[CANCER RESEARCH 32, 2753-2760, December 1972]

Radioimmunoassay for a ,-Fetoprotein in the Serum of Rats1

David D. Oakes, Joseph Shuster,2 í¡ndPhil Gold3

The Division of Clinical Immunology and Allergy and the McGill University Medical Clinic of the Montreal General Hospital, Montreal, 109,Quebec, Canada

SUMMARY

A radioimmunoassay for the detection of a ]-fetoprotein

(AFP) in the sera of rats is described. The procedure is basedon the preparation of purified and radiolabeled AFP,monospecific anti-AFP antiserum, and a modification of thecoprecipitation-inhibition technique in 50% saturatedammonium sulfate. A detailed description is given of thecombination of immunochemical and physiocochemicalmethods used in the purification of the rat AFP. The purifiedrat AFP is virtually identical to the native, circulating AFP byimmunological criteria and contains, at most, trace contamination with other materials. The radioimmunoassay has asensitivity that allows 25 ng AFP per ml of serum to bedetected with reproducibility. The assay requires 20 n\ ofserum for analysis and can be completed within a few hours.

INTRODUCTION

Since 1944, a variety of "fetoproteins" have been described

in several mammalian species (6). These constituents are ofhepatic origin and are distinguished by the fact that they arepresent in fetal sera but absent from the sera of thecorresponding adult animals.

In 1963, Abelev et al. (2) reported the synthesis, inchemically induced hepatomas of mice, of an a-globulin thatwas antigenically identical to that found in embryonic andneonatal mouse serum. Within the next few years,species-specific AFP's4 were detected in the sera of rats,

nonhuman primates, and humans harboring primary hepatomas (1,8).

In any 1 species, AFP production by primary liver tumorsdoes not appear to be a universal phenomenon. Thus, themajority of hepatomas that have been induced in rats by thefeeding of DAB produce AFP, while those induced by

'Supported by a grant from the Medical Research Council of

Canada, Ottawa, Ontario, Canada.- Scholar of the Medical Research Council of Canada.3Associate of the Medical Research Council of Canada.*The abbreviations used are: AFP, a, -fetoprotein; DAB, 4-dimethyl-

aminoazobenzene; anti-AFP, sheep anti-a,-fetoprotein antiserum; FF,rat fetal fluid; anti-FF, sheep anti-fetal fluid antiserum; SSC, sheepserum components; IEP, immunoelectrophoresis; RIEP, radioimmuno-electrophoresis; NRS, normal adult male rat serum; AFP-125!,radioiodinated purified rat AFP; anti-SSC, rabbit antiserum directedagainst sheep serum components; anti-NRS, sheep anti-normal adultmale rat serum.

Received July 10,1972; accepted September 5,1972.

aflatoxin are apparently not associated with AFP synthesis (4,14). This difference in biochemical expression may be due toalternative pathogenetic mechanisms in hepatoma development or, more simply, to quantitative variations in AFPproduction by tumors of different etiologies. In the majorityof studies concerning AFP synthesis by rat hepatomas,techniques based on double diffusion in gel media have beenused. Such procedures are highly specific but are, however,incapable of detecting antigen concentrations below 2.5 /¿g/mlof serum (10) and might well fail to distinguish betweenqualitative and quantitative variations in AFP production bydifferent tumors.

Hence, if the capacity for AFP synthesis is to be used as auseful criterion in distinguishing different classes of rathepatomas and the factors underlying their development, itwill be necessary to design assay procedures for circulatingAFP that are far more sensitive than those presently available.It was the objective of the present series of studies to developsuch a technique.

