Antigen and antibody detection by in vivo methods; A reevaluation of passive cutaneous anaphylactic reactions

Journal o f Immunological Methods, 14 (1977) 381--390 © Elsevier/North-Holland Biomedical Press

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A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY IN V I V O M E T H O D S ; A REEVALUATION OF PASSIVE CUTANEOUS ANAPHYLACTIC REACTIONS

NAOHIRO WATANABE ' and ZOLTAN OVARY

Department o f Pathology, New York University School o f Medicine, New York, N.Y. 10016, U.S.A. (Received 27 October 1976, accepted 31 October 1976)

INTRODUCTION

F o r d e t e c t i o n o f antigens a n d a n t i b o d i e s in vivo and in vitro m e t h o d s can be used. T h e actual t e n d e n c y is to use m o r e and m o r e in vitro m e t h o d s , h o w e v e r , in s o m e instances in vivo m e t h o d s are very useful a n d s o m e progress was a c h i e v e d in the p a s t few years; t h e r e f o r e , a r e e v a l u a t i o n o f in vivo m e t h o d s is t i m e l y . A r t h u s r e a c t i o n , s y s t e m i c a n a p h y l a x i s and active c u t a n e o u s a n a p h y l a c t i c r e a c t i o n s are o n l y of historical i n t e r e s t and for this reason will n o t be d e s c r i b e d . T h e use o f d e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n s is also s e l d o m e m p l o y e d as d e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n s are difficult to q u a n t i t a t e a n d their e x q u i s i t e specificity (i.e. the n e c e s s i t y to use n o t o n l y the s a m e carrier b u t also the s a m e d e t e r m i n a n t ) restricts t h e i r usefulness in antigen d e t e c t i o n . T h e o n l y r e a c t i o n still used for d e t e c t i o n o f antigens and a n t i b o d i e s in vivo is passive c u t a n e o u s a n a p h y l a x i s or the PCA r e a c t i o n . D e v e l o p m e n t and details of t e c h n i q u e w e r e d e s c r i b e d in p r e v i o u s m o n o g r a p h s ( O v a r y , 1 9 5 8 a , 1964). Reverse PCA r e a c t i o n s w e r e u s e d t o s t u d y the m e c h a n i s m of a n a p h y lactic r e a c t i o n s , b u t are n o t e m p l o y e d for d e t e c t i o n o f antigens or antibodies. In the p r e s e n t review, t h e r e f o r e , we shall o m i t historical details and we will describe o n l y t h o s e t e c h n i c a l details which allow one to p e r f o r m PCA r e a c t i o n s . H e t e r o l o g o u s a d o p t i v e c u t a n e o u s a n a p h y l a x i s m a y be used to d e t e c t m o u s e IgE a n t i b o d i e s . I t will also be d e s c r i b e d . BASIC ASPECTS

Passive c u t a n e o u s a n a p h y l a x i s (PCA) r e a c t i o n utilizes one of the f u n d a m e n t a l c h a r a c t e r i s t i c s o f the i m m e d i a t e t y p e h y p e r s e n s i t i v i t y r e a c t i o n s , i.e. the increase o f p e r m e a b i l i t y of the p o s t - c a p i l l a r y venules in the skin follow1Present address: Tokyo, Japan.

