Immunologic analysis of sheep pituitary gonadotropins

QENERAL

AND

cohf~m~m

ENDOCRINOUMY,

Immunologic

Analysis

SHELDON MARA Laboratories

Supplement

1, 12-21 (1962)

of Sheep Pituitary

Gonadotropinsl

J. SEGAL, KENNETH A. LAURENCE, PERLBACHS, AND SAFIA HAKIM

of the Population Council, The Rockefeller New York $1, New York, U. S. A.

INTRODUCTION

Institute,

for the refractoriness that develops in animals after an initial period of stimulation It haa long been known that heterologous by anterior pituitary extracts (Collip and protein hormones can engender the formaAnderson, 1934). Indeed, the serum of an tion of serologically detectable antibodies. animal that has been previously treated As more purified pituitary tropic hormones with a gonadotropin preparation may have have become available, it has been possible the property of neutralizing the hormone. to demonstrate that the hormones per se The exact relation of this neutralizing are antigenic and that the antibodies creagent to serologically detected antibodies, ated are specific for, and can inactivate, which constitutes the so-called “antihorthe biologically active hormonal moieties I-none” theory, has been the subject of much (for review see Hays and Steelman, 1955). investigation (for reviews see Zondek and This has aided considerably in elucidating Sulman, 1942; Thomson et al., 1941; the species variation in growth hormone (Geschwind, 1959). With respect to the Leathem, 1949). While not all workers in the same way, gonadotropins, it has been difficult to es- regard “antihormones” most would probably consider them to be tablish that the hormones themselves are antibodies with the property of antagothe specific antigens responsible for the nizing the action of the hormone. Since the formation of serologically detectable antihormonal action of gonadotropins is not bodies, because all preparations so far species specific, particularly within the available have proven to be mixtures of mammals, one would expect anti-gonadoseveral antigenic components. Even prepatropins to exhibit similar cross-reactivity rations with a great degree of homogeneity if their neutralizing action is attributed to as shown by physical and chemical methreaction with hormonally active sites on ods, prove to be multiply antigenic (Segal the molecule. An example that fits this et al., 1960). Van Dyke et al. (1950) propostulate is the report of Maddock (1949) duced precipitating antibodies in rabbits that after a course of injections of FSH against purified sheep FSH and swine from sheep anterior pituitary, men became FSH. Antisera have been prepared also refractory not only to exogenous gonadowith sheep ICSH (Henry and Van Dyke, tropins, but also to endogenous gonadotro1958). In both instances, the highly puripins. That the situation is more complified gonadotropin preparations employed proved to have several antigenic com- cated, however, is indicated by lack of species crosses in the serologic studies ponents so that it could not be ascertained mentioned earlier employing sheep FSH that the serologic antibodies were related and ICSH antisera (Van Dyke et al., specifically to the hormone molecules. Nev1950; Henry and Van Dyke, 1958). ertheless, it seems likely that this is the This report bears on these problems and case and that this phenomenon accounts describes experiments with antisera that have been prepared by immunizing rabbits ‘Aided by research grant RG-5280(C3) from with sheep anterior pituitary FSH and LH the National Institutes of Health, United States Public Health Service. preparat,ions that are known to contain 12

IMMUNOLOGIC

ANALYSIS

antigenic contaminants. The antisera have been analyzed immunologically in an effort to establish the relationship between the active gonadotropins in the antigens, and the antibodies produced. Lack of crossreactions support the view that a species difference exists in the chemistry of gonadotropic hormone, in spite of the broad specificity of action of these substances. Finally, immunologic evidence is presented to indicate that the two gonadotropins, FSH and LH, are chemically distinct antigen entities and that LH occurs as a particular contaminant in the FSH preparation used.

OF GONADOTROPINS

subsequent testing of absorbed antisera by hemagglutination and agar dausion. Antibody protein was determined in the region of slight antibody excess by the Folin-Ciocalteau method (Lowry et aZ., 1952). The biologic assay for LH activity was the weaver finch test (Segal, 1957) which is based on the direct action of LH on melanin deposition in the regenerating breast feathers of the female Euplectes afer. Total gonadotropins were assayed by a method based on the uterine weight response in immature female mice. Dose groups of 5 animals each received twice daily subcutaneous injections of test material for three days. Twenty-four hours after the last injection, uteri were removed, blotted dry and weighed on a torsion balance.

METHODS Male chinchilla and New Zealand white rabbits were immunized with gonadotropins purified from sheep pituitary glands. The preparations used were ovine FSH-Sl and ovine LH-Sl received from the Endocrinology Study Section, National Institutes of Health. Each rabbit received three weekly intradermal inoculations of 10 mg antigen per 1 ml saline, emulsified with 1 ml complete Freund adjuvant. The animals were bled at weekly intervals during the immunization procedure. One week after the third intradermal inoculation, an intravenous injection of 2 mg antigen in 1 ml saline was given. Blood was collected 4 to 10 days later and the animals were intermittently bled for several weeks thereafter. When antibody titer declined, additional intravenous boosters were given and finally several months after the initial immunization the anesthesized animals were exsanguinated from the heart. The weekly bleedings were used in the study of developing antibody, but the final bleeding was used exclusively for biologic tests, serologic reactions, and determinations of optimal combining ratios. Antisera were tested for number of antibody components and cross-reactions by the agar-gel double diffusion method as described by Feinberg (1957). Antibody titers were determined by the tanned erythrocyte hemagglutination method (Boyden, 1951). Optimal combining ratios were established using a quantitative precipitin test in which the quantity of antigen is titrated to determine the optimal conditions of precipitation with a fixed quantity of antibody. This method is described in detail by Dean and Webb (1926). Absorption of antisera to eliminate nonspecific antibodies was done at the proportions of optimal combination to achieve a slight antigen excess. This was confirmed bv

