preparation and evaluation of porcine sera against human immunoglobulins

Preparation and Evaluation of Porcine Sera against Human Immunoglobulins F. ~KVAI~IL, V. BI~IJMMELOV~, J. FRAGlXTER and J. ~INDEL.5_~

Research Institute of Immunology, Prague

Received February 6, 1968

ABSTRACT. Thirty-eight pigs were immunized with normal human yG-, yA- and yM-immunoglobulins, fragments of yG-immunoglobulin (Fab, Fe -p :F'e), type K and L Bence-Jones proteins and other antigens containing immunoglobulins or their fragments. The pigs formed antibodies readily against the heavy y, cc, iz aud ~ chains and also against x chains. No antibodies whatsoever were formed against the surface (to- torminants of X chains; the antibodies present reacted only with hidden determinants of those chains. In isolated cases, antibodies against the Fd fragments of yG-immunoglobulin were also identified. A quantitative precipitation test showed that porcine antibodies are of the "R", precipitin, type.

Specific precipitin antisera, because of their pronounced sensitivity and discriminative capacity, are increasingly used in the classification, typing and quantitative determination of immunoglobulins. Rabbit sera, a comprehensive evaluation of which has already been carried out (l~ahey & McLaughlin, 1963), are the most frequently used. The antisera of some pri- mates immunized with human proteins have likewise been adequately character- ized (Dray, 1960; Lichter & Dray, 1964) and their use has led to great advances, particularly in exposing the heterogeneity of the heavy yG-immunoglobulin chains.

The finding tha t pigs form antobodies both against protein antigens (Fran~k etal., 1965) and a~ainst diphtheria and tetanus anatoxin (Sterzl etal., 1966; Rej- nek et al., 1966) prompted the present study, in which the characteristics of the sera of pigs immunized with human immunoglobulins or their fragments were determined.

MATERIALS AND !VIETHODS

Antigens /or immunization and evaluation o[ antisera

Preparation o/ yG-immunoglobulin, yG- immunoglobulin was isolated from a pool

of normal human sera with rivanol (Ho~ej~i & Smetana, 1954) and was concentrated by salting out with ammonium sulphate at 45% saturation. Immune- chemically pure ~G-immunoglobulin was prepared by chromatography of the preparation on DEAE cellulose (Levy & So- ber, 1960).

In the same way (rivanol and ammonium sulphate fractionation), yG-immunoglobulin was isolated from myeloma serum (ZEN) containing type L paraprotein G.

Preparation el the _~ab ]ragment o~

~ G-immunoglobulin. A fragmented 16~/o herapeutie gamma globulin preparation,

after diluting to 3 0 , was precipitated with zinc sulphate to a final concentration of 50 mlV[ Zn 2+ ions. After two days' dialysis against 0.5~/0 sodium citrate, followed by dialysis against saline, the super- na tan t was lyophilized. Details of the preparation technique are given elsewhere (~kva~il & Brummelovs 1968).

Preparation el the Fc + F'c ]ragment of ~:G-immunoglobulin. A fragmented 16% therapeutic gamma globulin containing the subffagment :F'c (this preparation is denoted as GG-X), was precipitated with saturated ammonium sulphate solution to 35% saturation and after removing the

1968 P R E P A R A T I O N OF PORCINE SERA 483

precipitate (which mainly contained re- sidual non-fragmented yG-immunoglobulin and Fe fragment) to 50% saturation. The precipitate was spun off, dissolved in a minimum amount of water, dialyzed against saline and lyophilized. Details of the preparation technique are given elsewhere (~kva~il & Brummelov&, 1968).

Benee-Jones proteins. Urine samples containing Bence-Jones (BJ) proteins were concentrated by salting out with ammonium sulphate to 75% saturation. The precipitate was separated off, dissolved in a small amount of water and after dialyz- ing against water the solution was lyophilized. In all, three type K (VAV, KLI, VEJ) and two type L (VOS, KLO) BJ proteins were used. They were typed against s tandard pure type K and L BJ proteins*).