Department of Parasitology, Jikei University School of Medicine,

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ing antigen--antibody reaction. This local anaphylactic reaction, PCA, corresponds in every respect to systemic anaphylaxis (Ovary et al., 1963). However, it is easier to quantitate than systemic anaphylaxis, be it active or passive reaction. In order to produce the PCA reaction, antibody must get 'fixed' to the skin tissue constituents, probably to the mast cells. This process is termed sensitization. The only precise information we have on sensitization is that the structures necessary for this process are on the Fc fragment (Franklin and Ovary, 1963). Fc fragments differ according to the antibody class, and this is the reason why not every class of antibody is able to sensitize. For rabbit antibody it has been shown that the entire intact Fc region is necessary (Cebra et al., 1963), thus sensitization cannot be ascribed to one of the 'domains' of the Fc fragment (Ovary et al., 1976a). The term 'skin-sensitizing antibody' was used in the past, but this term is not quite correct as it is not the entire skin, but only some of its elements (mast cells) which 'fix' the antibody. Therefore it is better to avoid its use. The term ' h o m o c y t o t r o p i c a n t i b o d y ' was coined to group all the antibodies which sensitize the homologous species (Becker and Austen, 1966), in contrast to heterocytotropic antibody which can sensitize the heterologous species (e.g., rabbit IgG which can sensitize guinea pigs). This term also is incorrect and should be avoided as some antibody classes can sensitize both homologous and heterologous species (e.g., mouse IgE which can sensitize mice and rats) and the recent progress made it possible to know which class of antibody is involved. Therefore, it is better to name the class of antibody which sensitizes the homologous species, i.e. IgG, or IgE. Lastly, the term 'reagin' was also used to characterize in particular the human antibody responsible for immediate type hypersensitivity reactions. Today it is n o t necessary to use the term reagin. The progress made principally by the Ishizakas (1972) demonstrates that what was termed reagin is in reality an antibody of the IgE class. Before describing the technique of the PCA reaction, we will give some characteristics of the antibody classes involved in this reaction. Two antibody classes are important for PCA in the homologous species: IgE and IgG. IgE has the following properties: 1) Heat labile: does not sensitize the skin after heating at 56°C for 30 min to 4 h. 2) Persists in the skin for long periods of time (2--4 weeks at least) and the optimal sensitization period is long (48 h or longer). 3) Does not sensitize the skin after mild reduction and alkylation. 4) Higher molecular weight than IgG. IgG

The nomenclature is different according to the species; IgG, in the mouse (Nussenzweig et al., 1964; Barth and Fahey, 1965), IgG, in the guinea pig (Benacerraf et al., 1963; Block et al., 1963; Ovary et al., 1963), two sub-

383 classes have been described: IgGla and IgG:~ (Parish, 1970; Ovary and Warner, 1972), IgG a in the rat (Bloch et al., 1968) and the rabbit (Stux and Ovary, 1976). General characteristics of these antibodies are the following: 1) Heat resistance (after 2--4 h at 56°C and ability to sensitized the skin still remains). 2) Persist in the skin for short periods (3--8 days) and the optimal sensitization period is relatively short (1.5--16 h). 3 ) R e s i s t a n t to mild reduction and alkylation. 4) Almost the same molecular weight as other IgG classes and a sedimentation rate of 7S. IgE and IgG1 do n o t fix complement by the classical pathway. Finally, some complement-fixing IgG antibodies can be used for PCA reactions in heterologous species, f o r example, rabbit IgG antibody in guinea pigs (Ovary, 1964), mouse IgG2~ antibody in guinea pigs (Ovary, 1964), human IgG except for the IgG~ subclass in guinea pigs (Terry, 1964; Ovary et al., 1970), and dog IgG in guinea pigs (Rockey and Schwartzman, 1967). However, actually the most important example of the heterologous species is the use of rat for mouse IgE (Mota and Wong, 1969a; Ovary et al., 1975). It should be noted t h a t human IgE antibody is able to sensitize the m o n k e y , but because of the development of the radioallergosorbent test (RAST) (Wide et al., 1967) the actual tendency is to use in vitro techniques for human IgE detection. Another important point to emphasize is the sensitization period. With IgE antibodies, 24, or even better, 48 h, should be used in the homologous species. When mouse IgE is titrated in rats, a two-hour sensitization period can be used (Mota and Wong, 1969a; Ovary et al., 1975), although with 48 h the sensitivity increases (Ovary et al., 1975). The reason for this long sensitization period is not well understood. An enzymatic attack from the host cells on IgE was postulated (Bach and Brashler, 1975). With IgG antibodies the optimal period in most cases 3--4 h, except in mice where 1.5 h of sensitization is the best. PCA can be quantitated by the size of the reaction or by the endpoint dilution technique. The size of the reaction is roughly proportional to the antibody c o n t e n t (within certain limits). When very precise sensitization periods are observed and when the intradermal injection is perfect, the size of the diameter of the reaction can be used for quantitation. However, this technique requires many animals and very great skill. The more generally used m e t h o d is the endpoint technique, i.e. the last dilution giving a 5 mm colored spot in 2 out of 4 animals. It was thus shown that with rabbit IgG or guinea pig IgG1, the threshold in guinea pigs is less than 0.2 pg antibody protein/ml. For IgE even less is needed.

384 TECHNIQUE

Use of a dye T o visualize the increased p e r m e a b i l i t y , a d y e should be injected b e f o r e or at the t i m e o f the, challenge. T r y p a n blue, P o n t a m i n e sky blue, Evans blue, India ink and carmine m a y be used (Ovary, 1964). We use 1% Evans blue in 0.15 M NaC1 as it is n o t t o x i c and can be m a i n t a i n e d for weeks at r o o m temperature.