MATERIALS AND METHODS

Production of Monospecific Anti-AFP. Wistar rat fetuseswere obtained at 14 to 16 days of gestation by anesthetizingthe pregnant females and delivering the fetuses by Caesariansection. The fetuses were immediately coarsely minced in theirown amniotic fluid with stainless steel scissors. The preparation was then centrifuged at 20,000 X g for 30 min at 4°;the resulting supernatant was called "FF" and served as the

source of AFP. The FF was subjected to ascendingchromatography on a calibrated Sephadex G-200 column (5 x100 cm), equilibrated with 0.02 M Tris-0.15 M NaCl buffer,pH 8.2. Elution was carried out at 30 ml/hr with the samebuffer, and the eluate was continuously monitored spectro-photometrically at 280 nm. Two distinct peaks were obtained.The eluted fractions associated with the 2nd peak,corresponding to a molecular weight of 70,000, were pooledand concentrated in an Amicon ultrafiltration chamber fittedwith a PM-30 membrane. The concentrate was adjusted to aprotein content of 3 mg/ml and emulsified in an equal volumeof complete Freund's adjuvant. Sheep, weighing approxi

mately 20 kg, were given an initial i.m. immunizing injectionof this material, containing 1.0 mg of the fetal protein. Theseanimals received subsequent injections containing 0.5 mg ofprotein at 1- to 2- week intervals. After 3 months ofimmunization, the sheep were bled. One aliquot of serumremained untreated and was called "anti-FF." Each ml of the

remaining serum was absorbed with 200 mg of the whole,water-dialyzed, and lyophilized residue obtained from pooled

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David D. Oakes, Joseph Shuster, and Phil Gold

serum of normal adult male Wistar rats. The absorbedantiserum was called "anti-AFP."

Preparation of Purified Rat AFP. The method of AFPpurification was a modification ofthat described by Nishi (12)for human AFP. Aliquots of both fetal fluid and anti-AFPantiserum were initially centrifuged at 43,000 X g at 4°for 1

hr, and the supernatants were harvested. On the basis ofequivalence zone determinations in Ouchterlony plates 90 mlof anti-AFP antiserum were admixed with 6 ml of fetal fluid.The mixture was incubated at 37°for 1 hr and then at 4°for

48 hr. The antigen-antibody precipitate that formed wassedimented by centrifugation at 10,000 X g at 4" for 20 min.

The supernatant was removed, and the pellet washed byresuspension in approximately 10 times its volume of iced0.9% NaCl solution, followed by centrifugation at 10,000 X g.The supernatant was discarded and this process of washing wasrepeated 3 more times.

The washed antigen-antibody precipitate was then dissociated in 0.1 M glycine-HCl buffer, pH 1.8, and was kept insolution in this manner for 30 min at 4°. The acidifiedpreparation was then centrifuged at 10,000 X g at 4°for 20

min in order to sediment any acid-insoluble material. Thepellet was discarded and the supernatant was adjusted to pH7.0 with 0.4 M Na2 HPO4. The neutralization of the solutionresulted in the reappearance of the original antigen-antibodyprecipitate. Reassociation was allowed to proceed for 1 hr at37°and then at 4°for 24 hr.

After 4 complete cycles of washing in 0.9% NaCl solution,acid-dissociation, and neutralization-reassociation, a finalacid-dissociation was performed, and the solution was appliedto a Sephadex G-150 column (2.5x100 cm), which hadpreviously been equilibrated with the glycine-HCl bufferdescribed above. Ascending chromatography at 20 ml/hr wasperformed with the same buffer system. The Chromatographiecolumn was kept at 4°to minimize hydrolysis of the Sephadex

beads, and the procedure was continuously monitoredspectrophotometrically at 280 nm. Each 5-ml aliquot ofcolumn effluent was collected into 4 ml of 0.4 M Na2HPO4 inorder to achieve rapid neutralization of the eluate.

With the Mancini technique of radial diffusion in agar gel(see below) for the quantitative determination of sheep serumconstituents and rat AFP, the fractions of the eluatecontaining rat AFP but devoid of demonstrable SSC werepooled and concentrated by ultrafiltration in an Amiconchamber fitted with a PM-30 membrane. The concentrate wasapplied to a Sephadex G-200 column (0.9 x 100 cm)equilibrated with 0.02 M Tris-0.15 M NaCl buffer, pH 8.2.Descending chromatography at a rate of 4 ml/hr was carriedout with the same Tris buffer. The eluate was monitored at280 nm in a Zeiss PMQ-II spectrophotometer fitted with an8-fzl flowthrough cell. Two spectrophotometric peaks wereobtained, and the material represented in the 2nd peakdemonstrated only AFP activity by the Mancini reaction. Thismaterial was termed "purified AFP."

