CROSS-REACTIVE PROPERTIES OF ANTISERA PREPARED IN RABBITSBY STIMULATIONS WITH TELEOST VITELLINS

By FRED M. UTTER, Chemist and GEORGE J. RIDGWAY. Biochemist.1 BUREAU OF

COMMERCIAL FISHERIES BIOLOGICAL LABORATORY, SEATTLE. WASH.. 98102

ABSTRACT

Antisera were prepared by injecting rabbits with egg orserum vitellin preparations from seven teleost species be­longing to different families. The ranges of reactivity ofthese antisera were tested with sera from mature females ofnine teleost families as well as with sera from females ofspiny dogfish. Pacific lamprey. and white sturgeon. All ofthese antisera reacted with vitellins from all species testedfrom the homologous families. Antisera prepared against

Fishery researchers have used serologicaltechniques with increasing frequency since1950. Much of the work has been directedtoward identification of populations eitherthrough blood-grouping techniques or studiesof variable serum antigens (Cushing. 1964).Ridgway. Klontz, and Matsumoto (1962) ob.­served a characteristic antigen in the serum ofmaturing and mature female sockeye salmon(Ol/corhYl/chus lIerka). A review of the litera­ture and subsequent studies by our group hasrevealed that similar components occur notonly in all the teleosts but also in all vertebrateclasses were oviparity occurs (Urist andSchjeide, 1961; Drilhon and Fine. 1963). Theseantigens appear to have considerable practicalvalue in investigations of maturity in femalefish because of their connection with the proc­ess of sexual maturation (Ho and Vanstone,1961; Olivereau and Ridgway. 1962; Ridgway,1964: Utter and Ridgway, 1966).

The function of the blood semm as a trans­porting medium between the site of synthesisin the liver and the site of storage in the ovaryappears to explain the presence of yolk com­ponents in the blood (Vanstone. Maw. andCommon, 1955). The serum vitellins studiedhave displayed similar biochemical properties,are characterized as phospholipoproteins. and

FISHERY BULLETIN: VOLUME 66. NO.2

rockfish and flounder vitellins cross-reacted with sera frommature females of all teleost species tested. A greater anti­genic complexity in the vitellins of more taxonomicallyadvanced species than more primitive species is indicatedby the results of the reactions and absorption tests. Theresults are of practical importance in studies on maturity offishes and have theoretical implications in the field ofsystematics.

conform to the classical description of avianvitellin as the water-insoluble fraction of theegg yolk (Jukes and Kay. 1932; Vanstone.Maw, and Common, 1955).

This report is based on data obtainedthrough testing numerous antisera for cross­reactive properties. The antisera were pre­pared in rabbits against vitellins of teleosts.We intend to bring out points of both practicalsignificance and theoretical interest.

METHODS AND MATERIALS

IMMUNOLOGICAL TESTS

A microslide adaptation of the double dif­fusion method of Ouchterlony as modified byRidgway, Klontz. and Matsumoto (1962) wasused for all serological tests. The agar mediumconsisted of 1.5 percent Difco agar. 0.72 per­cent sodium chloride. 0.60 percent sodium ci­trate. 0.01 percent merthiolate. and 0.01 per­cent trypan blue. Wells were punched in theagar at 8-mm. intervals and filled to a volumeof about 0.01 cc. of reactant. Slides were eval­uated after 24 hours of incubation at 37° C.

PRODUCTION OF ANTISERA

Table 1 lists the antisera used in this study.Egg vitellin preparations were made by blend­ing and centrifuging one part eggs with three

203

TADLE I.-A nti.~crn used in this study

2 Traut:' 11811Jt:'S r('ferrell tv in thi;.,. 1,lIhli'.'otion do not irnpl~·

l'ntlr,rM!'III'1I1 Ilf f'hlllillp-r,·ial PTf)Ullf-t:-;.

IN umberDesignation \'it~Bin source ofI rahhits------ -------------------------------i-----

REACTIVE PROPERTIES OF THEANTISERA AND VITELLINS TESTED

Table 2 summarizes the data obtained

R~agent

All antisera reacted strongly with sera frommature females within the families that pro­vided the vitellin for antibody stimulation.Because of this high degree of cross-reactivitywithin families, the reactions of the antiseramay be considered mainly with regard to thefamily rather than the species from which thevitellin used for antibody stimulation origin­ated. The arbitrary designations given intables 1 and 2 refer to vitellin of any species ofthat family.

