION

The immune-adherence (IA) reaction (Nelson, 1953; Nelson, 1963} is a specific immunological reaction in which micro-organisms or other particulate antigens sensitized with antibody and complement attach to cell surfaces of, for example, primate erythrocytes, non-primate platelets, macrophages, and B-lymphocytes (Lay and Nussenzweig, 1968). IA is an adherence reaction between IA receptors and C3b and/or C4b molecules generated by activation of complement (Nishioka and Linscott, 1963; Cooper, 1969). IA is observed under the microscope as agglutination of particulate antigens and cells and as agglutination patterns at the bottom of tubes (Turk, 1958; Nishioka, 1963). Detailed kinetic and quantitative analyses of IA have not been undertaken to date, although some attempts have been made to investigate IA quantitatively by using colored proteins or radio-labeled reagents as antigens. Such attempts have failed because of non-specific reactions of antigen--antibody complexes and human erythrocytes in the centrifuged sediment without participation of complement (Taverne, 1975). This report presents a method for the quantification of IA and a kinetic analysis of the formation of IAactive antigen--antibody-complement complexes. Radio-labeled bovine serum albumin was used as antigen at low concentration (less than 200 ng! ml) with optimal antibody concentration. This avoided sedimentation of the

100 antigen--antibody complex and human erythrocytes without participation of complement. MATERIALS AND METHODS Bovine serum albumin (BSA) Crystallized BSA was obtained commercially (Armour Pharmaceutical Co., Illinois, U.S.A.). Radio-labeled B S A ([12SI]BSA) The labeling of BSA with 12sI was by the m e t h o d of Hunter and Greenw o o d (1962). The specific activity of the labeled BSA was approximately 0.6 mCi/mg of protein. Rabbit anti-BSA sera (anti-BSA ) Antisera were collected from hyperimmunized rabbits immunized with crystallized BSA in Freund's complete adjuvant. By quantitative precipitation the pooled anti-BSA sera contained 490 t~g of antibody N/ml. A complement fixation test was positive at 0.03 pg N/ml BSA and antiserum dilution 1 : 2560. The antiserum was heated at 56°C for 30 min and centrifuged at 15,000 rev./min for 30 min. Diluents GVB 2÷. Veronal buffered saline (pH 7.5) containing 0.15 mM CaC12, 0.5 mM MgC12, and 0.1% gelatin was prepared according to Kabat and Mayer (1961). O.04 M E D T A - G V B Veronal buffered saline (pH 7.5) containing 0.04 M Na3HEDTA (ethylene diaminetetraacetic acid trisodium) and 0.1% gelatin was prepared as described by Nelson et al. (1966). The sterilized GVB 2÷ and 0.04 M EDTAGVB were obtained from Ensai Co. (Tokyo, Japan). Guinea pig serum (GPS) The source of complement was fresh GPS obtained from Toshiba Chemical Co. (Tokyo, Japan). The GPS contained 250 CHs0 hemolytic units per millilitre and a very low titer of naturally occurring antibody against human and sheep erythrocytes. For controls, GPS at the same dilution as in the experiments was used, but heated at 56°C for 60 min. I

Human erythrocytes (HUE) Human blood from healthy adults was obtained with sterilized Alsever's solution. The blood was stored at 4°C and used within 4 weeks of donation. Reaction tubes Polypropylene test tubes, measuring 1.2 cm in diameter and 12 cm inside length were used for all reactions, for centrifugation and for counting.

101

Assay of radioactivity Radioactivity was assayed by using Automatic Gamma Well Counting Systems (Nuclear, Chicago, IL, U.S.A.).

Cell concentration HuE in Alsever's solution were washed 5 times with 0.04 M EDTA-GVB and suspended in the same buffer. The buffy coat was carefully removed. The cell concentration was measured photometrically (Nelson and Nelson, 1959). A scale reading of 0.190 at a wave-length of 541 nm was obtained when one volume of cell suspension containing 1 X 108 cells/ml was lysed by the addition of 9 volumes of 0.1% Na2CO3 solution.