Radioiodination of the Purified AFP Preparation. Thepurified AFP was radioiodinated by the chloramine-Ttechnique (7). The reaction mixture contained 50 ng of thepurified AFP in 250 Ailof 0.05 M phosphate buffer (pH 7.3),100 Me of chloramine-T in 100 /ul of the same diluent, and

1.85 mCi of 125I in 5 ßlof dilute NaOH (pH 8 to 9). The

reaction was permitted to proceed for 75 sec at roomtemperature (18°) and was then stopped by the addition of

200 /ng of sodium metabisulfite in 100 ¿/Iof the phosphatebuffer described above. Three drops of a 10% KI solution wereadded to the quenched reaction mixture which was thenapplied to a Sephadex G-25 column (1 x 45 cm), previouslyequilibrated with the same phosphate buffer. Chromatographywas performed by descending flow, and 1-ml aliquots werecollected into tubes containing 1 ml of 5% bovine serumalbumin in the same buffer system. The quantity of 125I in

each of the fractions was determined in a Nuclear-Chicago dualchannel automated scintillation -y-ray analyzer.

Immunochemical and Physicochemical Criteria of Purity.Ouchterlony and radio-Ouchterlony reactions, IEP and RIEP,were performed as previously outlined (15). Theradioprecipitin patterns were developed on Singul-XNon-Screen Safety Blue Base polyester X-ray film, EM No.220421 (AB Ceaverken, Strangnas, Sweden).

The Mancini technique was used as originally described(11). The anti-AFP was incorporated into the agar gel fordetermining relative concentrations of rat AFP, andhyperimmune rabbit antiserum (Behringwerke, CanadianHoechst Ltd., Montreal, Canada, Lot 1163 P) was incorporated into the agar gel for the detection of SSC.

Radioimmunoassay of Rat AFP. The radioimmunoassay wasbased on a modification of the coprecipitation-inhibitiontechnique described by Farr (5). NRS was diluted 1:20 in 0.05M borate buffer, pH 8.6, and was used as diluent for theanti-AFP antiserum. A 1:100 dilution of the same serum inborate buffer served as diluent for the AFP-12 51.

A titration curve of anti-AFP against AFP-12 s I wasobtained as follows. Serial dilutions of 500-/nl samples ofanti-AFP were prepared in duplicate. An aliquot of 500 fu ofAFP-1251, containing about 20,000 cpm and representing

approximately 1 to 2 ng of AFP, was then added to each tube.After 2 hr of incubation at 37°,the tubes were chilled in anice bath and 1 ml of saturated ammonium sulfate at 4°was

added to each tube with immediate vortex mixing. The tubeswere kept at 4°for 30 min and were then centrifuged at

15,000 X g for 15 min. Each supernatant was decanted, and itscontent of AFP-12 51 was determined.

A standard inhibition curve for the AFP assay was obtainedas follows. Doubling quantities of purified AFP in 20-/nlvolumes were added to a series of tubes in duplicate. Theanti-AFP antiserum was diluted to a final concentration of1:50,000, and 500 /LI!were added to each tube. The reactionmixture was incubated at 37°for 2 hr, after which 500 ;ul ofAFP-1251 were added to each tube and the incubation was

continued at the same temperature for another 2 hr. At theend of this period, 1 ml of saturated ammonium sulfate at 4°

was added to every tube with immediate vortex mixing. Thetubes were kept in an ice bath for 30 min and then centrifugedat 15,000 X g for 15 min at 4°.The supernatants weredecanted, and the AFP-12SI content of each was determined.

