29

SOME PHYSICAL PROPERTIES OF DIPHTHERIA ANTITOXICHORSE SERA.

R. A. KEKWICK AND B. R. RECORD.*

From the Lister Institute, London.

Received for publication January 20, 1941.

A FEW detailed physico-chemical studies of immune sera have been madein recent years. By the use of the electrophoresis apparatus Tiselius (1937)has demonstrated that the circulating antibody to egg albumin is associatedin the rabbit with the y-globulin of the serum. In antipneumococcus horseserum Tiselius and Kabat (1939) have shown that the antibody is associatedwith an electrophoretic component not present in normal horse serum. Usingthe ultracentrifuge Kabat (1939) has found that the molecular weight of anti-pneumococcus antibody varies considerably with the species of animal.

The therapeutic importance of antitoxic sera has encouraged a detailedinvestigation of diphtheria antitoxin prepared in the horse. As early as 1901Hiss and Atkinson showed that during the immunization of horses withdiphtheria toxin the pseudoglobulin content of the serum rises. This wasconfirmed by Ledingham (1907), who concluded that " in the horse the pseudo-globulin contains the greater part if not all the antitoxin." By means of theultracentrifuge McFarlane (1935) demonstrated the enhanced globulin contentof diphtheria antitoxic horse serum.

This paper is a report on some physical properties of a number of dipli-theria antitoxic horse sera, and attempts to correlate these properties withsuch characteristics as antitoxin titre, flocculation time and the in vivolin vitroratio.

EXPERIMENTAL.

JImmunization and Titration Methods.Horses were immunized by inoculation intramuscularly with diphtheria

toxoid according to the usual practice at this Institute for diphtheria antitoxinproduction. The dose of toxoid was doubled every two or three days up tothe maximum comfortably tolerated by the horse, and maintained at thislevel until a suitable titre was reached. In the special case of the horse" Patrol " (Table II) toxoid purified by the ferric chloride procedure of Moloneyand Orr (1935) and containing 500 Lf units/ml. was used as antigen. Aninitial dose of 0-1 ml. was injected; on the 14th day the dose had risen to4-0 ml., and from the 20th day to the final injection on the 34th day wasmaintained at 25 ml. The antitoxic sera produced showed no differences fromnthose resulting from the use of the ordinary toxoid.

The antitoxin titres of the sera were determined in vitro using the Ramon* Beit Memorial Research Fellow.

R. A. KEKWICK AND B. R. RECORD.

flocculation test and in vivo by the L+ test. The rate of flocculation in theRamon test is influenced by a number of factors. Temperatures up to anoptimum value hasten flocculation; a similar effect arises from convectioncurrents such as occur when the contents of-the tubes are only partiallyimmersed in the bath. Dilution delays flocculation considerably, and thepH and salt content of the mixtures also affect the flocculation time. Toeontrol these variables the following procedure was adopted. All tests werecarried out in a thermostatically controlled water bath at 50 i 0.10 C. Thecontents of the tubes were between one-third and one-half immersed in thebath water, under which conditions reproducible flocculation times wereobtained. In addition this procedure allowed the onset of flocculation to bemore easily discerned than when the tubes were completely immersed. Thetubes were illuminated by transmitted light, and observed against a deadblack background, direct rays being screened from the eye.

It is important that the concentration of toxin in each tube in the testshould have a constant value. The test toxin was titrated against the labora-tory standard antitoxin and diluted to a strength of 30 Lf/ml. After theaddition of varying amounts of the antitoxin under test to a constant volume,usually 10 ml. of standardized toxin, the volume was made up to 2 ml. withphosphate buffer pH 8 ,u _ 0.1. The serum or serum fractions to be testedwere dialysed to equilibrium with this buffer, so that variations in flocculationtime due to variations in pH and salt content were eliminated. At differingconcentrations small variations in salt content were introduced on accountof the composition of the toxin solution. These could have been eliminatedby the use of toxin dialysed against the standard buffer, but any advantagegained would have been offset by a resulting diminution in stability. Increasein flocculation time owing to ageing of the toxin was minimized as far aspossible by storing the toxin under toluene at 2° C. in the dark and discardingwhen more than a few months old.

Under the conditions adopted for carrying out the Ramon test, it wasfound that Kf, the flocculation time of the indicating mixture, is constant fora given serum, but that large differences may occur between the sera fromdifferent horses aild also between different bleedings from the same horse.