Calculation Results were calculated according to the following formula: Per cent of b o u n d [12sI] BSA (B%) after subtraction of background counts = count in precipitate -- count in control x 100 total count -- count in control

Experimental design The standard experimental procedures described below were followed in all experiments. The only deviations from the standard procedures were in time, temperature or concentration (Figs. 1--7b). The standard experimental procedures were based on results of preliminary experiments. In order to examine the formation of IA-reactive antigen--antibodycomplement complexes and the reaction of HuE with IA-active complexes, the following procedure was followed: 0.5 ml amounts of anti-BSA serum diluted with GVB 2÷ to 1 : 1000 were mixed with 0.5 ml amounts of [12SI]BSA diluted in GVB 2÷ to contain 200 ng/ml, 50 ng/ml, and 20 ng/ml. The mixtures were incubated in a shaker bath at 37°C for 120 min. Following incubation, 0.5 ml of fresh GPS diluted with GVB 2÷ to 1 : 25 was added (10 CHs0 hemolytic units per ml of diluted GPS). This mixture was incubated in a shaker bath at 37°C for 20 min. Next, 0.5 ml of HuE (1 X 109 cells/ml) in 0.04 M EDTA-GVB was added and the mixture was incubated in a shaker bath at 37°C for 20 min. Eight millilitres of ice-cold 0.04 M EDTA-GVB was then added and the cells centrifuged down (10 min, 700 X g, 4°C), and washed once with 10 ml of ice-cold 0.04 M EDTA-GVB. The radioactivity of the washed, packed cells was determined. GPS heated at 56°C for 60 min was used as complement-depleted control for non-specific reaction of HuE, [12SI]BSA, and anti-BSA complexes. In almost all experiments, the control values were below 5% of the initially added radioactivity.

102 RESULTS

Effect of incubation time of anti-BSA with [12SI]BSA o n the formation of IA-active complexes The antigen (200, 50, and 20 ng of [12SI]BSA/ml) was reacted with antiBSA diluted to 1 : 1000. The diluted anti-BSA contained 0.49 gg AbN/ml. [ 12SI]BSA binding to HuE increased with incubation time (Fig. 1). At higher antigen concentrations, the amount of IA-active complexes present after 4°C overnight incubation was not greater than after incubation at 37°C for 240 min. At the highest [12SI]BSA concentration (200 ng/ml), control values increased slightly with incubation time. However, control values after incubation for 240 min at 37°C or overnight at 4°C were approximately 4% of the initially added radioactivity. At the two other concentrations of [12SI]BSA (50 ng/ml, 20 ng/ml), the control values were below 2% of the initially added radioactivity and incubation time had no effect. Effect of [12SI]BSA and anti-BSA concentrations on control values The range of antigen concentrations shown in Fig. 3 was chosen because antigens at a concentration of less than 1 ~g/ml are generally not detectable by the conventional precipitin reaction (Kabat and Mayer, 1961). However, the complement fixation test (Kabat and Mayer, 1961), as well as the immune-adherence hemagglutination test (Turk, 1958; Nishioka, 1963; Kabat and Mayer, 1961) allows detection of antigens at such concentrations. [12SI]BSA (50, 100, 200, 400, and 800 ng/ml) was mixed with anti-BSA

1°°f

o
7.5

30

60

120

180

240

4°C overnight

Time in minutes

Fig. 1. Effect of incubation time of anti-BSA/[ 12 s I ]BSA on the formation of IA-reactive complexes. The incubation time of anti-BSA/[12SI]BSA was varied from a minimum of 7.5 rain to a m ax im u m of 240 min at 37°C, and overnight incubation at 4°C. (©) 200 ng/ ml [12SI]BSA; (El)50 ng/ml [12SI]BSA; ( x ) 2 0 mg/ml [12SI]BSA.

