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ENZYME-LINKED IMMUNOASSAY OF RICIN

N. KODA, T. SHIBATA and K. MOCHIDANational Institute of Hygienic Sciences, Osaka Branch, 1-1-43, Hoensaka,

Higashi-Ku, Osaka, Japan

(Acctpted jor publication 27 May 1980)

N. KoJe, T. SHIHATA andK. MOCN>DA. Enzymo-linked immunoassay of ricin . Toxicon 18, 611-618,1980.-Rabbit (anti-ricin) antibodies (reduced IgG and Fab' fragments) were conjugated with ß-o-galactoaidase using N,N'-o-pheaylenedimaleimide as a coupling reagent. Immunoassay ofricin withrabbit (anti-ricin) Fab'-ß-o-galactosidase comple: was more sensitivethan that with reducedIgG-ß-o-gelactosidax complex. Using the rabbit (anti-ricân) Fab'-ß-o-galacto®dase oamplex, it was possibleto deta~mine as little as 10fmole of ricin by the sandwich method technique. However, leas sensitivitywasobservedwhen thismethodwasutilized for the determinationofricinaddedto rabbit body fluids,and the smallest amount ofricin which could be assayed was 100 fmole. Results of immunossay ofooncanavalin A(Con A) using the rabbit (anti-Con A) Fab'-ß-o-galactosidase complex were similarto that in the ricin assay .

INTRODUCTION

IN rREVIOUS papers, we reported that ricin, which shows strong toxicity but very weakhemagglutiaating activity, had powerful inflammatory effects on rabbits (pyrogenicity,hemorrhagic activity, etc.). These effects were different from those ofGram-negative bacterialendotoxin andconcaaavalin A (Con A) which also showed inflammatory effects (KOJA et al .,1974, 1975, 1977). In order to examine the effects of ricin in detail, including its tissuedistribution and elimination, it is necessary to improve the method for routine assay ofricin .

Enzyme-linked immunossay has recently been proposed for the microanalysis of lowlevels of antigens sad antibodies in biological fluids . The sensitivity of this method iscomparable to that of radioimmtmoassay and the sandwich method technique is especiallysuitable for the determination of macromolecular antigens (ScHUUIes and VAx WesMAN,1977). The present paper describes the application ofrabbit antibody (reduced IgGandFab'fragments)-ß-Irgalactosidase complexes, developed by KATO et al. (1976), for immunoassayofricin and Con A. It is possible to determine as little as 10 fmole of each by the sandwichmethod.

MATERIALSAND METHODSRicin and ricinus hemagglutinin were extracted from castor bean (Ricinus comnwnLs L.) and puriflod using

Sephadex G-100 sad DEAF allukme column as described previously (KOJe and Moctt>De, 1974). They werehomogeneous on polyaaylamide gel elearophoresia and immunoelectrophoreais. The component polypeptidechains of ricin were prepared by the method ofOt.snt>3s and Pgt~ (1973) . ConAwas from Mibs Laboratories, Inc.,Indiana . ß-o-galactosidase (EC 32123. from E.ooli)wasfromBoehriuger Mannhmm, Mannheim,Germany, N,N'_o-p~heayleaedimabimide from Akh-ich Chemical Co, Inc. Milwaukee, wis, Sephade: G-25, G-150 and Sepharase4B from Pharmacdi Fine Chemicah, Uppsala, Sweden. Other chemicals were of analytical grade.