In the in vivo test the sera or serum fractions were titrated by mixingappropriate doses with the L+ dose of the test toxin, and after the lapse ofone hour at room temperature, injecting the mixtures subcutaneously intoguinea-pigs. The antitoxin titre was estimated from the mixture whichprotected the animal for five days.

Determination of Floccule Nitrogen.For the determination of floccule nitrogen, 10 ml. crude toxin, containing

30 Lf/ml., were mixed with an equivalent quantity of antitoxin (as determinedby the flocculation test), and partially immersed in a water bath at 500 C.The mixture was allowed to remain for 4 hours after the onset of flocculationand was then left overnight at 00 C. The floccules were centrifuged off,washed twice with 5 ml. 0 9 per cent. NaCl, and the total nitrogen estimatedby microkjeldahl.

30

PHYSICAL PROPERTIES OF DIPHTHERIA ANTITOXIC HORSE SERA. 31

Examination with the Ultracentrifuge and the Electrophoresis Apparatus.The experimental methods used in the treatment and examination of the

sera were similar to those used previously for human sera (Kekwick, 1939,1940). The samples were dialysed undiluted at constant volume againstphosphate buffer pH 8 ,u = 01 until ionic equilibrium was attained, andcentrifuged to remove the slight precipitate which usually formed. Therefraction due to the non-dialysable materials present was determined withthe dipping refractometer (A = 546 m,u.), and is recorded as n Protein inTables I and II. The figures given are the differences between the refractiveindices n1 and no of the protein solution and buffer respectively.

For examination in the ultracentrifuge the dialysed sera were diluted withbuffer to nl-n0 = 0 00300, and were subjected to a field 270,000 times gravity.Optical observations were made by the diagonal schlieren method, the lightsource being a high-pressure mercury arc from which monochromatic lightA - 546 mn. was isolated.

Electrophoretic measurements were made in the Tiselius (1937) apparatusat 0° C., a potential gradient of 5 V/cm. being applied. The optical obser-vations were made in the same manner as for the ultracentrifuge, but a higherprotein concentration, corresponding to nl-n0 = 0-00450, was used.

By optical projection of the recorded photographs with an enlargement ofeight diameters on to millimetre graph paper, tracings were made which wereanalysed for the quantities of the components present. The ultracentrifugalanalyses were obtained from the mean of two exposures in each case. Theelectrophoretic data are the mean values from a pair of exposures obtainedsimultaneously from the anode and cathode limb. In analysing the anodelimb diagrams allowance was made for the d boundary.

1. The physical behaviour of normal horse serum.In order to appreciate the changes occurring in horse serum during immuni-

zation with diphtheria toxoid, a knowledge of the variation in protein com-position of normal horse serum is necessary.

In the ultracentrifuge normal horse serum shows two main components,albumin and globulin, whose sedimentation constants S20, are 4-5 and 7*1 x10-13 respectively. In addition there is a small quantity of a molecularspecies having S20 = 18 x 10-13 (Svedberg and Pedersen, 1940). Four com-ponents are found by electrophoresis, albumin, o- 3- and y-globulins (Tiselius,1937). The globulins are believed to have the same sedimentation constant,S20= 7-1 x 10-13, but differ in charge; the component with S20 = 18 x 10-13probably migrates with the a globulin.

The quantitative composition of a number of normal horse sera is given inTable I, the amounts of the components being recorded as percentages of thetotal refraction. It will be noticed that there is a rather wide variation in thecomposition of the different samples. These deviations are greater than thoseobserved in a series of normal human sera (Kekwick, 1939).

It is known that horses whose serum possesses some natural diphtheriaantitoxin tend to produce sera of a higher titre when subjected to a course

R. A. KEKWICK AND B. R. RECORD.

of toxoid injections than horses whose sera have little or no natural antitoxicactivity. The antitoxin titre of the normal sera, determined by animal test,are included in Table I. Those horses giving sera with more than 1/10 unitantitoxin per ml. are usually found to produce satisfactory antisera, whereasthose with less than 1/50 unit per ml. are generally not used. There appearsto be no correlation between the composition of these normal horse sera andthe extent of the natural immunity shown.

TABLE I.-Normal Horse Serum: Ultracentrifugal and Electrophoretic Analysis.Electrophoretic.