In tradermal injection I n t r a d e r m a l injections should be carefully d o n e and a wheal m u s t be prod u c e d . T h e back skin is p r e f e r e n t i a l l y used and n o injection should be placed above the shoulders or b e l o w the base o f the sacrum. While the skin of the back c o n t a i n s slightly less h i s t a m i n e t h a n t h a t o f the a b d o m e n ( F e l d b e r g and Miles, 1 9 5 3 ) , the r e a c t i o n s are generally b e t t e r d e l i n e a t e d on the back. Each injection should be separated by 1 cm or m o r e f r o m the others. No injection should be d o n e on the midline of the back (lower reactivity).

Sensitization period We already stressed t h a t the challenge by the antigen should be d o n e a f t e r a sensitization period; we shall give indications later for each species and for each m o d a l i t y o f PCA r e a c t i o n .

Challenge T h e t o x i c i t y o f the antigen should be previously c h e c k e d . S o m e antigens p r o v o k e non-specific or t o x i c reactions; w h e n injected i n t r a v e n o u s l y a general blueing of the animal occurs. S o m e t i m e s r e d u c i n g the a m o u n t o f antigen prevents t o x i c reactions; if n o t , it c a n n o t be used for PCA. It is i m p o r t a n t to n o t e t h a t only soluble antigens are suitable.

Reading of the reaction T h e r e a c t i o n can be read 30 min after challenge; a circular blue spot appears (if Evans blue is used) in a few m i n u t e s and reaches the m a x i m u m in a b o u t 5 t o 10 min. T h e r e a c t i o n can be observed and the d i a m e t e r can be m e a s u r e d f r o m outside o f the skin. F o r precise readings, the animals should be killed, skinned, and the r e a c t i o n should be m e a s u r e d on the inside o f the skin. S p o t s larger than 5 m m in d i a m e t e r are c o n s i d e r e d as PCA reactions. T h r e e modalities o f PCA were described: 1) i n t r a d e r m a l injection o f antib o d y i n t r a v e n o u s challenge with the antigen; 2) i n t r a d e r m a l injection o f antib o d y and i n t r a d e r m a l challenge with the antigen; 3) i n t r a v e n o u s injection of

385 antibody and intradermal challenge with the antigen. Each modality has advantages depending on the purpose. Modalities (2) and (3) are generally used only in guinea pigs.

Intradermal injection o f antibody and intravenous challenge with the antigen This modality is the most frequently used, as it is suitable for comparing antibody titers. Dilutions of antisera are injected intradermally and, after an appropriate sensitization period, the animals are challenged intravenously with the antigen mixed with the dye. The dye can be injected intravenously 10 to 15 min before the challenging injection of the antigen. This precaution is particularly indicated when low dilutions of antisera are used. In fact, antisera can be irritating especially at low dilutions, even a few hours after intradermal injection and produce non-specific increase of permeability. Details of the methods for the different species will be described below. PCA is not inhibited by antigen excess. It is a molecular reaction; therefore, the a m o u n t of antigen needed is in inverse proportion to its molecular weight. With insufficient amounts of antigen, the threshold of PCA reaction needs more antibody than with excess of antigen (Ovary, 1964). Therefore it is advisable to use great excesses of antigen (e.g., 0.5 mg of hen egg albumin), though 0.01 mg would have been sufficient even with 0.2 pg a n t i b o d y / m l (which is the threshold). Intradermal injection o f antibody and intradermal challenge with the antigen This m e t h o d is used preferentially when only small amounts of antibody and antigen are available. As the antigen must be injected exactly in the same site were the antiserum has been already injected, it is a good precaution to mark the site with a dermographic pencil. The proper sensitization period depends on the antibody class. The dye should be injected intravenously before the intradermal injection of the antigen. Intradermal injection of antigen alone is necessary as control, because some antigens might produce nonspecific reactions. A small reaction with antigen alone is frequent, but at the site of challenge the reactions must be substantially bigger to have significance. Intravenous injection o f antibody and intradermal injection with the antigen This m e t h o d is used especially in the comparison of cross-reacting antigens, or for small amounts of antigens. Antibody will be injected intravenously. After at least 24 h the dye will be injected intravenously, then the intradermal antigen challenge will be made. As controls: the diluent of the antigen will be injected, and also intradermal injection of the antigen in nonsensitized animals.