Preparation of anNibodies to ricin and Con ARidn dissolved is saline solation was injected s.c . in a dose of 10 I+Bng into rabbits at weekly intervals during a

periodof2months.ConA dinolved in saline solation was injected i.v . in adoseof1 mg,/kg into rabbits accordingto

61 2

N. KOJA, T . SHIBATA and K . MOCHIDA

theschedule described above . Each serum was collected 1 wcek after the last injection. The IgGfractions from theseaatisera and nonspecific rabbit serum were isolated by a wmbination of sodium sulfate precipitation (KExwtcx,1940) and DEAE cellulose chromatography (LEVY and SoRaER, 1960).Preparation ofthe rabbit (anti-ricin and anti-Con A) Fab'-ß-o-galactosidase complexesAbout 20 mg of the IgG fractions in 0~1 M sodium acetate buffer (buffer B)was incubated with 0~4 mg pepsin in a

final volume of 1 ~5 ml at 37°C for 16 hr. After incubation, the reaction mixture was adjusted to pH 8 O with1 N NaOH and F(ab')~ fragments were separated from other components on a Sephadcx G-150 column(1 x 40 cm),with 0~ 1 M sodiumborate buffer, pH 89(buffer B). F(ab')z fragments wereconcentrated in a Collodionbag under a mild vacuum and dialyzed against buffer B . About 10 mg ofF(ab'), fragments in 2 ml ofbuffer B wasreduced by incubation with 0~2ml of 10mM 2-mercaptoethylamine at 37°C for 90 min as described by NtsoxoeFand RIVERS (1961).TheFab' fragments were separated fromcompoundsoflower mol . wton a Sephadex G-25 (1 x 40cm)with buffer

B. About 3 mg of Fab' fragments in 2 ml of buffer B was added dropwise to 1 ml of saturated solution of N,N'-o-phenykneditnaleimide (about075 mM) in buffer Bat4°Cfor20 min. Unreacted N,N'-o-phenylenedimaleimide wasremoved on a column of Sephadex G-25 (1 x 40 cm) with buffer B.About 1 mg of makimido-ttrated Fab' fragments was incubated with 20 fel (0-075 mg) of ß-o-galactasidase at 30°C

for 20 min . After neutralization with 1 N NaOH, 20 !tl of5~ bovine serum albumin and 2 pl of 1 mM MgC12 wereadded to stabilize the enzyme activity . The mixture was kept at 4°C for 24 hr and then applied to a column ofSepharose 4B (1 x 40cm) to separate Fab'-ß-o-galactosidase complex from Fab' fragments which were notcoupled to the enzyme. The mixture on the column was eluted with 0-01 M sodium phosphate buffer (pH 7~0)containing 0~1 M NaCI, 1 mM MgC12, 0~1% bovine serum albumin (buffer A) and 1 ml fracdona were collected .

Preparation of the rabbit (anti-ricin and anti-Con A) reduced IgG-ß-o-gakctostdase complexesAbout 10 mg of the IgG fractions in boner H were reduced by incubation with 0~2 ml of 10 mM 2-

mercaptoethylamine at 37°C for 90min and the reduced IgG was introduced with the tnaleimide residues and thenconjugated with ß-o-galactosidase by the same method as described above.

Preparation of the rabbit (anti-ricin) IgG- and (anti-Con A) IgG-solid phaseThe rabbit IgG-solid phases were prepared bythemethodofHAMAGUCHi et al . (1976) . Silicone rods (2~5 x 4 mm)

immersed in 0~1~ solution of rabbit (anti-ricin) IgG or (anti-Con A) IgG were incubated at 4°C for 24 hr . The IgGwas immobilized on silicone rods by physical adsorption .

Sandwich method techniqueThe rabbit (anti-ricin or aziti-Con A)IgG-solid phase wereincubated with various amounts of ricin or ConA with

shaking at 37°C in a finalvolume of0 ~ 15 mlofbufferA . After 4 hr incubation, the silicone rodswerewashed twicewith1 ml of buffer A and incubated with the corresponding Fab'- or reduced IgG-ß-o-galactosidase complexes in avolume of015 ml ofbuffer A with shaking at 37°C for 6 hr. The silicone rods were then washed twice with 1 ml ofbuffer A and the enzyme activities bound to the silicone rods were measured .