Serum Natural immunity n Protein Albumin. Ultracentrifugal.

a d y

3* 0-01197 446 . 3-5 . 21-8 . 201 65-4 . 3474* . .. . 0-01223 . 476 . 12.1 . 24-7 . 157 . 634 . 36-6. . >01 .01598 .349 . 160 . 328 . 163 499 . 50-16 . <0-02 . 001379 . 462 . 10-3 . 24-3 . 19-2 . 62-9 . 37.17* . >0*1 . 0-01521 43-.5 . 13.3 . 300 . 13*2 . 59.3 . 4(-88* . <0-02 . 001397 . 342 . 15-3 . 229 . 26-7 . 54-8 . 45.3* j3-globulin showed two components. Analytical values are percentages of the total refractiouis.

The discrepancy between the analysis for albumin and globulin by theultracentrifuge and electrophoresis apparatus has been observed and dis-cussed previously for human serum (Kekwick, 1939, 1940). The chief concernhere is with the electrophoresis data; the ultracentrifuge results have beenincluded for the purpose of completeness. This applies also to the data forthe antitoxic sera.

2. The serum changes during immunization.The changes occurring in the serum of the horse " Patrol " during the

course of immunization are illustrated by Figs. 1 and 2. The detailed analysisof these curves and other relevant data are given in Table II. These data aretypical, similar results having been obtained with other horses.

All samples showed the same components as normal horse serum, but withthe progress of immunization the quantitative relations changed. The bleed-ing taken previous to immunization of this horse, viz. sample 0, had a ratherlower percentage of 5-globulin than the other normal horse sera analysed.Quite early during immunization a hyperproteinaemia developed which becamemore marked and at the same time the percentage of globulin rose, as shownby the ultracentrifuge diagrams. Electrophoresis revealed that the globulinincrease was due to the 5-component, the percentage of cx- and y-globulinsremaining practically unchanged. Concomitant with these changes in com-position the antitoxin titre of the samples rose to a more or less limiting value.

In Table II are also included the Kf values for indicating mixtures con-taining 15 Lf/ml., and the in vivo/in vitro ratios for the various bleedings.The early bleedings are characterized by a short Kf; this increases as immuniza-tion proceeds, and approaches a limiting value which is very little affected by.further doses of toxoid and any increase in titre. The relatively short Kf of

32

PHYSICAL PROPERTIES OF DIPHTHERIA ANTITOXIC HORSE SERA. 33

* OX tcq* * *.

w -

(?n o C)- C*.

.00 >--4 -CC

0~~~~~~

mq* * 0* . .

. o cO or_ 4 Or

0 e Q

o 0000 nO

Oo + . . . -C

- -- 0- 0O

o.d -.0

,Q~ Q 0ew oo q 3 Cl -o -

co

v on C4040 000

O - - _0.

_ :'4

ri * * **

3

R. A. KEKWICK AND B. R. RECORD.

early bleedings during immunization was characteristic of all the horsesinvestigated. Locke, Main and Miller (1927) have stated in agreement withthis that early bleedings tend to flocculate rapidly.

It is often found that the Lf and L+ values of an antitoxin are not inagreement, the divergence, expressed as L+ /Lf, being referred to as the in vivo/in vitro ratio. A value exceeding unity is usually associated with sera havinga short flocculation time, though Glenny (1931) mentions exceptions to thisrule. The present series of bleedings illustrate this point, the in vivo/in vitroratio falling gradually during the course of immunization.

0 DAYS

14 DAYS

22 LJ/1&t

20 DAYS

24 DAYS m

360 Lfi/L.

41 DAYS ! |

930 if/mI'E

M A C B

FIG. 1.-Ultracentrifuge diagonal schlieren photographs of sera from the horse "Patrol"during immunization with diphtheria toxoid. M, meniscus; A, albumin; G, globulin; B,cell bottom; i, index.

The final titre obtained, the flocculation time, and the in vivo/in vitro ratiovary from horse to horse, though for the majority the characteristics of thesera lie close to those of the final bleedings of " Patrol."

The evidence suggests that the antitoxic activity of the sera is predomi-nantly associated with the p-globulin. In some of the normal sera described(Table I) the n-globulin consists of two components of very similar mobility,which may be referred to as (, and (32 in decreasing order of mobility. Alldiphtheria antitoxic sera show both ,l- and ,32-components, and it is the latterwhich appears to increase most during immunization. Owing to the smalldifference in the mobility of these components it has been impossible to makeindividual analyses.

Further evidence that antitoxic activity is associated with the P2-globulin

34

PHYSICAL PROPERTIES OF DIPHTHERIA ANTITOXIC HORSE SERA. 35

was obtained from the electrophoresis of the supernatant fluid from a balancedmixture of pure diphtheria toxin (Pappenheimer, 1937) and the serum " Patrol5." A comparison of the diagram obtained with that of the untreated serumrevealed a marked decrease in the 32-globulin. Refraction measurementsindicated that 10 per cent. ofthe serum protein was precipitated in the floccules.