Preparation o I y A-imm~tnoglobuIin. The method of Heremans et al. (1963), some- what modified, was used. A solution of the ethanol fraction I I I , isolated from placen- tal serum by a modification of the methods of Cohn et al. (1946) and Oncley et al. (1949) in saline was precipitated at :pH 7.2 with zinc sulphate to a final concentration of 50 mM. After adjusting to TH 6.85 and incubating for one hour at 22 ~ C, the precipitate was spun off. The supernatant was reincubated for 30 minutes at 28~ and the resultant sediment was again spun off and filtered. After adding glycine in a concentration of up to 2%, the proteins were salted out from the solution with solid ammonium sulphate at up to 42% saturation; the precipitate was then dissolved in water and filtered on a Se- phadex G 200 column (in 0.1 M Tris-HC1 quffer at ~oH 8 with 1 ~ NaC1). Samples in which yA-immunoglobulin were detected by immunoelectrophoresis were concentrated by salting out with ammonium sulphate under the above conditions, followed by dialysis and lyophilization.

Preparation o I y M-immunoglobulin. Nor-

mal pooled human serum was diluted with water in the ratio 1 : 20, adjusting the p i t to 5.9 (with 1 M acetic acid), and the mixture was incubated for one day at +4 ~ C. The resultant precipitate was spun off and diluted, first with saline to half the initial serum volume and then with water to twenty times the original serum volume. The precipitate was then spun off, dissolved in saline and lyophilized.

Human plasmin preparations. Since it was assumed tha t human plasmin pre- palations isolated from the ethanol fraction I I I would contain yG-immunoglobutin fragments, two types were used for immunization: (a) Fibrinolysin (Human) Lyovac -- Merck, Sharp & Dohme, Lot No. 1343 W a, and (b) Human plasmin, a Sevac (Imuna) preparation*.

Antisera

Immunization o/pigs. The scheme and doses described by Fwan~k et al. (1965) were used, with slight modifications.

Characterization o] antisera. Immuno- electrophoresis was carried out in a micro- modification on slides measuring 8.5 • 8.5 cm (~kva~il & Rejnek, 1958), in veronal- citrate-oxalate buffer at pH 8.6 on Oxoid Ionagar No. 2 at a potential gradient of 4 V/cm for 75 minutes, staining with amido black 10 B.

In the first characterization analysis, a s tandard scheme was used, in which the following antigens were separated by electrophoresis and were precipitated with a test antiserum: normal human .serum (for determining antibodies against the various immunoglobulins and other serum proteins), 1% fragmented ~G-immunoglobulin (for determining antibodies against Fab and Fc fragments), 0.5% solutions of purified :BJ K and BJ L protein preparations (for the detection of antibodies against the relevant types of light chains) and 1% GG-X solution containing the F'c fragment.

* Kindly provided by Dr. Burtin, Institut de l%echerches Scientifiques sur le Cancer, Vfllejuif (Seine), France.

*The plasmin was prepared and donated by Dr. ~t@pAnek of the Inst i tute of Sofa and Vaccines (Imuna).

484 F . ~ K V A I ~ I L E T A/ L Vol. 13

Antibodies against hidden determinants of x and ~ chains were detected only in sera in which antibodies were found in an analysis of BJ K or BJ L proteins. The test antiserawere absorbed in the troughs by 0.1 ~/o immunochemiealty pure, y(~-immunoglobulin solution (I-~irschfeld, 1960) and were used for the precipitation of electro- phoretically separated normal human serum and a solution of the relevant B$ protein.

The presence of antibodies against the Fd fragment of human yG-immunoglobulin was determined by immunoelectro- phoretie separation of fragmeted I(G- immunoglobulin precipitated by test sera absorbed in the troughs by a mixture of 0.5% B5 K and BJ L protein solution (1 : 1); the results were evaluated by the appearance of a line i~ the Fab fragment z o n e .