Four of the reagents also reacted stronglybeyond the immediate family group; two of

II) Pacific lam pre)'. Lampelra Iridmlala: I:?I spiny dogfish, 8,/uall/s aMlIthia.;(3) Whitc sturgcon, .·Iei".."s" lrall.monlallll.: 14.) shad, Alosa sapidi..;ma:Pacific herring, ('Iu",a h/lrenglL' palll/si: (5) ~oek~ye salmon, OllcorhYllrhllslluka; 101 e.arp, Cypril/lls carpio: northern sqlIawflsh Plyrhochtilll. orego/l­tnsis: largescale SlIcker, ('alosIMnll.• ma<'rocheill/S: (7) Pacific hake, lIlerlllccillsprodllcill.: Pacific cod. Gadlls macrocephallls: (8) higeye tuna, Thll./II1I1SolltslI.: Pacific mackerel, 8coml'tr ja"onirlls: IQ) copp~r rockfish, St"~aslodt8

callrhlllS: 110, cabezoll. Scorpamirhlhys marmoraills: staghorn sculpin,Lepiotollll. an/laills: (11) sand sole, Parllith/hy. mtianosliclll.s: Engli~h sole.Parophrys "elllills: starr)' t1oulld~r. Plalithlhys sidlaills: (121 northern mid­shipman, Porirhlhy, 1I01a11lS.

through testing of sera from mature females ofvarious fish species. All antisera were testedwith the same fish sera; this testing includedmales as well as females from most species.The only reaction with male serum occurredbetween the antirockfish reagent and male rock­fish serum. This reaction was very weak andwas most likely the result of nonvitelIin anti­gens present in the injected material. The re­action with male rockfish sera could not beconfused with the reaction with female rockfishsera.

TABLE 2.-Cross-reueti,'it.lJ of rabbit u-Il·titelrost "itellin sera withIli/eilius of Ji41 reprM('ntiug mriolls t(uoWllI/-i(' groups I

lX=strong reaction; W = weak r~action;0= no re.aetionl

I~;~~:~s:~ I I __~~~~~_I __I----

1~1~ I I I I I I:: Sj

'~Iili ~I!I~I-I~ ~ §Ii i. ~ .- ~ ~ - c ~ - - -l~ -

Ii jljl~l: ~ i j ~ iljl~< ~ ~ u W u ~ ~ 00 u ~ ~

------------1-- __1__ -- -- -- ------ -- --I--AntI· ICllIpeidICLMI. __ 0 0 0 X 0 0 0 0 woo 0

Salmollid ISM.'____ 0 0 0 0 X 0 0 0 woo 09·p~inid.(CM)---.I1 0 I 0 I X 0 W X ~ I q 0 0 0 I 0Gad.d I"M ,- --- - -- " " " " " 0 X Iwoo 0 USeomhrid ITM) __ ._I 0 '0 0 W W W X X X X,X 0Scorp~nid IRMJ __ I 0 I "I X X X IX 1 X X IX IX IX I XPlellron~ctidIHM)_, 0 0 0 X X X IX X X X X X

I3I

I135

parts 1 percent saline in a Waring Blender.~

After centrifugation, the addition of 11 partsof distilled water precipitated the vitellinsfrom the supernatant fluid. The precipitatewas dissolved again in saline, reprecipitatedand redissolved, and used for injections. Wholeserum from a mature female Pacific cod wasused to produce the anticod vitellin reagent.The resulting antiserum was absorbed at a 1:1ratio with male cod serum before testing.Usually the vitellin-bearing materials weresuspended in a bayol-arlacel mixture and in­jected into the rabbits intraperitioneally. Con­sistently uniform results were obtained whenother .injection procedures were used, but agreater mimber of injections was usually re­quired. Single bleedings were used for testingwith the exception of the reagent preparedagainst starry flounder vitellin which was apool of numerous .bleedings from five rabbits.The antisera produced in different rabbits in­jected with the same vitellin material werequalitatively very similar. This uniformity ofreagent indicates that the differences reportedlater are not due to variations in the immuneresponse of individual rabbits.

COLLECTION OF SERUM SAMPLES

Samples of fish serum were taken fromwhole blood that had been processed within 48hours after collection; the samples were thenstored at _35 0 C., a temperature at which thevitellin fraction appeared to be stable. Somesera had been stored as long as 8 years whentested.

Anti-CLM P"eifle herring lelll",a harOlglls pallasi) egg. _Auti·SM Chinook salmon IOl/corhYllchlls Ishall·ylscha) egg._Anti-CM ____ Northern squaw fish I.Plychochrillls oregollclI.i81

eg!!s.Autl·GM. ___ Pacific cod IOadus macrocepholll') serum _Auti·TM \. ,"cBowfln tuna IThllllllll. alhacarC81 eggs. _AnH·R M .. ___ Copper rf>Ckflsh (Sella,lod.., callrin ",\ ~ggs .. :Anti-HM

1

Starr)'lIouJ1(ler (PlalichlhY"'ella'll81 cggS---------i

204 U.S. FISH AND WILDLIFE SERVICE

[X= strong reaction; W= weak reaction; O~ no reaction. See figure 1J

TABLE 3.-Results of absorption~ of rabbit antirockfi·sh vitellin(RM) serum with sera of mature female teleosts