Assay ofß-o-galactosidase activityEnzyme activity wasdetermined by themethod ofK~TO et al. (1976) . Samples wereincubated with 3 x 10 - ` M 4-

methyl-umbellikryl-ß-o-galactoside at 25°C for 10 min in a final vohtme of O~15m1 of buffer A . The reaction wasterminated by adding 2'S ml of 0~1 M glycine-NaOH buffo, pH 103 and the fluorescence intensity of the 4methylumbelliferone released was measured . The wavelengths used were 360 nm for excitation and 450 nm foremissioa analysis . One unit of enzyme activity is deSned as the amount which is able to produce 1 pmole of 4-methylumbelliferone per min.

Assay ofsulJhydryl groupsSulmydryl groups were determined by the method ofGRnssern and MURRAY (1967) using4,4'-dithiodipyridine.

Adsorption ofthe rabbit (anti-ricin or anti-Con A) Fab'- and reduced IgG-ß-o-galactosidase complexeson ricin- orCon A- coupled Sepharose 4B a,~nity columnsAkin or Con A~oupled Sepharose 4Bwere prepared bythemethod ofP. CtiATREC~S (1970) . Each ofthe rabbit

(anti-ricin or anti-Con A) Fab'- and reduced IgG-ß-o-galactosidase complexes dissolved in buffer Awas applied tothe corresponding Sepharose 4B aeinity column (0"4 x 5 cm), and eluted with buffer A at a flow rate of about0~1 ml/Itr .

RESULTS AND DISCUSSION

Preparation ofthe rabbit (anti-ricin and anti-Con A) antibodies (Fob' and reduced IgG)-ß-o-galactosidase complexes.Fab' and reduced IgGß-n-galactosidase complexes used in this study were prepared by use ofN,N'-o-phenylendimaleimide and ß-D-galactosidase following the method of KATO et al.

Enzymo-linked Immunoassay of Ricin

613

(1976). In order to minimize oxidation of sullhydryl groups of Fab' fragments and reducedIgG, the buffer used for gel filtration and the N, N'-o-phenylenedimaleimide used for oomplex-forming were kept under an atmosphere of nitrogen .Theamount ofIgG andFab' fragments were calculated from their absorbance at 280nm

using Ei ~ = 15 (PALMER and NtSONOFF, 1964) and 148 (Mnxnv and NÎsoNOr~, 1963)respectively . The amount of sullhydryl groups were calculated from their absorbance at324nm using Ei~ = 19,800 (GxnsSE~-n and MURRAY, 1967). The average numbers ofsullhydryl groups were 098 and 130 per molecule for the (anti-ricin) Fab' fragments andreduced IgG and were 097 and 1~37 per molecule for the (anti-Con A) Fab' fragments andreduced IgG. Thenumbers ofmaleimide residues introduced were071and070for the (anti-ricin) Fab' fragments andreduced IgG, and were 075and073permolecule for the (anti-ConA) Fab' fragments and reduced IgG.

After the dimaleimido-treated Fab' fragments and reduced IgGwere conjugated with ß-n-galactosidase (11 ~2 SH/molecule), the complexes were chromatographed on a column ofSepharose 4B. Samples ofeach fraction were diluted 500-fold with bufferAandthe enzymaticactivity of 10 pl of the diluted sample was measured. The fractions containing maximumactivity were pooled for the assay of ricin or Con A.The free ß-o-galactosidase content in the preparation of the Fab' and reduced IgG-ß-n-

galactosidase complexes was examined by immunoa~nity chromatography. Each of theFab'- and reduced IgG-ß-n-galactosidase complexes was applied to the correspondingSepharose 4B amity column and eluted with buffer A. As controls, free enzyme andnonspecific Fab'-enzyme complexes were chromatographed in the same manner. Theenzyme activity in each sample applied to acolumn was2200 units in a volume of0~ 1 ml andthe enzyme activity in the eluate was measured.For the (anti-ricin) Fab'- and reduced IgG-enzyme complexes containing maximum