Radioimmunoassays for AFP were performed with 20-julaliquots on various dilutions of FF, maternal serum obtainedimmediately postpartum, and sera from rats bearing primaryhepatomas induced by the feeding of DAB. Dilutions were

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Radioimmunoassay for Rat AFP

always performed with NRS. The assay was carried out in amanner identical to that described for the standard inhibitioncurve, except that the 20 ß\of material to be analyzedreplaced the purified AFP standard.

RESULTS

Specificity of Anti-AFP. The precipitin pattern thatdeveloped between the FF and the corresponding untreatedantiserum is shown in Chart 1. A number of distinct precipitinlines formed. However, after absorption with normal adult

Chart 1. Ouchterlony plate. The single precipitin band between FFand anti-AFP shows a reaction of identity with one of the several bandsbetween FF and anti-FF but no identity with any of the heavy bandsbetween NRS (used to render anti-AFP monospecific) and anti-NRS.

male Wistar rat serum powder, the anti-AFP produced only asingle precipitin band against the FF. This finding wassupported by 1EP where a single arc formed, in the at -globulinregion, between the anti-AFP and the FF (Chart 2).

Preparation of Purified AFP. Chart 3 shows thespectrophotometric pattern, obtained upon Sephadex G-150chromatography, of the washed and acid-dissociatedprecipitate which had initially developed between theanti-ÕFP and the FF. Two peaks were detected. Mancinireactions revealed the maximal concentration of SSC in the2nd peak, with lesser amounts in the 1st peak. Rat AFP could,however, be detected only in and immediately after theshoulder of Peak 2.

The fractions that contained the AFP but were apparentlyfree of sheep serum (Chart 3) were pooled, concentrated, andapplied to the Sephadex G-200 column. The Chromatographiepattern obtained is shown in Chart 4. Of the 2 peaks detected,Mancini reactions indicated that the 2nd peak contained AFPbut no demonstrable SSC.

Chart 2. IEP. A single band appears between FF and anti-AFP in thea-globulin region, but there is no counterpart between NRS andanti-AFP. However, a corresponding a-globulin band appears betweenFF and anti-FF. Both albumin and 7-globulin marker bands are seenbetween FF and anti-FF and between NRS and anti-FF.

15r

oco

*

Elution Volume (ml)

Chart 3. Column chromatography on Sephadex G-150 of acid-dissociated AFP-anti-AFP.AFP (Mancini); hatched area, eluate containing AFP but no SSC.

200

, percentage absorption at 280 nm; »,SSC: o,

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David D. Oakes, Joseph Shuster, and Phil Gold

Purity of the Final AFP Preparation. The degree of purityof the final AFP preparation is indicated in theradio-Ouchterlony plate of Chart 5. The autoradiographrepresents the interaction of AFP-12 51, admixed with purifiedAFP as carrier, against anti-NRS, anti-FF, anti-AFP, andanti-SSC. Single precipitin and radioprecipitin lines developedbetween the purified and radiolabeled AFP and the antiseradirected against both AFP and FF. These bands showed areaction of identity. No reactions against the purified AFPwere, however, detected with either the anti-NRS or theanti-SSC.

Further tests of purity of the final AFP preparation, andcoincidentally the monospecificity of the purifiedAFP-anti-AFP system, are presented in Charts 6 and 7.Ouchterlony, radio-Ouchterlony, IEP, and RIEP procedureswere performed in which the AFP-12SI was added to FF, and

the patterns developed with anti-FF and anti-AFP antiserum.Although the Ouchterlony and IEP patterns developed withanti-FF demonstrated a number of precipitin arcs, thecorresponding radio-Ouchterlony and RIEP plates eachrevealed only a single radioactive precipitin line. Identicalradioprecipitin patterns were observed when monospecificanti-AFP was used. Moreover, with the monospecific

antiserum, the Ouchterlony and IEP plates also developedsingle arcs in the same positions.