The experiment also showed that the increase in globulin content of theserum during immunization is in part due to normal globulin, since the super-natant fluid contained a considerably higher percentage of globulin than theserum prior to immunization.

0 DAYS

o l/ml l______

20 DAYS I E I

e57 Ot

29 DAYS

610 Lf/mI.

41 DAYS5

930 Lf/mLAA 3 V s

FIG. 2.-Electrophoresis diagonal schlieren photographs of sera from the horsel" Patrol"during immunization with diphtheria toxoid. A, albumin; a, ,B, globulins; 8, boundary.

3. Isolation of components from the antitoxic sera.Although the observations of the previous section suggest that antitoxic

activity is associated predominantly with the ,3-globulin, it was considereddesirable to test all the electrophoretic components systematically for thepresence of antitoxin.

The electrophoretic preparation of albumin and y-globulin directly fromsera is a simple procedure. At pH 8 pure albumin can be obtained from theanode limb of the Tiselius U-tube, after a sufficient quantity of electricityhas passed and y-globulin similarly from the cathode. The isolation of pure

c- or ,B-globulins involves a two-stage procedure; for example the isolationof the x + y fraction followed by the separation of the P- and y-components.Unless a large scale U-tube is available this method is very tedious, severalprimary runs having to be made in order to accumulate sufficient material

R. A. KEKWICK AND B. R. RECORD.

for the secondary stage. Particularly for the isolation of ,3-globulin, the pre-liminary fractionation of the sera with salt has been found advantageous.

By dissolving 18 g. Na2SO4 in 100 ml. of a diphtheria antitoxic horseserum a precipitate is formed (Ppt. I), which consists of a-, P3- and y-globulins

SALT FRACTIONATION OF DIPHTHERIAANTITOXIC HORSE SERUM (FIDUS4j

SERUM88.ooo LFKFr,- 17

PROTEIN - 100/

18Z NA2504

3 PPT I - A712,TT . 1.

L&I.J 88~e.oooLF 3.800 LFPROTEIN:7I.27 I.~~~ PROTEIN =26

PPT. 2.54000 LFKF5I3=13PROTEIN 52.4X

x-GLOBULINKF3= i9

at-i SUPT. 2.2 500 LFKFr5 42PROTEIN, 15.770

/3- GLOBUILINKFI5= 39

FIG. 3.-Electrophoresis diagonal schlieren photographs of salt fractions from diphtheriaantitoxic. serum (Fidus 4). A, albumin; a, f, y, globulins; (-, boundary. Amounts ofeach fraction given as percentage of initial protein.

with a small amount of albumin and contains 95 per cent. or more of theantitoxin units, by the flocculation test, The supernatant fluid (Supernatant I)consists almost entirely of albumin, with traces of a- and 5-globulin. Solutionof Precipitate I in phosphate buffer pH 8 ,L= 0.1 to give 60 per cent. of thevolume of the initial serum and reprecipitation with 15 g. Na2SO4 per 100 ml.

36

PHYSICAL PROPERTIES OF DIPHTHERIA ANTITOXIC HORSE SERA. 37

gives Precipitate II, consisting of 3- and y-globulin with a trace of oc-globulin,and Supernatant II, which consists of some albumin, oc- and p-globulins, butcontains no y-globulin. The percentage of the original activity in thesefractions varies with the characteristics of the serum used; the shorter theflocculation time of the serum the greater the percentage of the activity foundin Precipitate II. Supernatant II was dialysed SO4" free against phosphatepH 8 p± = 01 and the ,-globulin isolated electrophoretically in the cathodelimb. Although Precipitate II was a good source of y-globulin the latter wasmore frequently isolated directly from the serum. A typical fractionationis illustrated by the diagonal schlieren photographs in Fig. 3.

It may be mentioned here that repeated precipitation of material fromPrecipitate II with 12 per cent. Na2SO4, although giving a product of highy-globulin content, did not produce pure y-globulin as in the fractionation ofnormal human serum (Kekwick, 1940). Fractionation of the total globulinfrom diphtheria antitoxic horse serum by a method similar to that used byGreen (1938) for normal horse serum did not give electrophoretically purecomponents.