The presence of anti- yD antibodies was demonstrated by immunoelectrophoresis of myeloma serum containing paraprotein D (JI)* and of serum from a patient with a high content of this immunoglobulin (VAV) (Rgdl, 1967).

Quantitative ~nethocls. Quantitive precipitation was carried out by Kabat and Mayer's modification (Kabat & Mayer, 1961) of the method of Heidelberger and Kendall (1935). After washing, the precipitate was dissolved in 0.01 ~ NaOH and absorbance of the solution was measured at 280 ~m.

R E S U L T S

~'on-absorbed 8era The antibodies found in the sera of 38

pigs immunized with different immunoglobulins or their fragments are shown in Table 1 -- will be found at the end of t ex t section - - p. 574.

As stated in the previous section, the first information on the quali ty of the antibodies in the test sera was obtained from the results of immunoelectrophoresis

carried out according to a s tandard scheme in which normal human serum, fragmented yG-immunoglobulin, BJ K and L proteins and a sample containing the F'c fragment of yG-immunoglobulin were precipitated by a test serum. A diagram illustrating the results of analysis of an "ideal" serum, i.e. one containing antibodies against the heavy chains of all three main classes of immunoglobulins (Y, ~, ~) and both types of light chains (k, x) and also precipitating all the three known yG-immunoglobulin fragments, (Fab, l~e and l~'e), is given in ~ig. 1. The results of analysis in the first strip can also be used to evaluate the presence of antibodies against other human serum proteins not included among the immunoglobulins (not shown in the diagram). All the antibodies given in Table 1 were thus identified by analysis according to the above scheme, with the exception of antibodies against the Fd fragment and the hidden determinants of the ~ and k chains.

Demonstrat ion of the presence of antibodies against the Fd fragment of ~,G-im-

~,G 3M .~A

3 Fa b o Fc

4 o

Fob Fc F'c

Fig. 1. Scheme of s t a n d a r d i m m u n o e l e c t r o p h o r e t i c ana lys i s of " i dea l " s e rum. A n t i g e n wells 1 - - n o r m a l h u m a n s e r u m ; 2 - - s ~ ' G - h n m u n o g l o b u l i n (1~o); 3 - - B J K pro te in (0.5%); 4 - - B J L p r o t e i n (0 .5%); 5 - - G G - X ( 1 % ) . T r o u g h s : " i dea l " a n t i s e r u m .

* S e r u m f rom t h e p a t i e n t J-i was k i n d l y p r o v i d e d b y Dr . L a p ~ k o v A of t h e B lood T r a n s f u s i o n Cent re , L o u n y , Czechos lovakia .

1968 P R E P A R A T I O N O F P O R C I N E S E R A 485

munoglobulin is illustrated in Fig. 2. In this analysis fragmented yG-immunoglobulin (containing the Fab abd Fc fragments and a small residue of non-fragmented yG-immunoglobulin) was separated by electrophoresis and was precipitated from the troughs by two test antisera (SwAHu/yG No. 3 and SwAHu/ /Fc -t-F'c No. 6) absorbed by a mixture of both types of BJ proteins. The apper- ance of a line in the Fab fragment zone can be attributed only to reaction of the :Fd fragment with the relevant antibody. In the ease of serum SwAHu/yG No. 3, which formed two lines in this zone, the presence of two antibodies reacting with Fd fragments can b0 presumed.

I o v ~ j

Fig. 3. I m m u n o e l e c t r o p h o r e t i e de t ec t i on o f an t i - bodies a g a i n s t h i d d e n d e t e r m i n a n t s o f X cha ins . A n t i g e n wells: 1 - - n o r m a l h u m a n s e r u m ; 2 - - B J L p ro t e in (0.5~/o). T roughs : 1 to 3 - - S w A H u / V O S No. 4; before filling w i t h a n t i s e r u m ,

0 .1% y G - i m m u n o g l o b u l i n so lu t ion was lef t to diffuse f r o m all t he t r oughs .