The Radioimmunoassay. The conditions used in theconjugation of 12s I to AFP by the chloramine-T technique

resulted in a radioiodination efficiency of approximately 60%.Hence, at the end of the labeling procedure, only 40% of therecoverable 125I was in the free form. The final radiolabeled

preparation of purified AFP, therefore, had a specific activityof 20 to 40 /iCi/jug. Thus, with the "y-ray analyzer efficiency of50%, the 20,000 cpm contained in each 500-^1 aliquot ofradiolabeled AFP added to each assay tube represented 1 to 2ng of AFP-1251.

The titration curve of anti-AFP against AFP-1251 is shown

in Chart 8. In the range of antibody excess, the maximalquantity of AFP-1 2SI specifically bound was 97% of the total

quantity of radiolabeled antigen added. At the other end ofthe spectrum, a 1:3 X IO6 dilution of the anti-AFP resulted in

a specific precipitation of only 4% of the radioactivity addedto each assay tube.

o00CM

OLO

-O<

fe*

36

Elution Volume (ml)Chart 4. Chromatography on Sephadex G-200 of material contained

in the hatched area of Chart 3. , percentage absorption at 280 nm;o, AFP (Mancini).

Chart 5. a, Ouchterlony plate. Single bands, showing a reaction ofidentity, appear between AFP-125I + AFP and anti-AFP, and betweenAFP-125I + AFP and anti-FF. However, there is no reaction of identity

between this system and the bands that appear between NRS andanti-NRS. b, autoradiograph of the same plate. All of the 1I5Iradioactivity is confined to the bands that form between AFP-12 51 +AFP and anti-AFP or anti-FF.

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Radioimmunoassay for Rat AFP

Anti-FF(UNDILUTED)

was approximately 75% of the AFP-12 51 in the reactionmixture, or 0.75 to 1.5 ng of AFP-12 s I.

A comparison of the patterns of coprecipitation-inhibitionachieved in the radioimmunoassay system with purified AFP,FF, maternal rat sera, and sera obtained from rats bearingprimary hepatomas that had been induced by DAB are shownin Charts 10 and 11. When the inhibition seen with purifiedAFP was compared with that effected by any of the otherreagents containing native AFP, which had undergone no

100r

so -

60

•¿�oC

m40

20 -

'/6,400 V31.200 V409.600 V3,276,800

Dilution of Anti-AFP

Chart 8. Titration curve of anti-AFP antiserum against AFP-IÕSI,

with 50% saturated ammonium sulfate as the coprecipitating agent.Approximately 75% of maximal net binding is observed at an antiserumdilution of 1:50,000.

Chart 6. a, Ouchterlony plate; b, autoradiograph of the same plate.All of the AFP-12 51 radioactivity is confined to the bands of identitythat appear between AFP-12 M + FF and anti-AFP, anti-FF (undiluted),and anti-FF (half-strength).

Chart 7. a, IEP; b, RIEP. These correspond to Chart 6. Singleradioactive arcs in the a-globulin region appear with both anti-FF andanti-AFP.

The standard inhibition curve shown in Chart 9 wasobtained with anti-AFP at a dilution of 1:50,000. At thisantiserum concentration, the maximal antigen-bindingcapacity of the 500 p\ of anti-AFP added to each assay tube

100

AFP ng/20 Ml

Chart 9. Standard coprecipitation-inhibition curve demonstrating thecapacity of AFP to inhibit binding of AFP-125I by anti-AFP at a

dilution of 1:50,000.

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David D. Oakes, Joseph Shuster, and Phil Gold

'Ja

'/OLMO"yTooo" "ViOÖ"

'/150,000 '/IS, 000 Vl.SOO

Vio

Dilution of Fetal Fluid

AFP ng/20 pi

•¿�---i Dilution of Hepaloma Sera

Neat

Chart 10. Comparative coprecipitation-inhibition curves with AFP (•),FF (o), and the sera from 2 rats bearing DAB-induced hepatomas (¿,*).The curves obtained are virtually parallel in their exponential portions.

'/i,000 '/IO.....< Dilution of Maternal Serum

Neat

AFP ng/20ul

Chart 11. Comparative coprecipitation-inhibition curves with AFP (•)and pooled maternal rat sera (*) obtained between 24 and 48 hrpostpartum. The curves obtained are virtually parallel in their exponential portions.