4. Comparison of the properties of the antitoxic components with those of wholeserum.

That there is no antitoxic activity associated with the albumin of the serawas demonstrated by flocculation tests using the method of blending. Similarlyan electrophoretic sample containing only albumin and oc-globulin causedno displacement in the indicating mixture on blending with a standard serum,and the in vivo test showed that any activity, if present at all, was of a verylow order. By direct determination on electrophoretically pure preparationsit was demonstrated that antitoxic activity is associated with both the P- andy-globulins, and moreover it became apparent that the antitoxins associatedwith these two globulins have distinctive characteristics.

In Table III the characteristics of a number of preparations of 3- and y-globulins from various diphtheria antitoxic sera are collected, the propertieslisted being specific activity, flocculation time and in vivo/in vitro ratio. Thespecific activity is referred to a basis of refractive increment, and is obtainedby dividing the Lf/ml. of the solution by the refractive increment of theprotein dissolved. A close approximation to the activity per g. proteinmay be obtained by multiplication of the activity values by the factor 0-2.

In general the specific activities of the y-globulin preparations are low,and as the y-fractions are also obtained in dilute solution from the electro-phoresis apparatus, it has been necessary to make activity tests at low toxinconcentrations. In order therefore to be able to compare y-globulin prepara-tions from the different sera, a curve showing the variation of flocculationtime of the indicating mixture with dilution was constructed (Fig. 4). Forthis purpose a y-globulin preparation was used from the serum " Fidus 4,"which was sufficiently active to enable a range from 1 to 10 Lf/ml. to becovered. Under the experimental conditions obtaining, the flocculationtimes of indicating mixtures at a level higher than 10 Lf/ml. is so short as tobe of little value for characterization.

38 R. A. KEKWICK AND B. R. RECORD.

It will be noticed that the Kf values for all y-globulin preparations lievery close to this curve, and it is justifiable to conclude that the y-globuhinantibody has reproducible flocculation characteristics which are independentof the horse.

In Fig. 4 is also included a Kf-Lf/ml. curve for ,-globulin, the experimentaldata being obtained from a preparation from "Hickory." The values for::X~ B:'~B~ ; .,- .

*I..'..* - I'................ .....

t4.;.' . :.\. . .. .

N ''',.;\.. L Fl'+';u+t3% : -4.." '.'.-'\ -'' ,' ''

FIG. 4.-Flocculation curves of purified antibodies. x, 5-globulin from "Hickory"; E,P-globulin from " Pool P. 4 "; V, P-globulin from " Fidus 4 "; +, y-globulin from"Fidus 4 "; Q, y-globulin from "Pool P. 4"; , y-globulin from " Patrol"; A,y-globulin from "Turin".

other 3-globulin preparations given in Table IV lie close to this curve, withthe exception of the n-globulin from " Turin 2."A comparison of the two curves shows marked differences in flocculation

time of the ,3- and y-antitoxins at all levels of toxin concentration. Thus at7-5 Lf/ml. the Kf values are 147 and 7 minutes respectively for the ,- and y-globulins.

Preparations covering a range of specific activity have been obtained forboth antitoxic globulins from different horses, and the flocculation times were

.j

PHYSICAL PROPERTIES OF DIPHTHERIA ANTITOXIC HORSE SERA. 39

found to be independent of the specific activity. The variation in specificactivity is attributable to admixture of antitoxic globulin with the corre-sponding normal globulin, the two being electrophoretically indistinguishable(cf. Pappenheimer et al., 1940).

TABLE Ill.-Properties of Antitoxic Globulins.Serum

(horse and bleeding).3-globulin:

Pool P. 4HickoryFidus 4Turin 2

y-glFobulin:

Lf/ml/3n 105.

1 442 641 401-77

Kf minutes.

37-54542

205

PoolP.4 . . 0 27 . 9(7)Fidus 3 . . 0 35 . 8-5 (75)Fidus 4 . . 041 . 19

(3)Turin 1 . . 0*085 . 20

(3)Turin 2 . . 0115 . 26

(3)Patrol 3 . . 0*114 . 26

(3)All the Kf values for P-globulin were determined at 15 Lf/ml.For the y-globulin the figure in brackets indicates the number of Lf/ml.

L+/Lf.

0 850 810 960 51

2 21-82-1

TABLE IV.-Nitrogen Content of Floccutes from Indicating Mixtures.

Sample.

Patrol 12

,, 34

,, 5,, 6

y-globulin (Fidus 4)5-globulin (Hickory)

Floccules.mg. N/Lf unit.

*00342*00238*00245*00251*00223*00217*00367*00204

Antitoxin.mg. N/Lf unit.