103

< -~

20

"x

D

i

O 50

100

200

400 ~I-BSA concentration

800

(ng/ml)

Fig. 2. Effect of [12SI]BSA and anti-BSA concentrations on control values. [12SI]BSA at concentrations from 50--800 ng/ml was mixed with anti-BSA [diluted to 1 : 100 (x); 1 : 300 (u); 1 : 500 (zx); 1 : 1000 (e);and 1 : 3000 (~)].

d i l u t e d t o 1 : 100, 1 : 300, 1 : 500, 1 : 1000, and I : 3000. A f t e r i n c u b a t i o n at 37°C f o r 20 min, h e a t e d GPS, d i l u t e d t o 1 : 25, was a d d e d t o each test t u b e . T h e s t a n d a r d p r o c e d u r e was f o l l o w e d and the results are s h o w n in Fig. 2. A t antigen c o n c e n t r a t i o n s o f 200 ng/ml or less, c o n t r o l values were l o w e r t h a n at h i g h e r antigen c o n c e n t r a t i o n s . T h e l o w e r a n t i b o d y c o n c e n t r a tions (1 : 1 0 0 0 and 1 : 3 0 0 0 d i l u t i o n ) , s h o w e d t h e l o w e s t c o n t r o l values, i n d e p e n d e n t l y o f antigen c o n c e n t r a t i o n . Since excess antigen in an a n t i g e n - a n t i b o d y c o m p l e x is k n o w n t o inhibit t h e r e a c t i o n with c o m p l e m e n t , antigen c o n c e n t r a t i o n s at 200 ng/ml or less were c h o s e n f o r this s t u d y .

Effect o f an ti-BSA concentrations on the formation of IA-active complexes T o e x a m i n e the e f f e c t o f anti-BSA c o n c e n t r a t i o n s o n the f o r m a t i o n o f IA-active c o m p l e x e s , anti-BSA in various dilutions (1 : 1 0 0 - - 1 : 3 0 , 0 0 0 ) was m i x e d with t h r e e c o n c e n t r a t i o n s o f [12SI]BSA (200, 50, and 2 0 n g / m l ) . Figure 3 shows the decrease o f [~2SI]BSA binding t o H u E at h i g h e r and l o w e r anti-BSA c o n c e n t r a t i o n s . Since a n t i b o d y o r antigen excess is t h o u g h t t o inhibit t h e c o m p l e m e n t r e a c t i o n , anti-BSA c o n c e n t r a t i o n s in 1 : 1 0 0 0 d i l u t i o n were c h o s e n f o r t h e s t a n d a r d p r o c e d u r e s .

Effect of HuE concentration on 12SI-BSA binding T o e x a m i n e t h e e f f e c t o f H u E c o n c e n t r a t i o n on the a m o u n t o f [12SI]BSA b o u n t to H u E b y IA, the H u E c o n c e n t r a t i o n was varied f r o m 6 × 107 t o

104 100

D

o
5O

1 :I00

I

,

1:300

1 :lOOO

i

Dilution

1:3000 of anti

1 :I 0000

1:30000

BSA

Fig. 3. Effect of anti-BSA concentration on the formation of IA-reactive complexes. AntiB S A at dilutions from 1 : 100--1 : 3 0 , 0 0 0 was m i x e d with three concentrations of [12SI]BSA [ 2 0 0 ng/ml (o); 50 ng/ml ([]); 20 ng/ml ( x ) ] .

6 X 10 9 cells/ml. As shown in Fig. 4, [12SI]BSA binding reached the highest level at about 6 X l 0 s cells/ml, independently of antigen concentration. Since it appears that a longer incubation time is necessary at HuE concentrations below 6 X 10 s cells/ml, a HuE concentration of 1 × 10 9 cells per ml was used in this study. At higher HuE concentrations, slightly decreased [I:SI]BSA binding was observed, the reason for which is not clear. At higher

100

5O m-e

6xlO ~

2xlO B Concentration

6xlO 8

2xlO 9

6xlO 9

o f Hu[ ( c e l l s / m l )

Fig. 4. Effect of HuE concentration on [12SI]BSA binding. HuE concentration was varied f r o m 6 x 107 to 6 x 109 cells/ml. HuE was added to mixtures prepared with three concentrations of [ 1 2 s I ] B S A [ 2 0 0 ng/ml (o); 50 ng/ml (a); and 20 ng/ml (X)]. Controls at 2 0 0 ng/ml [12SI]BSA (A).

