Immuno1loy, 1959, 4, 309-

Non-Precipitating Antithyroglobulin Studiedby the Ouchterlony Technique

R. B. GOUDIE, J. R. ANDERSON AND KATHLEEN G. GRAY

The University Department of Pathology, Western Infirmaty, Glasgow

Summary. Non-precipitating antibody reacting with human thyroglobulin hasbeen found in the serum of a patient with Hashimoto's disease. On Ouchterlonyplates this antibody causes a clear line to develop in the slightly cloudy agar insteadof the usual dense white precipitate: it is capable of blocking the action of certainprecipitating Hashimoto sera, whereas other 'enhancing' Hashimoto sera enablethe non-precipitating serum to take part in precipitate formation. A considerationofthis phenomenon suggests that the non-precipitating serum reacts at different siteson the same antigen molecule from the enhancing serum. This reaction providesa mechanism whereby two lines of precipitate may be formed in agar with a singlepure antigen.

INTRODUCTION

THE occurrence of 'incomplete' antibodies in human serum is most widely known inconnection with the Rh and other blood group systems. Non-precipitating antibodytowards soluble antigens has been described in the rabbit by Heidelberger and Kendall(I935), and in the horse by Pappenheimer (i94o) and Heidelberger, Treffers and Mayer(i 94o). The present study was undertaken to investigate a peculiar reaction observed onOuchterlony plates with serum F, obtained from a patient with Hashimoto's disease:this serum contained non-precipitating antibody which gave rise to two phenomena:(a) it inhibited or blocked the thyroglobulin precipitin reaction of certain precipitatingsera from patients with Hashimoto's disease, and (b) it increased the amount ofprecipitateformed by certain other such sera. Because of this behaviour we have regarded the anti-body in serum F as 'incomplete' or non-precipitating antibody. By this term it is notimplied that the antibody is univalent, but merely that it does not produce precipitatewith thyroglobulin, though it contains antibody closely akin to a precipitin.

MATERIAL AND METHODS

Sera were obtained from patients with Hashimoto's disease and allied conditions(primary myxoedema and certain cases of thyrotoxicosis) diagnosed by clinical andlaboratory criteria previously described (Goudie, Anderson, Gray, Clark, Murray andMcNicol, 1957). The sera were stored at - I5° C. Serum F, whose reactions form themain subject of this paper, was obtained from a 5I-year-old female who had had a goitrefor a year. She was hypothyroid and open surgical biopsy ofboth lobes ofthyroid revealedthe characteristic histological features of Hashimoto's disease with fibrosis, lymphocyticand plasma cell infiltration, lymphoid follicles and Askanazy cell change in the sparsethyroid epithelium. The thyroid skin test (Buchanan, Anderson, Goudie and Gray, 1958)was negative. Twelve samples of this patient's serum were obtained over a period ofi8 months.

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310 R. B. Goudie, J. R. Anderson and Kathleen G. Gray

The antigen preparations were crude saline extracts made as described previously(Goudie et al., I957). The radioactive thyroid extract was obtained from a patient withsimple non-toxic goitre who had been given 250 ,uc l""I 24 hours before operation. Asample of 'purified thyroglobulin' (Derrien, Michel and Roche, I948) 5 mg./ml. was alsoemployed as an antigen.

Precipitin tests were carried out by the Ouchterlony technique in 9 cm. diameter Petridishes. Each dish contained 20 ml. of I-5 per cent Difco 'Bacto' agar in o09 per centsaline with o i per cent sodium azide (added at 60° C.) as preservative. Wells ofappropriatesize were cut with a cork borer in the pattern required. In all tests serum was usedundiluted in 0o05-o I ml. quantities. The thyroid extracts were used diluted i in I6 ino09 per cent saline, but extracts of other organs were used undiluted. Plates were kept atroom temperature and were examined daily for the first 3 or 4 days, then twice a weekfor the next 2-8 weeks, and results were recorded with diagrams or by photography.