Antibodies in the test antisera against hidden determinats of the ~, and • chains were detected in a similar manner. Fig. 3 shows a representative analysis of serum containing this type of antibody against k chains (SwAHu/VOS 1~o. 4). Normal human serum and type L BJ protein were separated by electrophoresis and were precipitated from the troughs by a test antiserum absorbed by immunochemically pure yG-immunoglobulin. In the analysis of human serum, the antiserum did not precipitate any of the immunoglobulins (i.e. after absorption it did not contain antibodies against the heavy or the light immunoglobulin chains), but precipitated BJ protein. The presence of antibodies against transferrin and albumin, which were observed in the analysis of human

serum, did not interfere with this reaction in any way.

The relatively rare anti-yD-immunoglobulin antibodies (not given in Table 1) were found only in the sera of pigs immunized with an enriched yA-immunoglobulin preparation, and isolated myeloma D immunoglobulin.

Absorbed sera

Sera from immunized pigs for the preparation of specific anti-yG antiserum were chosen by the presence of antibodies against the Fc yG-immunoglobulin frag- meat, as components representing the y chain, and for the preparation of anti-yA or ant i-y~ antiserum by the results of immunoeleetrophoresis of normal human serum; the lines corresponding to these last two immunoglobulins show the presence of antibodies against the corresponding heavy chains, i.e. ~ or ~.

Absorption was carried out according to the generally accepted principle that unwanted antibodies can be absorbed by protein mixtures containing the corresponding antigens and definitely not con- raining antigens (or their fragments) corresponding to the required antibodies. The

I o

2 o

3 o

4

5

A 6

Fig. 5. I m m u n o e l e e t r o p h o r e t i e ana lys i s o f a b s o r b e d a n t i s e r u m w i t h an t ibod ie s a g a i n s t h i d d e n deter - m i n a n t s o f k cha ins . A n t i g e n wells 1 - - nor - m a l h u m a n s e r u m ; 2 - - f r a g m e n t e d y Q - i r n m u n o - g lobu l in (1~o); 3 - - B J K p ro t e in (0.5~o); 4 - - B J L p ro t e in (0.5%); 5 - - B J L p ro t e in (0.1~/o}; 6 - - B J L p ro t e in (0.05~o). T r o u g h s : a b s o r b e d S w A H u / B J L a n t i s e r u m (h.d.).

486 F. ~ K V A I ~ I L E T AL. Vol. 13

most difficult is the absorption of crude anti-TG serum which also contains antibodies against other immunoglobulins. As a rule, however, these immunoglobulins were adequately removed from antigen used for immunization; antibodies against

of human serum and of fragmented yG- immunoglobulin, the final serum precipitated type L Bff protein, but not 7G-immunoglobulin or the Fab fragment (Fig. 5).

The preparation of specific anti-~'D serum presents no difficulties if the crude

i o

2 o

3

:Fig. 6. I m m u n o e l e c t r o p h o r e t i c ana lys i s o f a b s o r b e d a n t i s e r u m SwAHu]TD. A n t i g e n wells 1 - - n o r m a l h u m a n s e r u m ; 2 - - s e r u m J I ( i : 64); 3 -- s e r u m J I (1 : 128). T r o u g h s : abso rbed S w A H u / T D a n t i s e r u m .

other containing proteins (e.g. transferrin) are absorbed relatively easily. Umbilical serum was normally used to absorb anti-~'A and anti-TM sera; for the preparation of anti-yM serum a prelimi- nary test was needed to find umbilical blood containing no yM-immunoglobulin As seen from Fig. 4, the absorbed sera reacted strictly specifically in an analysis of human serum.

When preparing antiserum against the light X or x chains, BJ protein of the opposite type (or myeloma serum containing paraprotein G of the opposite type) and also, in most cases, the Fc fragment of ~,G-immunoglobulin were used for absorption; the non-purified Bff proteins used for immunization contained an adequate amount of ~ - immunoglobu l in or its fragment for inducing antibody formation against these parts of the ~" chain (v. Table 1). In the case of anti-x serum (anti-BffK), this technique pro- duced monospecific serum. Absorption was not successful, however, in any of the eases in which anti-), antibodies were demonstrated in crude serum; in an analysis

0 . 7 - ~cr~'-~--.......~

").5 - '~.