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Radioimmunoassay for Rat AFP

physicochemical manipulation, virtually parallel lines wereobtained in the range of antigen concentration at whichexponential increases in coprecipitation-inhibition wereachieved.

DISCUSSION

This study describes the development of a sensitive andreproducible radioimmunoassay for rat AFP. The assay isdependent upon the preparation of monospecific anti-AFP,radioiodination of highly purified rat AFP, and a modifiedFarr procedure.

Purity of the AFP and Monospecificity of theAFP-Anti-AFP System Used in the Radioimmunoassay.Because the AFP was purified by a combination of immuno-

chemical and physicochemical techniques, the purity of thefinal product was, to a large extent, dependent upon themonospecificity of the anti-AFP used. The initial indicationthat the antiserum in question reacted solely with rat AFP wasobtained in the Ouchterlony and IEP reactions shown inCharts 1 and 2. Support for this conclusion was subsequentlyobtained by the far more sensitive techniques of radioprecip-itation used (Charts 5 to 7). In addition to demonstrating themonospecificity of the anti-AFP, however, these data revealedthat the only constitutent of the purified AFP preparationthat either was antigenic and/or could be labeled during theradioiodination procedure was AFP itself. This contention wassubstantiated by the finding that the anti-AFP was capable ofspecifically binding at least 93% of the total radioactivity ofthe AFP-125I preparation (Chart 8). In view of the possible

alteration of molecular structure that might have occurredduring the purification and radiolabeling procedures, and thekinetics of antigen-antibody interaction, it would appear thatthe purified AFP probably contained at most only tracecontamination of constituents other than the AFP moiety.

Sensitivity and Specificity of the Radioimmunoassay. Fromthe standard inhibition curve obtained (Chart 9), under theconditions of the assay used, quantities of purified rat AFPabove 0.5 ng/20 jul could be reproducibly demonstrated.Hence, a quantity of 25 ng of AFP per ml of serum could bedetected with confidence.

The close similarity between native AFP and the purifiedand radiolabeled AFP was initially demonstrated by theidentity of the precipitin reactions obtained between anti-AFPand the corresponding antigen in both FF and the purifiedAFP preparation (Charts 6 and 7). These observations weresubstantiated by a comparison of the coprecipitation-inhibition curves obtained in the radioimmunoassay betweenthe purified AFP and a number of sources of the nativematerial. Virtually parallel curves were observed in all caseswhen purified AFP, FF, maternal rat sera, and sera obtainedfrom rats bearing DAB-induced hepatomas were compared asinhibitors of the AFP-'2SI-anti-AFP system (Charts 10 and

11). This indicates that the AFP underwent little antigenicalteration in the process of purification and radioiodination,that levels of native AFP found in rat body fluids can beexpressed in terms of defined quantities of the purifiedantigen, and that the coprecipitation-inhibition reactions

observed were not due to nonspecific interactions (9). On thesame basis, it would also appear that the AFP moieties foundin FF, maternal rat sera, and the sera of rats bearingDAB-induced hepatomas are, at least, antigenically identical.

The ability to measure very low serum concentrations of ratAFP with a high degree of accuracy now provides the meansby which a number of problems may be investigated. Amongthese are the precise temporal relationship of AFP appearanceto the development of morphologically distinguishablehepatomas induced by certain carcinogens in rats (16), thekinetics of AFP metabolism under a variety of circumstances,and the possible appearance of AFP in rat sera underconditions other than those associated with primaryhepatomas.

Recent studies have suggested that sera obtained fromnormal humans are not completely devoid of the humananalog of AFP (13). The levels purported to exist in the sera ofnormal individuals were below 20 ng AFP per ml. Thedemonstration of this phenomenon required the use ofsolid-phase immunoadsorption, and the data obtained awaitconfirmation (3). The present series of experiments were notdesigned to assess this situation in rats, and it will probablyrequire some modification and increased sensitivity of theradioimmunoassay in order to do so. This should, however,prove possible by altering either or both of the specific activityof the AFP-12SI and the binding properties of the anti-AFP

antiserum used, as well as replacing the NRS in the diluents bya solution of 7-globulin from an unrelated species.