*0030-*0019*0020*0020*0018

I. *0017

-0032*0016

Another property which affords a means of distinguishing between the ,-and y- antitoxic globulins is the in vivo/in vitro ratio, which is close to 0 9and 2-0 (Table III) in the two cases respectively.

The floccule nitrogen per Lf unit has also been found to differ for the ,B-and y- antitoxic globulins (Table IV). From the floccule nitrogen, by sub-tracting the nitrogen content of 1 Lf unit of pure diphtheria toxin, which has

R. A. KEKWICK AND B. R. RECORD.

been determined as 4'6 x 10-4 mg. (Pappenheimer and Robinson, 1937;Pappenheimer, 1937), the antitoxin nitrogen per Lf unit has been obtained.The antitoxin nitrogen for the pure y-globulin is found to be twice that for theP-globulin.

Sedimentation constant measurements were made on solutions of 3-globulinfrom " Hickory," and the y-globulin from " Fidus 4," in a buffer containing

FiG. 5.-Flocculation curves for diphtheria antitoxic horse sera (bleedings from " Patrol ").O, 14 days; E3, 20 days; A, 24 days; V, 41 days; *-* , 3-globulin;- ,globulin.

phosphate pH 8 ,t = 0-09 and 0- 1 M/NaCl, the protein concentration correspond-ing to nl-no = 00100. The values obtained for the P- and y-globulins respec-tively were 820 7-18 x 10-13 and 6-87 x 10-13, these being in reasonableagreement with the accepted value for normal horse-serum globulin, viz.S20 = 7-1 x 10-13. Thus the antitoxic globulins, as Pappenheimer et al.(1940) have already shown for the 5-globulin, have approximately the samemolecular dimensions as normal horse-serum globulin, and there appears tobe no antibody activity associated with any high molecular species as inantipneumococcal horse serum.

40

PHYSICAL PROPERTIES OF DIPHTHERIA ANTITOXIC HORSE SERA. 41

The flocculation curves, in vivo/in vitro ratio and floccule nittogen forthe bleedings of the horse " Patrol " may now be exainined in comparisonwith the data obtained for the (3 and y antitoxic globulins.

The flocculation curves for several bleedings of the horse " Patrol " aregiven in Fig. 5, and these all lie between the limits demarcated by the floccu-lation curves for the P- and y-globulins. The early bleedings give curveslying close to that of y-globulin, and as immunization proceeds there is a pro-gressive shift towards the 5-globulin curve. The curves for bleedings 4, 5and 6 are practically superimposable.

A reference to Table II shows that the in vivo/in vitro ratio of these seratends towards the value found for the 5-globulin in the later bleedings. Thevalues of the floccule nitrogen per Lf unit for the sera (Table IV) all lie between36 x 10 mg.- for y globulin and 20 x 10- mg. for 5-globulin.

These trends are all consistent with the supposition that in early bleedingsa high percentage of the total antitoxic activity is associated with the y-globulin, but that in later bleedings the activity is predominantly due to the5-globulin. By a method explained in detail in the following section (5) ithas been found that for bleedings 2, 3 and 5 the activity associated with they-globulin was 25 per cent., 11 per cent. and 5 per cent. of the totals respectively.

5. The activity balance 8heets for some sera.The evidence of the previous sections indicates that antitoxic activity is

associated with both the ,- and y-globulins, and it should therefore be possibleto account for the total number of antitoxin units in a serum from its electro-phoretic analysis and the specific activities of the (- and y-globulins.

The data for five sera are presented in Table V, the sera being chosen tocover as wide as possible a range of flocculation time. The significance of anumber of the properties tabulated has already been discussed in connectionwith other sera. The electrophoretic analyses of the sera are given in lines5-8, the quantities being expressed as percentages of the total refraction.Fromi lines 4, 7 and 8 the refractive increment of the (- and y-globulins isobtained and given in line 9. In line 10 the experimentally determined

TABLE V.-The Activity Balance Sheet for Several Sera.Serum. Turin 1. Fidus 4. Pool P. 4. Hickory. Tturin 2.