0.3- ~

@d-

,~o J6oo J~oo ~6oo A280 ,ug of ~G immunoglobulin added

0.7-

0.5-

0.3-

O.i-

o.5 ,~o ,15 i.o ml of onotoxin added

r i g . 7. P rec ip i t a t i on r eac t ions o f poreine, r a b b i t a n d horse a n t i s e r u m . A b o v e : p rec ip i t a t ion cu rve o f porc ine a n t i s e r u m ( u n b r o k e n line) a n d r a b b i t an~i. s e r u m (broken line) w i t h n o r m a l h u m a n . ~ - i m m u n o g l o b u l i n . Below: p rec ip i ta t ion cu rve o f ho r se d i p h t h e r i a an t i t ox i e s e r u m wi th d i p h t h e r i a aria. toxin (200 Lf/ml).

antiserum contains a sufficient amount of antibodies. Unwanted antibodies were absorbed by umbilical serum and the re- maining anti-3,A antibodies were then absorbed by myeloma serum with paraprotein A; the resultant serum reacted strictly specifically (Fig. 6).

1968 P R E P A R A T I O N OF P O R C I N E SERA 487

Type of porcine antibodies The question of whether porcine anti-

protein antibodies correspond to "R" (precipitating) or "H" (flocculating) antibodies was resolved b y quanti tat ive precipitation of anti-yG porcine serum and comparison with rabbit anti-yG serum (representing " R " type antibodies) and with a diphtheria anatoxin -- horse anti- toxin system ("H" type antibodies). The results are given in Fig. 7.

DISCUSSION

A superficial evaluation of the results shows quite clearly tha t human immune- globulins are an effective antigen when administered to pigs.

In immunization with al] the test antigens (purified yG-immunoglobulin or enriched y/k-preparations and TlYLimmuno- globulins), antibodies were formed most frequently against the y chain, as determined by means of antibodies against the Fc or F'c fragments and less often against the light chains. A minute amount of yG-immunoglobulin or its fragments, constituting contamination of the antigens employed -- particularly of B J proteins - - was sufficient to induce the formation of anti-y antibodies. The inci- dence of antibodies against the :F'c fragment was small and evidently depended on the animals' individual reactions, especially in cases in which non-fragmented $G-immunoglobulin was used for immunization. The relatively frequent finding of these antibodies in the sera of pigs immunized with B J proteins was evidently due to the presence of this fragment in the urine (Turner & Rowe, 1966), from which it was isolated together with B J proteins; the formation of anti-F'c antibodies in two sera following immuniza- ation with plasmin can likewise be attributed to the presence of this fragment in the plasmin preparations.

Anti-y/k-immunoglobulin antibodies were likewise found relatively often in the sera of animals immunized with other

immunoglobulins; only serum obtained by immunization with an enriched yA-immunoglobulin preparation was suitable for the preparation of monospecific anti- -fA serum, however. Ant i -y~ antibodies were found in the sera of animals immunized with yM- and yA-immunoglobulins and also in the group immunized with the Fc ~-F 'c fragment; in ammonium sulphate fractionation, par t of the y-M-immunoglobulin originally present in the therapeutic gamma globulin preparation (expired batches were used as a source of fragments) passes into the fraction isolated between 35--50% saturation, which was used as a source of the Fc ~ F ' e fragment.

In the basic analysis, antibodies against the Fab fragment were often seen as a very faint line (in the relevant column in Table 1 these sera are marked ~:) and in these cases their low concentration did not allow detailed identification. In the other cases, in which the sera clearly precipitated the :Fab fragment, it was possible to determine whether this was a reaction of x or X chains or of the Fd fragment, or a simultaneous reaction of all the reactants concerned. The only doubtful finding was the line corresponding to the Fab fragment in two SwAHu/yl~I sera, in which no antibodies against either the light chains or the Fd fragment could be determined.