'Buffered plates' were made by adding two parts of 0 15 M phosphate buffer pH 5, 7and 9 to one part of 4 5 per cent agar in o 9 per cent saline, all at 60° C. When an indicatorwas required B.D.H. Universal Indicator was added to the molten agar at 6o0 C., givinga final concentration of 2 per cent indicator.The plates were stained, without preliminary extraction of soluble protein, by flooding

for 5 minutes with a filtered solution of naphthol black (solvent, mcthyl alcohol 5o parts,distilled water 50 parts, acetic acid I0 parts). The stain was then poured offand the platewashed with several changes of solvent for 24 hours. A reasonably clear background wasobtained only when diffusion was prolonged for 4-6 weeks before staining.

Quantitative precipitin curves were performed using o- i ml. quantities of each dilutionof radioactive thyroid extract, o- I ml. of each serum, and o- i ml. of i per cent sodiumazide solution. Before analysis the precipitates were washed twice in saline at 40 C. anddissolved in 2 ml. of o' I M. Na2CO3. The radioactivity was measured in a well-typescintillation counter and the protein nitrogen by digestion and estimation of ammonianitrogen with Nessler's reagent (Paul, I958). The material available was insufficient forduplicate estimation.The electrophoretic mobility of antigen-antibody complexes was assessed by mixing

equal volumes of antiserum and a i in 5 dilution of radioactive thyroid extract. Half anhour after mixing, 0-02 ml. of each mixture was applied to a 5 cm. wide Whatman No. 3filter paper strip. Horizontal electrophoresis was performed for i8 hours using barbitonebuffer pH 8- 6 (ionic strength o- I), current 0-4 mA/cm. width, and the migration of theradioactive antigen determined by contact autoradiography on an X-ray film. With theapparatus used it was established that simultaneous electrophoresis ofthe same preparationon different paper strips resulted in identical migration on each strip.

RESULTS

REACTIONS OF SERUM F ON OUCHTERLONY PLATES

Instead of the usual dense opaque line of white precipitate formed between the wellscontaining Hashimoto serum and thyroid extract, all twelve samples of serum F gaverise to a sharply defined zone of clearance in the slightly cloudy agar (Fig. I). With thefirst ten samples ofserum obtained, the clear line appeared on the fourth day and persistedtill the plates were discarded, i.e. after diffusion for 2-8 weeks, but the latest two samplesobtained after 14 and I8 months of thyroxine treatment produced more diffuse and

Non-Precipitating Antithyroglobulin Studied by the Ouchterlony Technique

weaker clear lines which persisted for only a few days and then disappeared. The clearline was best seen when viewed against a black background with bright oblique illumina-tion. When freshly withdrawn serum F was tested, the clear line was not replaced byprecipitate, but when the serum was retested after storage for some weeks or months at- 15 C., the clear line appeared as usual after 4 days' diffusion, but after a further 4 days

FIG. i. Ordinary white precipitate (left) and the dear line formed by serum F (right).The central well contains antigen. ( x 2)

it was partly obscured by a fine line of white precipitate which formed in contact with it,usually on the same side as the antigen (see lower part of precipitate ofserum F in Fig. 6A).The clear line was not doubly refractile and microscopic examination disclosed noparticulate material in the agar. Staining of the plate with naphthol black showed thatthe clear line corresponded to a localized zone of high protein concentration.

TABLE ISPECIFICITY OF REACTION OF SERUM F AND OF PRECIPITATING HASHIMOTO SERUM

Number of Reaction withAntigen preparations Precipitating

tested Serun F Hashimoto serum

Purified thyroglobulin I Clear line PrecipitateNormal human thyroid extract

(autopsy) .. .. 3 Clear line PrecipitateThyrotoxic human thyroid extract

(operation) 4 Clear line PrecipitateSimple non-toxic goitre (opera-

tion) .. .. Clear line PrecipitateNormal human liver extract

(autopsy) .. 2 Nil NilNormal human kidney extract

(autopsy) .. .. 3 Nil NilNormal human adrenal extract

(autopsy) .. 3 Nil NilNormal bovine thyroid extract

(autopsy) I. .. Nil Nil

Table i shows that serum F produced a clear line in agar only with organ extractswhich precipitate with ordinary Hashimoto sera: that is, with the antigens tested, thereactions of serum F had the same specificity as precipitating Hashimoto sera.When dilute hydrochloric acid was painted over part of the clear line this became

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R. B. Goudie, J. R. Anderson and Kathleen G. Gray

opaque in a few minutes (Fig. 2). The same effect was produced by exposing the plateto the fumes of hydrochloric acid, and subsequent treatment with ammonia rendered theline transparent again: this procedure could be repeated several times. Further experi-ments using plates with phosphate buffer incorporated in the agar showed that an acidpH did not affect the diffusibility of the reactants, although it again produced a denseopaque precipitate in place of the clear line (Fig. 3).