1. Lf/mI . . . 75 . 935 . 975 . 2050 . 7902. Kf15 . . 105 . 19 22 . 43 . 613. L+-/Lf . . . . 167 . 117 . 0.97 . 097 . 0-804. n Protein . . . 001575 . 001600 . 0-01490 . 001601 . 0-016105. Alb. . . . . 364 . 247 . 24.2 . 211 . 31-66. oc-glob .. 158 127 . 16-8 . 161 12-77. P-glob. . . . 25.5 . 308 . 37.4 . 467 . 3218. y-glob. . . . . 224 . 31-8 . 217 . 16-2 . 23-69. n" P 0-00402 0 00493 0.00557 0-00748 0-00517

* y1 Y . . . .0 00353 0-00509 0-00323 0-00259 0-0038010. Lf/ml/n. 105 015 . 1-40 . 144 264 1-77*^ y . . 0.084 . 0-41 . 0-27 . 033 . 0-115

11Lf'1t'~ 360 2690 802 1975 91530 . 209 87 85 . 44

12. Lf/lml . . 90 . 899 . 889 . 2060 . 959

R. A. KEKWICK AND B. R. RECORD.

specific antitoxic activities of the ,B- and y-globulins appropriate to each serumare given, from which by combination with the data of line 9 the number ofLf units per ml. of serum for each component is derived (line 11). The totalactivity thus calculated is given in line 12. This treatment involves noassumptions with regard to the specific refractive increments of the f- and y-globulins.

A comparison of lines 1 and 12 ofthe table shows that the calculated activityis in reasonable agreement with the value determined directly, having regardto the possible errors in the electrophoresis curve analysis and the flocculationtitration. It is reasonable to conclude that the antitoxin titre of the seracan be quantitatively accounted for on the basis of the activity of the ,3- andy-globulins. If there is any activity associated with the cx-globulin, whichother methods failed to detect, the number of antitoxin units must be verysmall.

A further point of interest arising from the data is that as the flocculationtime of the serum increases, the proportion of the total activity associated withthe n-globulin rises. The sera also show the same relation between flocculationtime and in vivo/in vitro ratio as found for the serum samples from the horse"Patrol."

Some comment is necessary concerning the serum " Turin 2." Amongst31 samples of serum examined from 11 different horses this was the onlyserum whose flocculation curve lay outside the limits of the ,3- and y-globulincurves of Fig. 4. Further the 3-globulin isolated from it has an abnormalflocculation time (cf. Table IV), and the calculated activity of the serum ishigh.

DISCUSSION.

The experimental evidence has demonstrated that in diphtheria antitoxichorse sera there are two antitoxins, one associated with the 3- and the otherwith the y-globulin. In addition to a difference in electrochemical constitutionwhich has made possible their isolation, the antitoxins show quantitativedifferences in respect to flocculation time, in vivo/in vitro ratio and the amountof antitoxin nitrogen contained in the floccules formed with toxin.

It has been found (Pappenheimer et al., 1940) that the floccules from indi-cating mixtures of 5-globulin and toxin have the constitution (TA2),,, whereT represents one molecule of toxin and A one of antitoxin. A comparisonof the antitoxin nitrogen precipitated in the floccules from indicating mixturesof ,3- and y-globulins has shown that approximately twice as much nitrogenper Lf unit is precipitated from the y- as from the ,B-globulin. Consequentlythe floccules from the y-globulin indicating mixtures have the constitution(TA4)n.

From the therapeutic point of view the y-globulin antitoxin may havesome advantage in that it combines more rapidly with toxin than the ,-globulinantitoxin; there are however some indications that it is less stable.

Concerning the changes occurring during the course of immunization theimmediate reaction in the horse is the production of the y-globulin antitoxin.As further injections are given increasing quantities of n-globulin antitoxin are

42

PHYSICAL PROPERTIES OF DIPHTHERIA ANTITOXIC HORSE SERA. 43

formed, but the number of units of y-globulin remain fairly constant at thelow level of 50-100 Lf/ml. in most horses. These changes are reflected in theincreasing flocculation time and decreasing in vivo/in vitro ratio of the serumsamples. The floccule nitrogen also tends to fall.

The high in vivo/in vitro ratio of early bleedings and the general tendencyfor this ratio to fall as immunization proceeds has been observed by Glennyet al. (1925) for a number of horses.

It has been shown that it is possible to account quantitatively for the totalnumber of Lf units in a serum on the basis of the content and specific activitiesof the (- and y-globulins. In view of the fact that the in vivo/in vitro ratioof the ,- and y-globulins is approximately 0*9 and 2-0 respectively, it followsthat no serum should be found having an in vivo/in vitro ratio outside theselimits. Glenny et al. (1925) have reported a serum with a ratio as high as3 0, and " Turin 2 " investigated here had a ratio of 0*80. These observationsare difficult to explain on the basis of the evidence presented here. The factthat the least soluble globulin fractions have the highest in vivo/in vitroratios (Barr and Glenny, 1931) is in accord with the observations made herethat such fractions tend to contain a higher proportion of y-globulin, whilstmore soluble fractions contain increasing amounts of (-globulin.