Unabsorbed.._... X X X X X XRockfish ......... 0 0 0 0 0 0sOle··············

1

X 0 W 0 0 0Tuna. ___________ X 0 0 0 0 0Cod.............. X X X 0 0 0Carp·············1 X X X

IX 0 X

Salmon··········1

X I X X X 0 0

I Carp iSalmon

Fish sera tested

I II Tuna CodRockfish Sale

Fish sera usedfor absorption

ponents. The SM vitellin cross-reacts com­pletely with the antibodies directed against oneof these components. Two components arevisible in figure 2b that react wi.th the anti­SM antiserum. The RM vitellin cross-reactspartially and very weakly with the antibodiesdirected against one of these components.

Comparisons similar to those presented infigure 2 were made between the broadly cross­reactive antisera (anti-RM and anti-HM), theless cross-reactive antisera (anti-CLM, anti­SM, anti-CM, and anti-GM), and the cor­responding vitellins. All results were similar.The weak or negative reactions of the RM andHM vitellins with the less cross-reactive orgroup-specific antisera contrasted with thestrong reactions of the anti-RM and anti-HMantisera with the vitellins which elicited theless cross-reactive antisera present an interest­ing serological phenomenon. The cross-reactiveantibodies of the anti-RM and anti-HM anti­sera apjear to have a considerably greateravidity for the vitellins which elicit group­specific antisera than the group-specific anti­bodies have for the RM and HM vitellins.

2

3

FIGURE l.-Reactions of sera from mature females ofsix teleost species with rabbit anti-RM serum. Peri­pheral wells contain sera from 1, copper rockfish;2, sand sole; 3, bigeye tuna; 4, Pacific cod; 5, carp;6, sockeye salmon.

female teleosts of six different families whentested with the anti-RM reagent. The strongestreaction was with the female rockfish serum.The degree of cross-reactivity regularly de­creased through the somewhat distantly relatedsalmonoids and cyprinoids. Even in thesegroups, however, the reaction was clear.

them reacted distinctly with sera from maturefemales of all teleost species tested. Figure 1illustrates the reactions of sera from mature

Table 3 gives the results of absorptions ofthe anti-RM reagent with the fish sera of figure1. It is evident from both figure 1 and table 3that the anti-RM reagent contains antibodies ofnumerous specificities.

Figure 2 presents a more detailed examina­tion of the relationship betvveen SM and RMvitellins. It is evident from figure 2a that theRM vitellin has at least three distinct com-

Both the homologous and cross-reachingheterologous antisera gave uniform resultswhere tested with a larger number of individ­uals. Randomly selected sera from 48 sockeyesalmon were tested with anto-SM, anti-RM, andanti-HM reagents; 48 halibut sera were testedwith the anti-RM and anti-HM reagents. Re­sults were identical regardless of the antiserumused, including two weak but positively react­ing halibut sera.

CROSS-REACTIVE PROPERTIES OF ANTISERA 205

a.

FIGURE 2.-The relation bebyeen 8M and RM vitellinsdetected (a) by rabbit anti-RM serum and (b) byrabbit anti-8M serum. Arrow in (b) indicates the

PRACTICAL APPLICATIONS FORSTUDIES OF MATURITY

The broad cross-reactive range of antiseraproduced against the vitellins of rockfish andstarry flounder is of practical importance. It islikely that these reagents react with serumvitellins from mature female teleosts at leastthrough the taxonomic range of this study. Aninvestigator wishing to include serological datain maturity studies may therefore use a singlereagent throughout a range of teleost speciesrather than produce different antisera forrelatively limited taxonomic groupings. Thenecessity of obtaining vitellin-bearing materialfor immunizations from species where suchmaterials would be difficult to obtain or processis also eliminated.

The data suggest that vitellins of the mosttaxonomically advanced species stimulate thehighly cross-reactive antisera. Some theoreticalimplications of this apparent trend are dis­cussed below. As a practical consideration,however, it appears that vitellin from Perci­form or closely allied species may be most likelyto stimulate antisera which have broad cross­reactive properties.

206

b.

partial cross reaction between RM vitellin and theanti-8M reagent.

SYSTEMATIC CONSIDERATIONS

Nuttall (1904) in an early immunologicalstudy, observed that the quantities of precipi­tates formed by specific antigen-antibody inter­actions decrease as the taxonomic relationshipsbecome more distant from the materials usedin antibody stimulation. The present studyagrees generally with this observation. Asillustrated by figure 1, the broadly cross-reac­tive antisera reacted most strongly with thehomologous vitellin and least strongly with themost distantly related cpyrinid and salmonidvitellins. A notable exception is the reaction ofsturgeon vitellin with anti-cyprinid vitellin, anantiserum which fails to react with vitellins ofnumerous, more closely related, groups. Theother execptions include the weak cross-reac­tions of antisalmonid vitellin with rockfishvitellin but not with herring or carp vitellin,and the similarly weak cross-reaction of anti­clupeid vitellin with rockfish vitellin but notwith salmon vitellin.