activity, about 51 and 55% of the activity applied to the amity column were recovered . Forthe (anti-Con A) Fab'-and reduced IgG-enzymecomplexesabout 32 and 40'/ ofthe activityapplied to the column were recovered. In contrast, almost all the enzyme activity ofthe freeenzyme and nonspecific Fab'-enzymecomplexwasrecovered (98 and 9T/)from the appliedcolumn . The results indicate that about 500'/ofß-o-galactosidasewasconjugated with atleast one mole of Fab' fragments and reduced IgG.Assay ofricin and Con A in buffer A with the rabbit (anti-ricin and anti-Con A) antibodies(Fab' and reduced IgG)-ß-n-galactosidase complexes

In order to assess the optimum amount ofthe Fab'- and reduced IgG-enzyme complexesfor assay, the rabbit (anti-ricin) IgG- or (anti-Con A) IgG-solid phase were incubated with(0~1 and 1 ~0 pmole) and without ricin or Con A, and then with various amounts of thecorresponding antibodies (Fab' and reduced IgG)-ß-n-galactosidase complexes using thesandwich method . After measurement ofenzyme activity, the ratio of the activities bound tothe solid phase with/without ricin or ConA were calculated (Table 1) . Theenzyme activitiesbound to the solid phase tended to increase in proportion to the amount ofcomplexes addednot only in the presence ofricin or ConAbut also in its absence, and the amount ofthe Fab'-enzyme complex which gave the maximum ratio was smaller than that of the reduced IgG-enzyme complex. The enzyme activity which gave the maximum ratio was5500 and 6000 Ufor the (anti-ricin) Fab'- and reduced IgG~nzyme complexes respectively while for the(anti-Con A) Fab'- and reduced IgG~nzyme complexes it was 4500 and 5000 Urespectively.The larger the ratio was, the higher the sensitivity ofthe immunoassay. Therefore, ricin or

Con A was assayed by the sandwich method in buffer A with optimum amount of each

614

N. KOJA, T. SHIBATAand K. MOCHIDA

TABLE 1. CORRELATION BETWEEN THE ADDEDAMOUNT OF THE RABBIT (AN'rI-RICIN AND ANTI-CON A) ANTIBODIES(Fab' ANDREDUCED IgG}-ß-D"GALACrOSIDASECOMPLEXPS ANDTHEENZYME ACf1VITIES BOUNDTOTHE SOLID PHASE

IMMOBILIZING THE CORRESPONDING RABBIT IgG

' Amount of ricin or Con A added : (a) none, (b) 0~1 pmole, (c) 1 pmole.t The enzyme activities bound to the solid phase are shown as a mean of two experiments.

antibody~nzyme complex (Fig . l). The smallest amount of ricin and Con A which could beassayed were 10 fmole each by use of these complexes and steeper dose-response curveswere observed in the assay of Con A.

03

arnourr5, pmde~assay tube

FIG. 1 . SOLID PHASE SANDWICH IMMUNOASSAY OF RICIN ANDCON A IN BUFFER A.Ricin or Con A was dissolved in a final volume of 015ml of buffer A. The enzyme activity of theantibody-ß-o-galactosidase complexes added to individual assay tubes was 6000 U for the rabbit(anti-ricin) IgG~llzyme complex(~-~~5500 Ufor the rabbit (anti-ricin) Fab'-enzyme complex(O-O~ 5000 Ufor the rabbit (anti-Con A) IgG~nzyme complex (/-/) and 4500 U for therabbit (anti-Con A) Fab'~nzyme complex (p-p). The activity of free ß-o-galactosidase was2500 U (D-O)" Each point represents the mean tS.D . of five experiments. One unit of enzymeactivity is defined as theamount whichwasable toproduce 1 pmok of4methylumbelliferone permin

from 4-methylumbelliferyl-ß-o-galactoside.