FIG. 2. Effect of painting clear line of serum F with HCa. Theopaque area above is the painted area. Left well, serum F;right well, antigen. ( x 3)

FIG. 3. Effect of buffering agar on reaction of serum F. Top wells, serum F; lower wells, antigen. Left pH 9,middle pH 7, rightPH 5. ( x 2)

REACTIONS OF SERUM F IN THE PRESENCE OF OTHER SERA

The reactions of serum F with thyroid antigen were not influenced by the presence ofnormal human serum. Fig. 4 shows a phenomenon seen when serum F reacted besidecertain precipitating Hashimoto sera: the 'normal' white precipitate curves slightly toblend into the clear line ('reaction of identity') and the white precipitate is asymmetrical,much less apparently being produced on the side of the clear line. This appears toindicate that serum F blocks precipitation of antigen by the precipitating serum.

Fig. 5 illustrates the effect of certain other Hashimoto sera on the clear line ofserum F:

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Non-Preciptating Antithyroglobulin Studied by the Ouchterlony Technique 313in the proximity of the precipitating serum N a zone of dense white precipitate replacesthe clear line of serum F. We have termed this phenomenon 'enhancement' and haveshown that it occurs only when all three reagents are present (thyroid antigen, serum F,and an enhancing serum which may or may not contain precipitating antibody). The'cut off' appearance of the lower end of the enhanced area corresponds to an almost

FIG.4. 'Reaction of identity' and 'inhibi-tion of precipitation' with serum F. Leftwell, normal serum; right well, serum F;middle well, antigen; top well, precipita-ting Hashimoto serum. ( x I)

FIG. 5. Enhancement of clear line of serum F. Top well,serum N, a precipitating Hashimoto serum; left well, normalserum; right well, serum F; middle well, antigen. Note thatthe 'cut off' effect of the enhanced area corresponds to analmost invisible clear line formed by serum N. ( x 2)

FIG. 6. Complex a ents between serum F (stored s ) and serum L. Top well,serum L; bottom left well, antigen; bottom right well, serum F. (a) Unstained, (b) stainedwith naphthol black. ( x 2)

invisible clear line of serum N which could be rendered opaque with HCI and stainedwith naphthol black. Enhancement was demonstrated also when purified thyroglobulinwas used as the antigen.More complex interactions were found with other Hashimoto sera, e.g. serum L

(Figs. 6a and 6b); this serum produces two lines of precipitate with thyroid antigen, and

R. B. Goudie, J. R. Anderson and Kathleen G. Gray

when it reacts beside serum F enhancement occurs apparently in the zone of antigenexcess, where it forms an opacity extending radially from the antigen well.Enhancement occurred in buffered agar plates at pH7 and 9, and when universal

indicator was incorporated in unbuffered plates no change ofpH was seen in or aroundthe enhanced precipitate.

TABLE 2THE OCCURRENCE OF ENHANCEMENT WITH SEVEN HASHIMOTO SERA

TESTED EACH BESIDE THE OTHER

Srum L N E NL F H ML - + + +N - - + + +E - - + + +NL - + + +FHM

- = no enhancement.? = doubtful enhancement.

+ = enhancement.