Fine, Buffa, and Drilhon (1964) found acomponent in mature female marine lampreysanalogous to the teleost vitellins described inthis report. The spiny dogfish egg, unlike those

U.S. FISH AND WILDLIFE SERVICE

of many sharks, is provided with an abundanceof yolk material. A Saline extract of the dogfishyolk material was tested in addition to the serafrom numerous adult dogfish; this extract alsofailed to react with any of the antisera used inthis study. The lack of reactivity observed herewith yolk materials from females of dogfish orlampreys appeal'S to reflect the phylogeneticgap between the teleosts and these more primi­tive vertebrates.

The vitellin substances of the advanced tele­osts that stimulate pl'oduction of the broadlycross-reactive antisera appears to be biochemi­cally and antigenically more complex than thoseof the more primitive teleosts. It is evidentfrom figure 1 and table 3 that only a smallfraction of the total number of anti-RM: anti­bodies react with SM vitellin; the major anti­genic vitellin component of SM: is detected,however, by the anti-RM reagent (Figure 2).Possibly the vitellin antigens of more primitiveteleost species have been retained in certainadvanced species without extensive modifica­tion during the evolution of additional vitellinsubstances.

This study further demonstrates the useful­ness of serological methods to determine ma­turity in oviparous vertebrates. The results arealso of significance in systematics. The exist­ence of antigens in the sera of maturing fe­males which do not occur in the sera of malesand immature females must be taken into ac­count in studies that attempt to apply serologyto problems of taxonomy. These antigens them­selves, as was demonstrated here, also offeradditional materials for more detailed examina­tions of systematic relationships.

LITERATURE CITED

CUSHING, JOHN E.1964. The blood groups of marine animals. Adv.

Mar. BioI. 2: 85-131.

CROSS-REACTIVE PROPERTIES OF ANTISERA

DRILHON, A., and J. M. FINE.1963. Dimorphisme sexuel dans les proteines se­

riques de Sa.lmo salar: Etude electrophoretiqueCompt. Rend. Soc. BioI. 157(9): 1897-2000.

FINE, J. M., G. A. BOFFA, and A. DRILHON.1964. Etude electrophoretique et immunologique

des proteines seriques de la lamproie marine(Petrom.yzooll marinus L.) Compt. Rend. Soc.BioI. 158 (10): 2021-2025.

Ho, F. CHUNG-WAI, and W. E. VANSTONE.1961. Effect of estradiol monobenzoate on some

serum constituents of maturing sockeye salmon(Oncorhynchus nerka). J. Fish. Res. Bd. Can.18: 859-864.

JUKES, T. H., and H. D. KAY.1932. Egg-yolk proteins. J. Nutr. 5: 81-101.

NUTTALL, G. H. F.1904. Blood immunity and blood relationship.

Cambridge Univ. Press, New York, 444 pp.OLIVEREAU, MADELEINE, and GEORGE RIDGWAY.

1962. Cytologie hypophysaire et antigene seriqueen relation avec la maturation sexuelle chexOncorhynchus species. Compt. Rend. SeancesAcad. Sci. 254: 753-755.

RIDGWAY, GEORGE J.1964. Salmon serology. In U.S. Bureau of Com­

mercial Fisheries, Report on the Investigationsby the United States for the International NorthPacific Fisheries Commission-1962, pp. 107-110.Int. N. Pac. Comm. Annu. Rep. 1962.

RIDGWAY, GEORGE J., G. W. KLONTZ, and C. MATSUMOTO.1962. Intraspecific differences in serum antigens

of red salmon demonstrated by immunochemicalmethods. Int. N. Pac. Fish Comm., Bul. 8: 1-13.

URIST, MARSHALL R., and ARNE O. SCHJEIDE.1961. The partition of calcium and protein in the

blood of oviparous vertebrates during estrus. J ..Gen. Physiol. 44: 743-756.

UTTER, FRED M., and GEORGE J. RIDGWAY.1967. A serologically detected serum factor as­

sociated with maturity in English sole, Paro­phyrys vetulus. and Pacific halibut, Hippoglos­sus stenolepis. U.S. Fish. WildI. Serv., Fish.Bull. 66: 47-58.

VANSTONE, W. E., W. A. MAW, and R. H. COMMON.1955. Levels and partition of the fowl's serum

proteins in relation to age and egg production.Can. J. Biochem. Physiol. 33(6): 891-903.

207