386 GUINEA PIGS Three h u n d r e d g albino guinea pigs should be used. The volume of intradermal injection should be 0.1 ml. If necessary, this can be increased to 0.2 ml. The best are 26 gauge, short-bevel needles (bevel turned down). An assistant must hold the animal when they are injected intradermally. The m a x i m u m n u m b e r of injection sites is sixteen. For challenge, the volume could be 1 or 2 ml. For intravenous injection the easiest veins are those which run on the hind foot. Those on the f o r e f o o t are more difficult. The feet should be shaved. A t ur ni quet can be used to make the veins m ore apparent. Twenty-seven gauge normal-bevel needles are the best, the bevel t u rn ed upward. The guinea pig can be held by an assistant or in a medium size rat holder (Metro Industries, Long Island City, N.Y.). Ear veins can also be injected if necessary (30 gauge needles are the best). As there is some variability between the animals, 3 to 5 guinea pigs should be used for each experiment. With rabbit IgG (Ovary, 1964), human IgG (Ovary et al., 1954, 1960, 1970; Terry, 1964), or mouse IgGua (Ovary et al., 1965), 3 to 6 h sensitization period is optimal. With guinea pig IgGt a somewhat longer sensitization period is o p t i m u m (IgGla 4 h, IgGlb 16--48 h) (Parish," 1970; Ovary and Warner, 1972). With guinea pig IgE 24 to 48 h are necessary, but longer periods can be used (Ovary et al., 1976b). Guinea pig IgGla can persist in the skin up to 4 days, guinea pig IgGl~ up to 7 days, guinea pig IgE for several weeks. The possibility of distinguishing IgG1 and IgE is the real actual advantage of PCA: a n t i b o d y which persist for weeks but c a n n o t sensitize after heating is certainly IgE. See com pl e t e reference in Ovary (1964), Catty (1969), Parish (1970) and Ovary et al. {1976b). RABBIT

Albino rabbits, such as New Zealand white, weighing approxi m at el y 2.5 kg are suitable for PCA. Shaved rabbits will be injected intradermally with 27 gauge needles (bevel upwards). Anaesthesia is not given but rabbit holders can be used. F o r t y injection sites are the m a x i m u m per rabbit. After the desired sensitization period the challenging antigen will be injected intravenously in the ear vein w i t h o u t anaesthesia. Two mg protein antigen per 2.5 ml mixed with the same volume of 1% Evans blue in 0.15 M NaC1 is usually used. Titration of antisera will be done using 2 or 3 rabbits per group. For rabbit IgE 48 to 72 h of sensitization period should be used (Zvaifler and Becker, 1966; Lindquist, 1968; Zvaifler and Robinson, 1969; Revoltella and Ovary, 1969b; Ishizaka et al., 1970; Stux and Ovary, 1976). For rabbit IgG~ 4 h is the o p t i m u m (Stux and Ovary, 1976). MOUSE Eight to 12 week-old female SJ L / J mice are the best for PCA reactions. Males fight t o o m uch and the skin is full of scars. Other white mice can also

387 be used; h o w e v e r , s o m e strains are less sensitive ( O v a r y et al., 1 9 7 5 ) . S h a v e d m i c e will be i n t r a d e r m a l l y i n j e c t e d using 27 gauge n e e d l e s (bevel t u r n e d u p w a r d s ) . T h e v o l u m e s h o u l d be 0.03 m l o f a n t i s e r u m . H e r e it is i m p o r t a n t to use 0.25 ml t u b e r c u l i n syringes. Six i n j e c t i o n p o i n t s are the m a x i m u m in t h e m o u s e . Shaving a n d i n t r a d e r m a l injections s h o u l d be d o n e u n d e r light e t h e r a n a e s t h e s i a . A f t e r a sensistization p e r i o d , 0.2 t o 0.3 m l o f a n t i g e n a n d Evans blue m i x t u r e (final c o n c e n t r a t i o n o f Evans blue s h o u l d be 0.5%) will be i n t r a v e n o u s l y i n j e c t e d f r o m t h e tail vein using a m o u s e h o l d e r w i t h o u t anaesthesia. T h e r e t r o - o r b i t a l sinus u n d e r w e a k a n a e s t h e s i a can also be used. T h e dose o f a n t i g e n s h o u l d be a r o u n d 0.5 mg. T w e n t y to 30 m i n a f t e r the challenge t h e m i c e are killed, s k i n n e d a n d t h e n t h e r e a c t i o n can be r e a d on t h e inner side o f t h e skin, a n d g r a d e d f r o m 0 to 4+ a c c o r d i n g to the d i a m e t e r ( O v a r y , 1 9 5 8 b ) . T h r e e t o 6 m i c e s h o u l d be used in e a c h e x p e r i m e n t . T h e e n d p o i n t is t a k e n w h e n at least 1+ r e a c t i o n is p r o d u c e d in t w o o u t o f t h r e e animals. F o r IgG1 1.5 to 2 h is the o p t i m a l sensitization p e r i o d ( M o t a et al., 1 9 6 8 ; O v a r y et al., 1975). F o r IgE a n t i b o d y the o p t i m a l sensitization p e r i o d is 48 h or m o r e ( M o t a a n d P e i x o t o , 1 9 6 6 ; M c C a m i s h , 1 9 6 7 ; M o t a et al., 1 9 6 9 b ; R e v o l t e l l a a n d O v a r y , 1 9 6 9 a ; P r o u v o s t - D a n o n e t al., 1 9 7 2 ; O v a r y et al., 1975). RAT