Complexadded

Enzyme activity of complexes (U/assay

bound

(a)' ro)' (~)'

tube)

ro/a)

Ratio

(~/a)

(Anti-ricin}IgG-enzyme 5000 1~4t 1 ~8 5~2 1 ~3 3~76000 1~6 2~1 6-0 13 3~87000 2-0 2~4 7~0 1~2 3~5

(Anti-ricin}Fab'~nzyme 4000 1~6 2~1 7~5 1~3 4~75500 1~6 b4 9~0 1~5 5~66000 1~8 2~7 9~4 1~5 5~2

(Anti-Con A}IgG~nzyme 3500 1~1 1~8 3~8 li 3~55000 1~5 2~5 8-0 1~7 537000 2-0 3~0 82 1~5 4~1

(Anti-Con A}Fab'~nzyme 2000 0~8 2~1 8~2 2~6 10~34500 1~2 32 16~7 2~7 13~96500 1~7 4~2 21~0 2~5 12~4

Enzymo-linked Immunoassay of Ricin

615

Amounts, pmole~assay tube

FIa. 2 . COMPARISONOF SOLTD PHASE SANDWICHIMMUNOASSAY OFRICINUS HEMAOGLUTININ ANDTHECOMPONENT POLYPEPTH)E CHAINS OF RICIN WITH RICIN.

To examine the specificity of the rabbit (anti-ricin) Fab'-enzyme, the rabbit (anti-ricin) IgG solidphase and rabbit (anti-ricin) Fab'-ß-o-galactosidase complex (5500U) were used for the assay ofricinus hemagglutinin (O-O~Achain (/-/), B chain (~-~) ofricin and ricin (p-p).

Each point represents the mean tS.D . of three experiments.

Spec city of the rabbit (anti-ricin) Fab'-ß-n-galactosidase complexTo examine the specificity of the rabbit (anti-ricin) Fab'-enzyme, ricinus hemagglutinin

and the component polypeptide chains of ricin in buffer A were assayed by the sandwichmethod (Fig . 2) . The smallest amount which could be assayed were 0~1, 0~1 and 1 pmole forricinus hemagglutinin, A chain (the smaller peptide chain) and B chain (the larger peptidechain) ofricin, respectively. The percentage cross-reaction ofthe Fab'-enzyme complex wascalculated at 50'/ inhibition of immune reaction by the method of ABRAHAM (1969) . Theresults indicate that the percentage cross-reaction measured with the (anti-ricin) Fab'-enzyme complex were 10 ~9, 3 ~5 and 100 for ricin Achain, Bchain and ricinus hemagglutinin .Therefore, it was considered that the cross-reaction in this groupwas low and the specificityof the Fab'~nzyme complex for ricin was high.

Assay ojricin in rabbit serum and cerebrospinalfluid with the rabbit (anti-ricin) Fab'ß-~galactosidase complex

Ricin dissolved in rabbit body fluids such as serum and cerebrospinal fluid was assayed.These fluids are important samples to investigate because ofthe possibilityofpassage ofricinthrough the intact blood-brain barrier which is the first barrier for pyrogenic substance suchas Gram-negative bacterial endotoxin (Moct-uDn et aL, 1974).

Ricin solution in buffer A was diluted with various amounts ofserum and cerebrospinalfluid until the total volume was 015ml, and assayed by the method described above. Asshown in Fig. 3, the smallest amount of ricin which could be assayed was 100 fmole. Thesensitivity of the assay decreased corresponding to the added amount of body fluids. .When10 pmole of ricin was assayed in 100 ~l of body fluids, the enzyme activity in serum sampleand cerebrospinal fluid sample showed about 48 and 62% decreases from that in buffer A.