Although conspicuous enhancement is not commonly found it is not a unique propertyofserum F (Table 2). Four sera (L, N, E, NL) known to enhance serum F and three sera(F, H and M) whose reactions could be enhanced by serum L were tested, each besideall the others. The results showed clearly that all the sera which enhanced serum F alsoenhanced sera H and M, whereas sera F, H andM did not enhance each other. Since thesera like serum L produced opaque lines it was difficult to assess enhancement when they

FIG. 7. 'Hurdle plate.' Centre well, antigen; inner two wells left, normalserum; inner two wells, right, a precipitating Hashimnoto serum which canenhance serum F; extreme left and right wells, recently obtained freshserum F. Note that serum F produces an opaque white precipitate outsidethe 'hurdle' precipitate, but there is no visible reaction on the left side of theplate, the transient clear line having now disappeared. ( x I)

were tested beside each other, but probable enhancement did occur in some instances,in the form of increased opacity where the lines overlapped.An alternative method of studying the interaction of sera in agar is the use of a 'hurdle'

technique (Fig. 7). In this procedure the central antigen well is surrounded by an innerseries offour wells containing the 'hurdle' serum. A second series ofwells containing othersera is placed outside. In effect the inner serum provides a hurdle between the antigen

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Non-Precipitating Antithyroglobulin Studied by the Ouchterlony Techniqueand the sera contained in the outer cups. In Fig. 7 the 'hurdle' serum on the left is normalserum and that on the right is a precipitating Hashimoto serum known to enhance serumF. A recently obtained fresh sample of serum F (which produces only a transient clearline) placed outside the 'hurdle' of normal serum shows no visible effect, whereas outsidethe precipitate of the Hashimoto 'hurdle' serum, serum F forms an opaque precipitate.This effect is also seen when serum L forms the 'hurdle'. When serum F forms the hurdle,the precipitation of most Hashimoto sera is inhibited, but serum L forms a weak lineoutside the 'hurdle'.

ELECTROPHORESIS OF MIXTURES OF SERUM F AND RADIOACTIVE THYROID EXTRACT

Three mixtures of a I in 5 dilution ofradioactive thyroid extract were made with serumF, serum L and normal serum, and allowed to stand at room temperature for 30 minutesbefore being subjected to electrophoresis at pH 8x6. The antigen (detected by auto-radiography) migrated in the same direction with all three sera, but the distance migratedwas greatest with normal serum. The complexes formed in 30 minutes between serum Fand serum L and a I in 5 dilution of antigen thus have the same electric charge atpH 8.6.

40 o 40

0 G~~~~~~~~~~C 2.

0 ~~~ .. 0 .'

0 20 40 60 00 100 0 20 40 60 80o 0Antigen (count/mi,n) Antigen (counts/1M1)

Thyroid + Normal Serum + LaurieThyroid + Fullerton Serum -+ Laurie

FIG. 8. Precipitin curves of serum L; radioactive thyroid antigen pretreated with normalserum and with serum F.

REACTIONS OF SERUM F IN FLUID MEDIA

Several unsuccessful attempts were made to obtain precipitate by adding constantvolumes of serum F to closely spaced increasing quantities of thyroid extract. No preci-pitate was formed, but it was possible to demonstrate indirectly the reaction of serum Fwith antigen by allowing the mixtures to stand overnight at 40 C., then adding constantamounts of serum L to the serum F-antigen mixtures. A parallel experiment in whichnormal human serum was used instead ofserum F served as a control. Table 3 shows thatserum F prevented or delayed visible precipitation by serum L. After IO days at 40 C.the precipitates obtained were washed and the amount of precipitate (protein nitrogen)and precipitated antigen (radioactivity) were measured. The results are shown in Fig. 8.It is seen that serum F inhibited precipitation ofserum L with the lowest concentrations ofantigen: in the presence of higher concentrations of antigen, serum F increased theamount of precipitate formed by serum L and the amount of antigen precipitated, anenhancing effect comparable to that seen on the Ouchterlony plate (Figs. 6a and b).

VOL. II. 4. 3

315

316 R. B. Goudie, J. R. Anderson and Kathleen G. Gray

a o ~~~IIE1HE-4+

0 +IEIEEE+z~~ | IIIE111> +++

g .} k 11II11II1 A64

.I, co i,Il

'+tE+++++

o 6

a~~R 1>J

' ++++++++

a E s'IO ++++++++

g *t s++++++++S X w++++++++

|~~~~~~~~0. co 0A;Hbb>H

2N

iCI ti>X°n

Non-Precipitating Antithyroglobulin Studied by the Ouchterlony TechniqueDISCUSSION

The evidence that the clear line which serum F produces in agar is the result of anantigen-antibody reaction is as follows:

(i) The specificity of the reaction is that of precipitating sera obtained from otherpatients with Hashimoto's disease.