105 100

o 50

_~__ 1/800

1/400

t. __ i i 1/200 1/100 1/50 (2.5CH ~/m] )

i

1/25

1/12.5

1/6.2 (40CHso/ml)

C o n c e n t r a t i o n of complement

Fig. 5. Effect of complement concentration on the formation of IA-active complexes. The concentration of complement was varied as shown at each antigen concentration level [200 ng/ml [12SI]BSA (o); 50 ng/ml [12SI]BSA (D); 20 ng/ml [12SI]BSA (X)].

HuE c o n c e n t r a t i o n s , c o n t r o l values were slightly higher, but at a concentrat i o n o f 2 0 0 n g / m l o f [12SI]BSA, were as small as 3.5% at.6 X 10 ~ cells/ml and 4.9% at 6 X 109 cells/ml.

100

[]

O

D

t3

6(]

120

180

5C

I

15

30

)

<

-

Time i n m i n u t e s

Fig. 6. Kinetics of formation of IA-active complexes with [12SI]BSA--anti-BSA complexes and complement. The complement was added to pre-incubated [12SI]BSA/antiBSA mixtures and incubated at 37°C for various periods. The experiments were performed at three concentrations of antigen [200 ng/ml [12SI]BSA (o); 50 ng/ml [12sI]BSA (D); and 20 ng/ml [12SI]BSA (X)].

106

Effect of complement concentration complexes

on the formation

of IA-active

After incubation of the anti-BSA with [125I]BSA, complement, at concentrations ranging from 0.31 CHs0/ml (GPS dilution 1 : 800) to 40. CHs0/ml (GPS dilution 1 : 6.2), was added to the mixtures. In control experiments, heated GPS at the same dilutions was added. Fig. 5 shows that the greatest amount of IA-active complexes is seen at a complement concentration of 2.5 CHs0/ml ( 1 : 1 0 0 dilution). At the higher antigen concentrations (200 ng/ml, 50 ng/ml), a slight decrease in bound [12SI]BSA occurred with increasing complement concentration. At the lowest antigen concentration

1 O0

o o 5~

f x / x /

15

30

120

60

180

Time in minutes 10(]

---0~

"t:)

g 5(

--:;4.

15

30

120

60

]80

Time in minutes

Fig. 7. Kinetics of the reaction of HuE with IA-reactive complexes at 37°C and 0°C. The HuE was added to preincubated mixtures of [12sI]BSA [200 ng/ml (©); 50 ng/ml (D); 20 ng/ml (×)], anti-BSA and complement and incubated for various periods at 37°C and 0°C.

107

(20 ng/ml), the amount of bound ['2SI]BSA detected at 5 CHs0/ml (1 : 50 dilution) increased slightly with increased complement concentration. Control values did not change with changes in complement concentration. Kinetics of the formation of IA-active complexes with 12SI-BSA anti-BSA complexes and complement The effect of incubation time of ['~SI]BSA anti-BSA complexes and complement on the formation of IA-active complexes are shown in Fig. 6. The IA-active complexes formed rapidly at three different antigen concentrations. At the lowest antigen concentration (20 ng/ml), the amount of bound ['2SI]BSA increased slightly thereafter with incubation time. It is possible that this was due to the formation of new ['2SI]BSA anti-BSA complexes during the extended incubation period (Fig. 1). Kinetics o f the reaction o f HuE with IA-active complexes at 37°C and O°C The reaction of HuE with IA-reactive complexes was examined at 37°C and 0°C. Rapid reactions occurred at 37°C (Fig. 7a), but not at 0°C (Fig. 7b). This confirms that the reaction of IA receptors and IA-reactive complexes is temperature-dependent. The experiments were performed at excess HuE for adsorption of IA-active complexes. Control values did not increase with longer incubation periods. DISCUSSION