616

N.KOLA, T. SHIBATA and K. MOCHIDA

da0v0tUG15C70

15

10

5

0

i"

FIG . 3. INHIBITORY EFFECTS OF BODY FLUIDS ON IMMUNOASSAY OF RICIN.Ricindissolved in buffer was diluted with indicated amounts ofrabbit cerebrospinal fluid and serum,and assayed with the rabbit (anti-ricin) Fab'-ß-o-galactosidase complex (5500 U). Each point

represents the mean fS.D. of three experiments .

Similar tendencies were observed in the assay of Con A.It is well known, particularly in radioimmunoassay experience, that serum and plasma

may interfere with the immune reaction . An additional problem in enzyme immunoassay isthe possibility that fluids affect the enzyme reaction rates. However, in the sandwich method,these body fluids were removed from the assay medium prior to the measurement ofenzymeactivity, so itcan be assumed that theinhibition ofthebodyfluidson the enzymereaction was

a'

m0

wO

1111111

I I I I IIIII

I

I 11111110.05 O.I 0.5 I

5 10

loo

F~cin/assay tube, pRwle

O 0.2 0.4 0.6 O.B I

Galactose/assay tube, prlwle

FIG. 4. INFLUENCE OF GALAC OSE ON IMMUNOASSAY.Ten pmole of ricin was assayed with indicated amount ofgalactose in a final volume of0" 15 ml ofbuffer A using the rabbit (anti-ricin) Fab'-ß-D-galactosidase complex (5500 U). 100'/ in this

experiment is 43 U and each point represents the mean tS.D. of flue experiments.

negligible. From the amity of ricin for certain types of sugars, the interference with theimmune reaction in this method may be explained by binding sites of ricin to the antibodymolecule previously occupied with glycoprotein or glycopeptide in body fluids. Theinhibitory effects of galactose, a carbohydrate moiety of glycoprotein or glycopeptide, onhemagglutinin reactions are well known (GOLDSTEIN et al., 1965) ; therefore, the possibleinhibition ofthe assay reaction by galactose was investigated. Tenpmole ofricin was assayedwith various amounts ofgalactose in a final volume of0~15 ml ofbuffer A(Fig. 4). As expected,the enzyme activities bound to the silicone rods decreased corresponding to the amount ofgalactose.

In order to remove interfering substances in body fluids, serum and cerebrospinal fluidsamples containing 10 pmole of ricin were dialyzed against buffer A, for substances of lowmol. wt, and fractionated with ammonium sulfate to obtain macromolecule substances (Fig.5) . Theenzyme activities in the dialysate were approximately equal to that measured in bodyfluids before dialyzing, but the enzyme activities in the supernatants after treatment with 10and 20'/ ammonium sulfate precipitation decreased slightly because of coprecipitation ofricin with macromolecule substances in body fluids . Therefore, further investigations areneeded to minimi~E the difference between the assay results in body fluid samples and inbuffer A. At present, the best way may be the use of the corresponding body fluid as astandard solution .

This study indicates the feasibility ofimmunoassay for ricin and Con Ain vitro . Attemptsto apply this method to the determination ofricin and Con A in rabbit's body fluids after itsadministration will be described in the near future.

Acknowiedgeinent-The authors thank Professor E. ISFIIKAWA of Medical College of Miyazaki, Miyazaki, for histechnical advice .

Pretreatments(10 pmole of ricin)

Buffer A

CSF

Dialyzed CSF

Sup't of ( NH4 )z SOq

(10q0)

Sup't of (NH4 1 zS04

Serum

Dlolyzed serum

Sup' t

of

l NH4 )zS04

( IO °Y0)

Sup't Of

l NH4 )z SO4 (20 9'e1

Enzyme-linked Immunoassay of Ricin

61 7

(209'01

~- D- Galactosidase activity, U/assay tube

FIa. S. IMMUNOASSAY OF AKIN IN BODY FLUIDS AFTEA VARIOUS TREATMENTS.Ten pmoleofricindissolved in 50 pl of bufferA wasdiluted with 100 pl of rabbit cerebrospinalfluidorserum anddialyzed againstbufferAor subjected to 10and 20'/ ammonium sulfateprecipitation.Thedislyzate and the supernatants wero assayed with the rabbit (anti-ricin) Fab'-ß-o-galactosidasecomplex (5500U) by the sandwich method. Eaeh point reprnsenta the mean tS.D . of flue

experiments.