(2) Serum F exhibits inhibiting and enhancing effects in agar and in fluid media withselected precipitating-Hashimoto sera.

(3) The staining properties of the clear line indicate that it coincides with a zone ofhigh protein concentration similar to that found in the usual opaque white precipitateformed by other Hashimoto sera.

(4) Tanned red cells coated with thyroglobulin were agglutinated by serum F to adilution of at least io million (single pipette).

It is therefore postulated that fresh serum F contains antibody which combines speci-fically with thyroglobulin- to form complexes too small to cause the white opacity of anordinary precipitate but too large to diffuse readily from the zone of optimal proportionswhere these complexes are formed. It is probable that the high protein concentration ofthe clear line is responsible for the optical effect produced..The occurrence of a clear line in agar has already been described as a manifestation of

an immunological reaction by Silverstein, Feinberg, and Flax (1958) using antiserumprepared in the horse. In this connection it is relevant that Roitt, Campbell and Doniach(I958) have observed that certain human antithyroglobulin sera behave like horse anti-serum in showing precipitation curves with solubility in zones of antigen and antibodyexcess.

Indeed, most of the immunological properties of serum F have been described at onetime or another, though in different contexts. The acquisition of the ability of serum Fto precipitate in agar after storage at - i5' C. is similar to the phenomenon described byCoca and Kelly (I921), who found a non-agglutinating antibody in the serum of arabbit immunized with H. inftuenzae: this serum was at first only capable of specificallyblocking the action of specific agglutinating sera for the same organism, but after storagefor 6 weeks the blocking serum suddenly acquired specific agglutinating properties.Boyd (I947) has described an antibody against arsanilic acid azocasein which formedprecipitate only at acid pH. Most striking of all is the similarity of behaviour betweenserum F and the serum of a horse immunized with egg albumin (Pappenheimer, 1940):this serum did not precipitate when mixed with egg albumin, but was capable of blockingthe precipitating action of an anti-egg albumin serum produced in a rabbit. UsingPappenheimer's reagents, Heidelberger, Treffers and Mayer (I940) subsequently showed,by altering the conditions of the experiment, that the presence of the horse serum enabledmore precipitate to be formed by the rabbit serum in the antigen excess zone.The enhancement illustrated in Fig. 9 shows that this phenomenon occurs not only

with sera which contain a precipitating antibody, but also with sera which form novisible precipitate. By enhancement of the clear line of serum F it has been possible todetect antithyroglobulin in four of forty precipitin-negative sera obtained from patientswith Hashimoto's disease and allied conditions. These four sera produced neither a clearline nor a stainable line in agar. Enhancement thus afforded a method of demonstratingthe presence of antibody which could not otherwise be detected using the agar diffusionmethod.

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R. B. Goudie, J. R. Anderson and Kathleen G. Gray

It is of interest to consider the reactions of serum F in the light of Marrack's latticehypothesis (I938). As shown above there is strong evidence that the clear line representsthe site of optimal proportions of an antigen-antibody reaction. The complexes formeddo not appear as visible precipitate, but yet do not readily diffuse from the zone in theagar in which they are formed. The absence of precipitation could be attributed tounivalence of antigen or of antibody. It seems likely that the antigen is multivalent forat least some antibodies, for ifour interpretation of Fig. 4 is correct, the clear line ofserumF is 'identical' with the opaque line ofthe precipitating serum and the antibody in serum Fblocks the action of the precipitating serum, presumably by reacting with the samemultivalent antigen molecules. It is difficult to know whether fresh serum F containstrue univalent antibody; our interpretation of enhancement outlined below suggests that

Nii

FiG. 9. E nancentofserumF by precipitin negative Hashi-moto serum. Centre well, antigen; top well, serum F; left andright wells, duplicate contann precipitin negative Hashi-moto serum; o'wer well, normal serum. The ends of theclear line formed by serum F are enhanced. X 2)

this is not so; certainly, after storage, precipitate is produced by serum F and this indicatesthe presence of multivalent antibody. It seems probable that both antigen and antibodyare multivalent, but for an unknown reason they do not produce visible aggregates, asimilar phenomenon to that seen when excess horse antibody is added to protein. antigens.