No antibodies against the surface determinants of the X chains were determinep in any of the tes t sera, as they usually were in the case of • chains. Wherever antibodies were demonstrated in immunoelectrophoresis of type L B J protein, precipitation in most cases was very indistinct and in the few cases in which further identification of the antibodies was possible, they were found to be directed against hidden determinants of the k chains; they were thus reminiscent of the antibodies obtained from rabbits immunized with isolated light chains (Tan & Epstein, 1965). This type of ant ibody did not appear in any of the sera containing anti-

488 F. ~KVAI~IL E T AL. VoI. 13

bodies against • chains. Since it proved impossible to prepare antibodies of the usual quality against k chains by immunization with normal immunoglobulins, type L BJ proteins and isolated type L pars- protein G, we must conclude that a given relationship exists between these chains in pigs and man; similar conclusions were recently drawn from finger-print analyses of isolated porcine k chains and their comparison with analyses of type L BJ proteins (Fran~k & Zorina, 1967).

The shape of the precipitation line of porcine anti-yG serum with the corresponding antigen, which was especially characteristic in the excess antibody zone, showed that porcine antibodies are of the precipitin, or "R", type. This finding was verified in numerous immunoelectropho- retie analyses of the sera with all classes of myeloma proteins; although these sera, apart from a large amount of myeloma protein, frequently contain a low, or only minute, amount of the other immune- globulins, even these amounts can be demonstrated by porcine antisera. An analysis of undiluted myeloma serum, in cases in which the pathological protein reacts, actually represents a reaction based on a large amount of excess antigen and in cases in which the other immunoglobulins

react, a reaction based on a large amount of excess antibody.

The results of orientation tests showed that absorbed anti-yG and anti-yA sera posses a given discriminative cal~acity in relation to the heterogeneity of the heavy chains of the relevant classes. On the basis of present analyses, these findings cannot be correlated with similar properties of monkey or rabbit antisera (Dray, 1960; Lichter & Dray, 1964; Terry & Fahey, 1964; Frangione & Franklin, 1965; Terry, 1966); this problem requires further study.

From all the above results it can be concluded that porcine sera against human immunoglobulins are equivalent, as re- gards sensitivity and discriminative capacity, to the rabbit sera hitherto used. Their only possible deficiency is the absence of antibodies against surface determinants of the ~ chains.

The authors would like to t h a n k Dr. Tr~vntSek of the Ins t i tu te of Microbiology, Czechoslovak Academy of Sciences, Prague, for his active help with the methods in the init ial stage of immuniza- t ion of the pigs. They would also like to express thei r appreciat ion to Mrs. Sedmihorsk~ for the excellent immunoelcctrophoretie analyses and to Mrs. Karlikov~, Mrs. Vohnoutov~ and Mr. Sokol for technical assistance with immunizat ion of the pigs.

R e f e r e n c e s

Cohn, J . E., Strong, L. E., Hughes, W. L., Mul- ford, D. J . , Ashworth, J . N., Melin, M., Taylor, A. L.: Preparation and properties o] serum and plasma proteins. J . Am. chem. Soc. 68 : 459, 1946.

Dray, S.: Three v-globulins in normal human seru~ revealed by monkey precipitins. Science 132 : 1313, 1960.

Fahoy, J. L., McLaughlin, C.: Preparation of antisera specific ]or 6,6 S y-globulins, fl~A.globullns, 71-macroglobulins, and ]or type I and 1I com~non y-globulin determinants. J. Immunology 91 : 484, 1963.

Frsn~k, F., ~imok, L., Kot~nok, 0 . : An$idinitro. phenyl antibodies in pigs and bulls. Fol. microbiol. 10 : 335, 1965.

Frau~k, F., Zorina, O. M.: On proteins. GX. Isola- tion and characterization o] X- and 7~-chains representing structural types o] pig yG-globulin ligh~ chains. Collection Czechoslov. Chem. Comm. 32 : 3229, 1967.