618 N. KOJA,T. SHIBATA and K. MOCHIDA

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polyacrylamide beads. J. 6b1. Crepe. 245, 3059.Got.nsrEnv, L, HOLLF1tMAN, C. E. andSMmi,E. E. (1965) Protcin~arbohydrate interaction. II. Inhibition studies

on the interaction ofconcanavalin A with polysaccharides. Bbcranistry 4, 876.GR~ssE~-n,D. R. andMuRRw,Jr., J. F. (1967) Determination ofsulfhydrylgroupswith 2,2- or 4,4~ithiodipyridine .

Arcrs Bbdrare. Bbprys. 119, 41 .HAMAGUCtü, Y, Kem, K., ISHIKAWA, E., KoenvtisHt, K. and KATUNUMA, N. (1976) Enzymo-linked sandwichimmunoassay ofmacromolecular antigens usingtherabbit antibody-loadedsilicone piece asa solid phase. FEESLeft, 69, 11 .

K~~ro, K., Fuxt,H., HAMAGUCHI,Y. andISHIKAWA, E. (1976)Enzymo-linked immunoassay :conjugationofthe Fab'fragnent of rabbit IgG with ß-o-galactosidase from E. coH and its use for immunoassay. J. Immun. 116, 1554 .

KEwtcx, R. A. (1940) The serum proteins in multiple myelomatosis. Bbcrem . J. 34, 1248 .Kw~,N. and Moctüne, K. (1974) Studies ofthe physiological activity of ricin from castorbean . I. Purification andPyrogmic activity of ricin. J. ryg. Creme. 20, 204.

KOJ~,N., Mocxro~, K. and YOSFIroA,M. (1975) Studies ofthe physiological activity of ricin from castor bean. III.Vascular permeability of peerified ricin. J. hyg. Crem . 21, 337.

KO~~,N., MOCFIroA, K. and YOSHroA, M. (1977) Studies of the physiological activity of ricin from castor bean . Iy .Pyrogenic activity and effects on rabbit erythrocytes acetylcholinesterase of concanavalin A and ricin. J. ryg.Crem. 23, 27 .

LEVY,H. B. and SoRBER,H. A. (1960) A simple chromatographic method forpreparation ofgammaglobulin . Prac .Soe. exp. Bbl. Med. 103, 250.

M~rmv, W. J. and NtsotvoFF, A. (1963) Effect of reduction of several disulfide bonds on the properties acidrecombination of emivalcmt fragments of rabbit antibody . J. 6b1. Chem . 238, 206.

Mocwro~, K.,Oo~we, Y. and Kruvoe, S. (1974) Studies on thepyrogenicfactorappearing in thecerebrospinalfiuidof febrile rabbits. Fol. Prarnwc. Jap. 70, 359.

NiSONOFF, A. and RtvERS,M. M. (1961) Recombination of a mixture ofunivalent antibody fragments of differentspecificity. Arcrs Bbcran . Bbprys . 93, 460.

OtsxES, S. and Ptet, A. (1973) Different biological properties of the two constituent peptide chains of ricin, a toxicprotein inhibiting protein synthesis. Bbcremistry 12, 3121 .

PALMER, J. L. and NtsoxoF-~,A. (1964) Dissociation ofrabbit y-globulin into half-molecules after reduction ofonelabile disulfide bond . Biceremistry 3, 863.

SCIiUURS, A. H. W. M. and VAN WP.EMAN, B. K. (1977) Enzymo-immunoassay . Clip. Cram. Acts 81, 1.