ACKNOWLEDGMENTS

The authors are indebted to Dr. Samuel O. Freedman for his valuablesuggestions in the preparation of the manuscript. Sera from rats bearingDAB-induced hepatomas were kindly provided by Dr. Roger Daoust ofthe Montreal Cancer Institute, Montreal, Quebec, Canada.

REFERENCES

1. Abelev, G. I. Production of Embryonal Serum a-Globulin byHepatoma: Review of Experimental and Clinical Data. Cancer Res.,28: 1344-1350, 1968.

2. Abelev, G. I., Perova, S. D., Khramkova, N. I., Postnikova, Z. A.,and Irlin, 1. S. Production of Embryonal a-Globulin by Trans-plantable Mouse Hepatomas. Transplantation, /.' 174-180, 1963.

3. Alexander, P. Foetal "Antigens" in Cancer. Nature New Biol., 235:

137-140, 1972.4. Editorial. Fetoproteins. Lancet, 1: 397-398, 1970.5. Farr, R. S. A Quantitative Immunochemical Measure of the

Primary Interaction between I* BSA and Antibody. J. Infect.Diseases, 103: 237-262, 1958.

6. Gitlin, D., and Boesman, M. Fetus-Specific Serum Proteins inSeveral Mammals and Their Relation to Human a-Fetoprotein.Comp. Biochem. Physiol., 21: 327-336, 1967.

7. Greenwood, F. C., Hunter, W. M., and Glover, J. S. ThePreparation of '3 ' I-labelled Human Growth Hormone of High

Specific Radioactivity. Biochem. i.,89: 114-123, 1963.8. Hull, E. W., Carbone, P. P., Gitlin, D., O'Gara, R. W., and Kelly, M.

G. ot-Fetoprotein in Monkeys with Hepatoma. J. Nati. Cancer Inst.,42: 1035-1040, 1969.

9. Hunter, W. M. The Preparation of Radioiodinated Proteins of High

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Activity. Their Reactions with Antibodies In Vitro: The Radio- 13. Ruoslahti, E., and Seppala, M. a-Fetoprotein in Normal Humanimmunoassay. In: D. M. Weir (ed.), Handbook of Experimental Serum. Nature New Biol., 235: 161-162, 1972.Immunology, pp. 608-654. Philadelphia: F. A. Davis Publications, 14. Stanislawski-Birencwajg, M., Uriel, J., and Grabar, P. Association of1967. Embryonic Antigens with Experimentally Induced Hepatic Lesions

10. Kabat, E. A., and Mayer, M. D. Experimental Immunochemistry, in the Rat. Cancer Res., 27: 1990-1997,1967.Ed. 2, p. 88. Springfield, 111.:Charles C Thomas, Publisher, 1964. 15. Thomson, D. M. P., Krupey, J., Freedman, S. O., and Gold, P. The

11. Mancini, G., Carbonara, A. O., and Heremans, J. F. Immuno- Radioimmunoassay of Circulating Carcinoembryonic Antigen ofchemical Quantitation of Antigens by Single Radial Immuno- the Human Digestive System. Proc. Nati. Acad. Sei. U. S., 64:diffusion. Immunochemistry, 2: 235-254,1965. 161-167, 1969.

12. Nishi, S. Isolation and Characterization of a Human Fetal 16. Watabe, H. Early Appearance of Embryonic a-Globulin in Rata-Globulin from the Sera of Fetuses and a Hepatoma Patient. Serum during Carcinogenesis with 4-Dimethylaminoazobenzene.Cancer Res., JO: 2507-2513, 1970. Cancer Res., 31: 1192-1194, 1971.

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1972;32:2753-2760. Cancer Res   David D. Oakes, Joseph Shuster and Phil Gold 

-Fetoprotein in the Serum of Rats1αRadioimmunoassay for

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