Frangione, B., Frankl in , E. C.: Structural s~ud~es of human immunoglobulins. Difference8 in the -~d ]ragments o] the heavy chains o/ G myeloma pro- reins. J . exptl. Med. 1 2 2 : 1 , 1965.

Hofldelberger, M., Kendall , F. E.: The precipitin reaction between type 1 H 2rneumocoeeus poly- ~ascha~ide and homologous antibody. J. exptl . Med. 61 : 563, 1935.

Heremans, J. :F., Vaerman, J . P., Carbonate , A. O., Rodhain, J . A., Heremans, M. Th.: 71A-gtobulin: Its isolation, properties, /unctions and pathology. In: Proceedings o/the lOth Colloquium Pratldes of the biological fluids. Ed. H. Peeters. Publ . Else- vier Publ. Co. Amsterdam 1963.

Hirschfeld, J . : Characterization o] preeipitaJing components in normal hunmn sera obtained by an immunoeleetrophoretir technique. Act s ps th . mi- crob. Scsnd. 4 9 : 2 5 5 , 1960.

Ho~ejw J. , Smetana, R.: The effect of rivanol or,

1968 P R E P A R A T I O N OF PORCINE SERA 489

plasma proteins. (In Czech.) Chem. listy 48 : 758, 1954.

Kabat , E. A., Mayer, M. M.: Experimental Ira. munochenvistry. Publ. Charles C. Thomas, Spring- field, Illinois, ~rSA, 1961.

Levy, B. H., Sober, H. A.: A simple chromato- graphic method ]or preparation of gamma-globulin. Prec. See. exp l . Biol. Med. 103 : 250, 1960.

Lichter, E. A., Dray, S.: lmmunoetectrophoretic characterization o/ human serum protein~ with primate antisera. J. Tmmunology 92 : 91, 1964.

Oneley, J . L., Melin, M., Rieher~, D. A., Cameron, g. W., Gross, P. M. Jr . : The separation of the antibodies, isoagglutin/ins, prothrombin, plasmino- gen and betaz-lipoprotein into subfractions of human plasma. J . Am. chem. Soc. 71 : 541, 1949.

l ~ d l , J. , Masopust, J. , Laekovg~, E.: Selective by. perimmunoglobulinemia A and D in a case with chronic generalized eczema and prolonged eepsis. Helv. Paediat. Ac t s 3 : 278, 1967.

Rejnek, J. , Kostka, J . , Tr~vnl6ek, J . : Stud/es on the immunoglobutin spectrum of porcine serum and caloatrum. Fol. mierobiol. 11 ; 173, 1966.

~kva~l, F., Rejnek, J . : Our experience with micro- modification o] immunoelectrophoresis. (In Czech.) ~s. epidemiol, microbiol, immunol. 7 �9 414, 1958.

~kva~il, F., Brtmamelovs V.: Isolation o] com- ponent.9 Item ]ragmented normal human 7G-ira. munoglobulin Collection Czeehoslov. Chem. Comm. 33 : 2316, 1968.

~terzl, J. , Rejnek, J . , Tr~vn/6ok, J . : Impermeal~ili- ty o] pig placenta /or antibodies. Fol. microbiol. 11 : 7, 1966.

Tan, M., Epstein, W. V.: A direct immunologio assay ol human sera for Benee Jones proteins (L-ehain..s). J . lab. elin. Med. 66 : 344, 1965.

Terry, W. D., Fahey, J . L : Heterogeneity of the H chains of human 7 S 7-globulin. Fed. Prec. 23 : 454, 1964.

Terry, W. D : Antlgengc heterogeneity o/human immunoglobulin A proteins. Information Exeh. Gr. No. 5 (Tmmtmopathology), so. memo 189, 1966.

Tm'aer, M. W., Rowe, D. S.: A naturally occurrir~g ]rayment related to the heavy chedns of ~mmuno- globulin G ~n normal human urine. :Nature 210 : 130, 1966.

The plates and Table 1 will be found at the end of the tex t section - - p. 574

preparation and evaluation of porcine sera against human immunoglobulins

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