Until the structure -of the serum F-antigen complexes in the clear line is known,enhancement cannot fully be understood. The following possibilities, however, have beenconsidered:

(i) Serum F and the enhancing serum react with different antigenic molecules, andenhancement is non-specific co-precipitation. This possibility is considered uniely:non-specific factors which could cause enhancement have not been demonstrated, e.g.enhancing sera do not cause marked pH changes, and co-precipitation is not due to the

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Non-Precipitating Antithyroglobulin Studied by the Ouchterlony Techniqueinteraction of oppositely charged complexes. Furthermore enhancement may occurbetween two sera neither of which alone react to form precipitate.

(2) The possibilities that serum F or the enhancing serum contain anti-antibody (seeLing, 1958) or that serum F combines with the same antigenic groupings as the enhancingserum, can be dismissed, for they do not explain all the appearances shown in Figs. 5, 6,7 and 9.

(3) Serum F combines with the same antigen molecules as the enhancing serum, butreacts with additional antigenic groupings. This produces a mixed precipitate incor-porating two antibodies which react with different parts of the same antigen molecule.This hypothesis explains the blocking and enhancement seen in Fig. 8, and the formationof opaque white precipitate by serum F in the zone of antibody excess of the 'hurdle'serum (Fig. 7). It also explains, in terms of specificity of antibody to individual groups onthe antigen molecule, why only a limited number of antibody-containing Hashimoto seraare capable of enhancing the clear line of serum F.

(e) (h) (c)Rabbit 4l4orse Horse TypeType' @Type Enhancinqti Antibody Antibody

Aq. presentNo Aq. as soluble

complex ini : ::: nARACedAb. excess . . :,O..

Antiqen i Antiqen Antigen

FIG. IO. Simple scheme to show how two lines of precipitate may be formed by amixture of antibodies and a single antigen.

From a consideration of the above points we feel that the most acceptable view of thereactions ofserum F is that it contains potentially multivalent antibody which for unknownreasons does not precipitate with homologous antigen. Because of this antibody, serum Fcan block the reactions of precipitating antibody of identical specificity, and can formspecific co-precipitate with other sera which contain antibody specific for the samemolecule but reacting with different antigenic groupings. A comparable, and perhapsclosely similar, situation has been described by Pauling, Pressman and Campbell (I944),who obtained precipitate with mixtures of two antisera, each of which reacted specificallywith separate, different, univalent haptenic groups on the same composite dihaptenicantigen molecules.

If the above views are correct, they have important bearings on the interpretation ofreactions seen on the Ouchterlony plate and in other agar diffusion methods. It has oftenbeen suggested that a single antigen can result in only a single line of precipitate and thattwo lines of precipitate indicate the presence of two antigens in the preparation. Withantibody of 'rabbit type', in which antigen precipitation is complete in the equivalencezone, and in which antigen-antibody complexes are insoluble in antibody excess, only oneline of precipitate is possible on the Ouchterlony plate (see Fig. ioa). Quantitative

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320 R. B. Goudie, J. R. Anderson and Kathleen G. Gray

precipitin curves with radioactive thyroglobulin have shown, however, that with someHashimoto sera precipitation of antigen is incomplete at all dilutions (possibly due to thepresence of blocking antibody), and furthermore we have recently been able to show thata specific precipitate can be redissolved by addition of an excess of the antibody serum('horse type' antibody). With such an antibody, antigen-antibody complexes may diffusethrough the precipitate on an Ouchterlony plate and pass into the antibody excess zone(Fig. iob). If an antiserum contains a mixture of antibodies of 'horse' type which, byreacting with different sites on the same antigen molecule are capable of enhancement,then a possible mechanism is provided for the occurrence of two lines with a single 'pure'antigen preparation (Fig. ioc); two lines occur because the 'antigen' contains in itselfreactive sites of differing specificity. It should be mentioned that the above use of theexpressions antibody of 'horse' and antibody of 'rabbit' type is for convenience of descrip-tion: it has long been known that rabbit serum may contain non-precipitating antibody(Heidelberger, 1935).