A l b i n o rats, such as S p r a g u e - - D a w l e y or Wistar strains, weighing 2 5 0 to 300 g are t h e b e s t f o r PCA. Well-shaved rats can be i n t r a d e r m a l l y i n j e c t e d TABLE 1 PCA reactions in homologous and heterologous species. Species

Class of immunoglobulin

Optimum sensitization period Homologous species

Guinea pig Rabbit Mouse Rat Human

IgGla IgGlb IgE IgG IgG a IgE IgG 1 IgG2a IgE IgG a IgE IgG 1 IgG 3 IgG4 IgE

Heterologous species

4h 16--48 h 48 h or longer 3--6 h (guinea pig) 4h 48 h or longer 1.5--2 h 48 h or longer 4h 48 h or longer

see text

3--6 h (guinea pig) 24 h (rat) see text 3--6 h (guinea pig) 3--6 h (guinea pig) 3--6 h (guinea pig) 48 h or longer (monkey)

388 with 0.1 ml volume of antisera using 26 gauge short-bevel needles (bevel downwards). Shaving and intradermal injection should be done under light ether anesthesia. Thirty points are the maximum for intradermal injection of the rat. After an appropriate sensitization peroid, the rats can be challenged intravenously with about 1 ml of antigen and Evans blue mixture (in equal volumes) from the tail vein, foot vein, or the dorsal vein of the penis under light ether anesthesia. One mg protein antigen in 0.5 M NaC1 is usually used. A n t i b o d y titers will be determined using 2 to 4 rats. The sensitization period should be 4 h for rat IgG~ (Bloch et al., 1968; Morse et al., 1968, 1969), and 2 days or longer for rat IgE (Binaghi and Benacerraf, 1964; Mota, 1964; Stechschulte et al., 1970; Tada et al., 1975). Mouse IgE detection and titration is the most important use of the rat for PCA (Mota and Wong, 1969a; Ovary et al., 1975), and the same procedure is used as that when rat IgE is titrated. For the detection of mouse IgE in rat skin, the sensitization period may be 2 h; the titers are then the same as in mice with 1--2 day sensitization period. However, longer sensitization increases the sensitivity of the reaction (Ovary et al., 1975). The reading is generally done as in guinea pigs. OTHER SPECIES

Human The first detection of Prausnitz (the Prausnitz because of the danger of sensitive in vitro technique

antibody in vivo was made in 1921 by Carl and Ktistner: PK reaction). Today, especially hepatitis and because of the development of the (RAST), this test is not used anymore.

Monkey PCA reaction in m o n k e y skin has been described using m o n k e y sera (Malley et al., 1968; Weiszer et al., 1968) and human sera (Layton et al., 1961; Buckley and Metzgar, 1965). The cross-reaction between human IgE and monkey IgE has been demonstrated (Ishizaka and Ishizaka, 1968; Ishizaka et al., 1969). However, here again, the in vitro RAST method makes in vivo detection obsolete.

Dog PCA reaction in the dog has also been studied (Patterson and Sparks, 1962; Rockey and Schwartzman, 1969). (For more detailed studies see Block, 1967.) In other species PCA is not frequently used. It was investigated in cows (Hammer et al., 1971), sheep (Hogarth-Scott, 1969), pigs (Barratt, 1972), and chickens (Celada and Ramos, 1961; Kaplan and Freeman, 1969).