Some attempts to determine IA quantitatively have previously been made. Nelson (1953) described a 'clearance test' detecting Treponema pallidum as antigen in the supernatant following incubation of antigen with anti-serum complement and HuE. In this test antigen bound to HuE was calculated from the amount of antigen remaining in the supernatant. In another context, Taverne (1957) used radio-labeled T2 phage as an antigen and determined the radioactivity in the HuE pellet. Both these attempts to measure IA failed because non-specific reactions led to the appearance of the antigen-antibody complexes in the centrifuged erythrocyte sediment without participation of complement. Furthermore, control values were unreliable. The immune-adherence hemagglutination test (IAHA) has been used to detect various antigens such as bacterial cells, albumins (Nelson, 1963), tumor antigens (Nishioka et al., 1969), viral antigens (Ito and Tagaya, 1966; Mayumi et al., 1971; Krugman et al., 1975; Tsuda et al., 1975) and antibodies. Because of its extreme sensitivity and specificity, the immuneadherence hemagglutination test is of greater value than the complement fixation test. However, the mechanism underlying immune adherence hemagglutination has not been completely analyzed, and no common procedures have been established to detect various antigens and antibodies. In the present investigation, a detailed analysis of IA is presented. The procedure outlined permits measurement of IA without non-specific reactions.

108

In vivo, IA is important for phagocytosis. Also, complement receptors which involve IA are found on B l y m p h o c y t e s (Lay and Nussenzweig, 1968). These were named 'complement receptor l y m p h o c y t e s ' and are essential for 19 S antibody formation (Mason, 1976). However, the biological functions of IA receptors on HuE, unlike those on leukocytes in vivo, have n o t been fully examined. Measurement of IA reactivity of HuE is reported by Nelson and Uhlenbruck, 1967; Klopstock et al., 1965; Gozin and Soulier, 1972; and R o t h m a n et al., 1975, but the results are confusing. In these reports, IA reactivity was measured by hemagglutination which does not provide a quantitative m e t h o d and is susceptible to variation due to experimental conditions. The present m e t h o d facilitates quantitative measurement of IA reactivity of HuE and makes possible more detailed analysis of the mechanism underlying the IA reaction. ACKNOWLEDGEMENTS

The author thanks Ms. Petralia for her editorial assistance in the preparation of this manuscript. This investigation was supported by Research Grants from the Ministry of Health and Welfare. REFERENCES Cooper, N.R., 1969, Science 165,396. Gozin, D. and J.P. Soulier, 1972, Vox Sang. 23,472. Hunter, W.M. and F.C. Greenwood, 1962, Nature 194,495. Ito, M. and I. Tagaya, 1966, Jap. Med. Sci. Biol. 19, 109. Kabat, E.A. and M.M. Mayer, 1961, Experimental Immunochemistry, 2nd edn (Charles C. Thomas, Springfield IL)p. 113. Klopstock, A., J. Schwartz, Y. Bleiberg, A. Adam, and A. Szeinberg, 1965, Vox Sang. 10,177. Krugman, S., H. Friedman, and C. Lattemer, 1975, New Engl. J. Med. 292, 1141. Lay, W.H. and V. Nussenzweig, 1968, J. Exp. Med. 128,991. Mason, D.W., 1976, J. Exp. Med. 143, 1111. Mayumi, M., K. Okochi, and K. Nichioka, 1971, Vox Sang. 20,178. Nelson, D.S. 1963, Immune Adherence. Advances in Immunology, Vol. 3 (Academic Press, New York and London) p. 131. Nelson, D.S. and R.A. Nelson, Jr., 1959, Yale J. Biol. Med. 31,185. Nelson, D.S. and G. Uhlenbruck, 1967, Vox Sang. 12, 43. Nelson, R.A. Jr., 1953, Science, 118,733. Nelson, R.A. Jr., J. Jensen, I. Gigli, and N. Tamura, 1966, Immunochemistry 3,111. Nishioka, K., 1963, J. Immunol. 90, 86. Nishioka, K. and W.D. Linscott, 1963, J. Exp. Med. 118,767. Nishioka, K., R.F. Irie, T. Kawana, and S. Takeuchi, 1969, Int. J. Cancer 4, 139. Rothman, I.V., J.A. Gelfand, A.C. Fauci, and M.M. Frank, 1975, J. Immunol. 115, 1312. Taverne, J., 1957, Br. J. Exp. Pathol. 38,377. Tsuda, F., T. Takahashi, K. Takahashi, Y. Miyakawa, and M. Mayuni, 1975, J. Immunol. 115,834. Turk, J.L., 1958, Immunology 1,303.