Finally, it is ofinterest to consider why these peculiar reactions should have been formedwith the antibody present in the serum of patients with Hashimoto's disease. Theexplanation is probably twofold; namely that extensive studies of human precipitatingantibodies have not hitherto been possible. The long-continued presence of an antigengenerated within the patient's own body provides excellent opportunity for prolongedimmunization and a good antibody response. Second, it is known that the ability todiscriminate between closely related tissue antigens generally increases as the species oforigin of the antigen approaches the species used for antibody production; isoantibodiesare more discriminating than hetero-antibodies. If this trend can be extended further itseems likely that auto-antibodies will be the most discriminating of all, and this mayaccount for the peculiar 'intramolecular specificity' which we have postulated above,

ACKNOWLEDGMENTS

The authors wish to thank Dr. J. Paul of the Biochemistry Department, The University ofGlasgow, for the estimation of the protein content of precipitates, and Dr. I. M. Roitt of theMiddlesex Hospital, London, for providing the purified thyroglobulin and performing the tannedred cell agglutination test. We are greatly indebted to the Medical Research Fund of theUniversity of Glasgow for a contribution towards the expenses of the work. The investigationwas assisted by a financial grant to one of us (J.R.A.) by the Department of Health for Scotland.

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BUCHANAN, W. W., ANDERSON, J. R., GouDnm, R. B.and GRAY, K. G. (I958). 'A skin test in thyroiddisease.' Lancet, 2, 928-31.

COCA, A. F. and KELLY, M. F. (i92i). 'A serologicalstudy of the bacillus of Pfeiffer.' J. Immunol.,6, 87-IOI.

DERRIEN, Y., MICHEL, R. and RocHE, J. (i948).'Recherches sur la preparation et les propri&1ss dela thyroglobuline pure-I.' Biochim. biophys. Acta,2, 454-70.

GouDm, R. B., ANDERSON, J. R., GRAY, K. G.,CLEitx, D. H., MuRRAY, I. P. and McNicoL, G. P.(I957). 'Precipitin tests in thyroid disease.' Lancet,2,976-9.

HEIDELBERGER, M. and KENDALL, F. E. (I935) .'A quantitative theory of the precipitin reaction.-III. The reaction between crystalline eggalbumin and its homologous antibody.' J. exp. Med.,62, 697-720.

HEIDELBERGER, M., TREmRs, H. P. and MAYER, M.(i940). 'A quantitative theory of the precipitinreaction.-VII. The egg albumin-antibody reactionin antisera from the rabbit and horse.' 3. exp. Med.,71, 27I-82.

IJNG, N. R. (1958). 'The backbone of the antibody.'Lancet, 2, I28I-2.

MARRACK,J. R. (I 938). 'The chemistryofantigens andantibodies.' Spec. Rep. Ser. med. Res. Coun. (Lomd.),No. 230.

PAPPENHEIMER, A. M. (i94o). 'Anti-egg albuminantibody in the horse.' 3. exp. Med., 71, 263-8.

Non-Precipitating Antithyroglobulin Studied by the Ouchkterlony Technique 32I

REFERENCES (continued)PAUL, J. (1958). 'Determination of the major con-

stituents of small amounts of tissue.' Analyst, 83,37-8.

PAULNG, L., PRESsMAN, D. and CAMPBELL, D. H.(I944). 'The serological properties of simplesubstances.-VI. The precipitation of a mixtureof two specific antisera by a dihaptenic substancecontaining the two corresponding haptenic groups;evidence for the framework theory of serologicalprecipitation.' J. Amer. chum. Soc., J., 66, 330-6.

Romr, I. M., CAmpmmi, P. N. and Domecs, D. (i958).'The nature of the thyroglobulin auto-antibodiespresent in patients with HMashmoto's thyroiditis(lymphadenoid goitre).' Biocm J., 69 248-56.

SHLvmsRmN, A. M., FBnwzRG, R. and FLAx, M. H.(I958). '"N tive precipitin" bands in antigen-antibody gel diffusion techniques.' Fed. Proc., x7,535-