389 ADOPTIVE CUTANEOUS ANAPHYLAXIS This m e t h o d is b a s e d on the f a c t t h a t a n t i b o d y - p r o d u c i n g cells t a k e n o u t f r o m i m m u n i z e d a n i m a l s m i g h t survive a n d p r o d u c e a n t i b o d y w h e n t h e y are injected i n t o a n o n - s e n s i t i z e d r e c i p i e n t ( R o s e n b e r g et al., 1 9 5 8 ; B a t t i s t o , 1 9 6 6 ) . This m e t h o d was p r i n c i p a l l y used t o d e t e c t m o u s e IgE f o r m a t i o n ( K i n d a n d M a c e d o - S o b r i n h o , 1973). This m e t h o d has also s o m e p o t e n t i a l ities f o r s t u d y i n g e s t a b l i s h m e n t o f t o l e r a n c e . We will n o t describe the p r e p a r a t i o n of cell s u s p e n s i o n as it is a r o u t i n e p r o c e d u r e . A w a s h e d suspension of l y m p h o i d cells o b t a i n e d f r o m i m m u n i z e d d o n o r s (mice) is injected i n t r a d e r m a l l y in rats (106 to 107 cells in 0.1 ml are suitable). T w e n t y - f o u r h o u r s later the rats are c h a l l e n g e d w i t h the antigen a n d d y e as d e s c r i b e d a b o v e for PCA. As c o n t r o l s : 1) cells f r o m n o n - s e n s i t i z e d animals can be injected in o n e site, a n d 2) the positive c o n t r o l , i.e. I g E - c o n t a i n i n g s e r u m o f t h e s a m e s p e c i f i c i t y , can be injected i n t o a n o t h e r site. T h e r e a d i n g o f the r e a c t i o n is d o n e as f o r PCA; s o m e q u a n t i t a t i o n is possible c o r r e l a t i n g the d i a m e t e r o f the r e a c t i o n a n d n u m b e r o f cells injected. FINAL CONSIDERATION Specific anti-IgG~ sera are difficult t o o b t a i n a n d t h e r e f o r e in vitro m e t h o d s are rarely used f o r d e t e c t i o n o f I g G l . F o r the s t u d y o f IgGl, PCA on h o m o l o g o u s species is still v e r y useful. IgE m y e l o m a p r o t e i n s w e r e n o t y e t o b s e r v e d in m i c e a n d guinea pigs; h o m o l o g o u s PCA r e a c t i o n s in guinea pigs a n d h e t e r o l o g o u s PCA r e a c t i o n s in rats w i t h m o u s e a n t i s e r a r e m a i n the m o s t useful t o o l s for d e t e c t i o n a n d q u a n t i t a t i o n o f the r e s p e c t i v e IgE antibodies. REFERENCES Bach, M.K. and J.R. Brashler, 1975, Fed. Proc. 34, 1001. Barratt, M.E.J., 1972, Immunology 22,601. Barth, W.F. and J.L. Fahey, 1965, Nature 206, 730. Battisto, J.R., 1966, J. Immunol. 97,939. Becker, E.L. and K.F. Austen, 1966, J. Exp. Med. 124,379. Benacerraf, B., Z. Ovary, K.J. Bloch and E.C. Franklin, 1963, J. Exp. Med. 117,937. Binaghi, R.A. and B. Benacerraf, 1964, J. Immunol. 92,920. Bloch, K.J., F.M. Kourilsky, Z. Ovary and B. Benacerraf, 1963, J. Exp. Med. 117,965. Bloch, K.J., 1967, Prog. Allergy 10, 84. Block, J., H.C. Morse and K.F. Austen, 1968, J. Immunol. 101, 650. Buckley, R.H. and R.S. Metzgar, 1965, J. Allergy 36, 382. Catty, D., 1969, Monogr. Allergy 5, 1. Cebra, J.J., B. Bloom, H. Jaquet, Z. Ovary, 1963 in: 143rd Meeting of the American Chemical Society p. 3A. Celada, F. and A. Ramos, 1961, Proc. Soc. Exp. Biol. Med., 108, 129. Feldberg, W. and A.A. Miles, 1953, J. Physiol. 120, 205. Franklin, E.C. and Z. Ovary, 1963, Immunology 6,434. Hammer, D.K., B. Kickhofen and T. Schmid, 1971, Eur. J. Immonol. 1, 249.

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