These investigations indicate that the properties of the majority of diph-theria antitoxic horse sera may be interpreted in terms of their content of( and y antitoxic globulins, the characteristics of which remain sensiblyconstant and independent of the individual horse. Occasionally abnormalsera occur, which probably owe their properties to the presence of atypicalantitoxic globulin components.

SUMMARY.

The physical behaviour of a number of normal horse sera has been studied,and the changes occurring in horse serum during the course of immunizationwith diphtheria toxoid are described.

It has been demonstrated that diphtheria antitoxic horse sera contain twoantitoxins associated with the (3- and y-globulins respectively. These anti-toxins are sharply distinguishable from one another by a number of propertieswhich for preparations from most sera remain sensibly constant. The anti-toxic properties of diphtheria antitoxic horse sera may be interpreted in termsof their content of ( and y antitoxic globulins.

The authors wish to express their indebtedness to Dr. C. R. Amies for hisinvaluable co-operation in the supply of sera and in carrying out the animaltests.

One of us (R. A. K.) is indebted to the Medical Research Council for agrant in aid of personal and research expenses.

REFERENCES.BARR, M., AND GLENNY, A. T.-(1931) Brit. J. exp. Path., 12, 337.GLENNY, A. T.-(1931) Med. Res. Council, 'A System of Bacteriology,' Vol. VI,

106.

44- A. KLECZKOWSKI.

Idem, POPE, G. G., WADDINGTON, H., AND WALLACE, U.-(1925) J. Path. Bact., 28,463.

GREEN, A. A.-(1938) J. Amer. chem. Soc., 60, 1108.Hiss, P. H., AND ATKINSON, J. P.-(1901) J. exp. Med., 5, 47.KABAT, E.-(1939) Ibid., 69, 103.KEKWICK, R. A.-(1939) Biochem J., 33, 1122.-(1940) Ibid., 34, 1248.LEDINGHAM, J. C. G.-(1907) J. Hyg., Camb., 7, 65.LOCKE, A., MAIN, E. R., AND MILLER, F. A.-(1927) J. infect. Dis., 41, 32.MCFARLANE, A. S. (1935) Biochem. J., 29, 1175.MOLONEY, P. J., AND ORR, M. D.-(1935) Ibid., 29, 1525.PAPPENHEIMER, A. M.-(1937) J. biol. Chem., 120, 543.Idem AND ROBINSON, E. S.-(1937) J. Immunol., 32, 291.Idem, LUNDGREN,.H. P., AND WILLIAMS, J. W.-(1940) J. exp. Med., 71, 247.SVEDBERG, T., AND PEDERSEN, K. O. -(1940) 'The Ultracentrifuge,' Oxford

(Clarendon Press).TISELIUS, A.-(1937) Biochem. J., 31, 1464.Idem AND KABAT, E.-(1939) J. exp. Med., 69, 119.

QUANTITATIVE STUDIES ON THE SEROLOGICAL REACTIONSOF SOME PLANT VIRUSES AND OF A PEA NODULE BAC-TERIUM (RHIZOBIUM LEGUMINOSARUM).

A. KLECZKOWSKI.From the Rothamsted Experimental Station, Harpenden, Herts.

Received for publication January 22, 1941.

IN both agglutination and precipitation, combination between antigenand antibody is followed by the separation of an antigen-antibody complex.The most striking difference between the two reactions is quantitative; inprecipitin reactions small amounts of antigen combine with comparativelylarge amounts of antibody, whereas in agglutination reactions large amounts ofantigen react with small amounts of antibody. This difference seems to becontrolled by the sizes of the antigens; those giving precipitin reactions aresolutions of finely dispersed particles, whereas those giving agglutinin reactionsare suspensions of large particles.

The ratio in which a given antigen combines with its antibody in precipitinreactions is a function of the proportions in which the two are mixed (Marrackand Smith, 1931b; Heidelberger and Kendall, 1935a). However, when theproportions are kept constant at those corresponding to equivalence (i.e.when neither antigen nor antibody is left in the supernatant after the precipitatehas been removed by centrifugation), the ratio varies with different sera(Marrack and Smith, 193 lb). In the serum of one animal it even varies duringthe progress of immunization, the antibody/antigen ratio becoming larger witheach successive immunization (Heidelberger and Kendall, 1935a). In addition