13

RESULTS SEROLOGIC TESTS WITH FSH-Sl

ANTISERUM

FSH-Sl is multiply antigenic, giving rise to 4 precipitin bands in agar diffusion plates (Fig. 1). Each antigen-antibody system has its own optimal proportions for forming visible precipitation; dilution of antigen results in a gradual disappearance of the precipitin bands, one after the other. With undiluted antiserum, at an antigen concentration of 2 mg/ml 4 bands appear; TABLE .bTlBODY

1

TITER OF FSH-Sl ANTISERUM HEMAGGLUTINATION TEST

Antigen

Con~$r$ion

FSH-Sl FSH-Sl FSH-Sl FSH-Sl Sheep serum protein Sheep serum protein l In this and subsequent reported, the reactions were plastic trays. The granular reaction is graded from 1 f tions are reported. b Reciprocal of the highest a positive (4+) reaction.

1.0 0.1 0.01 0.001 14.0 2.8

AB Titer-

IO,2406 10,240 5,120 1,280 20,480 20,480

hemagglutination tests carried out in disposable appearance of a positive to 4 -I-. Only 4 f reacdilution

of serum giving

only a single band occurs when the antigen concentration is 0.25 mg/ml (Fig. 1). The maximal antibody titer detected by the

14

SEGAL, LAURENCE, PERLBACHS,

hemagglutination test is 1: 10,240 (Table 1). This titer is achieved when erythrocytes are modified by FSH-Sl at a concentration of 1.0 or 0.1 mg/ml. That the antibodies are partly nonspecific is indicated by the reaction of the antiserum at a dilution of 1: 20,480 with erythrocytes modified with sheep serum protein (2.8 mg/ml). The reaction with sheep serum protein is discussed in more detail below. CROSS-REACTIONS WITH OTHER TROPIC HORMONES

No precipitin appears when the antiserum to FSH-Sl is placed in agar-diffusion plates with ovine luteotropic hormone.

AND HAXIM

bovine growth hormone, bovine luteinizing hormone, hog pituitary lipotropic factor, human chorionic gonadotropin or ovine thyrotropic hormone. The antigen concentrations employed in these tests are indicated in Table 2. A cross-reaction occurs with LH purified from sheep hypophysis (Fig. 2). Antiserum absorbed with LH-Sl, centrifuged and subsequent,ly tested in geldiffusion plates with FSH-Sl produces only 3 precipitin lines. When FSH-Sl is absorbed with LH-Sl antiserum, 3 precipitin lines appear when tested against FSH-Sl antiserum (Fig. 3). The deleted reaction corresponds to that given by LH antigen and unabsorbed FSH-Sl antiserum.

FIG. 1. FSH-Sl AS in center well test.ed against dilutions of FSH-SI: 2.0 mg/ml, 1.0 mg/ml, 0.5 mg/ml, 0.25 mg/ml. In this and subsequent figure explanations, the peripheral wells are listed counter-clockwise starting from the notch. AS refers to antiserum. FIQ. 2. FSH-Sl AS in center well tested against FSH-Sl 2.0 mg/ml, sheep serum protein 14.0 mg/ml, LH-Sl 2.0 mg/ml. FIG. 3. FSH-Sl AS in center well tested against FSH-Sl 2.0 rhg/ml incubated with LH-Sl, AS, FSH-Sl 2.0 mg/ml incubated with NRS (normal rabbit serum). FIG. 4. LH-Sl AS in center well tested against LH-Sl ‘0.2 mg/ml, 2.0 mg/ml, FSH-Sl 2.0 mg/ml.

IMMUNOLOGIC

ANALYSIS

agar-diffusion with FSH-Sl antiserum, this combination fails to give a positive hemagglutination reaction, regardless of the antigen-antibody proportions employed. Negative results were obtained with LH-Sl at concentrations of 0.33 mg/ ml, 0.02 mg/ml, and 0.002 mg/ml.

CROSS-REACTIONSWITH HETEROLGGOUS PREPARATIONS HAVING FOLLICLESTIMULATING

ACTIVITY

Some evidence for the independence of antigenic and hormonal properties of the FSH molecule is indicated by the absence of cross-reactions when the sheep FSH-Sl antiserum is tested with various heterologous preparations with follicle-stimulating activity. Rat hypophysial extract with proven FSH activity (4 mature male pituitaries per ml), pregnant mare

CROSS-REACTIONS WITH SHEEP SERUM PROTEINS

Two bands of precipitation develop when FSH-Sl antiserum is tested against

TABLE SEROLOGIC

REACTIONS

15

OF GONADGTROPINS

WITH

2 FSHSl

ANTISERUM

Agar-Diffusion Conmegcetm~tion Antigen

Hemagglutination %%!!*

AB Titer

FSH-Sl

2.0

0.1

10,240

LH-Sl

2.0

0.33 (not tested)

0 -

0.33

0

0.33 (not tested)

0 -

LH (bovine) LTH

(PC 6-105A)a

2.0 2.0

(ovine) (Squibb 4B74978)

GH (bovine)

2.0

(Somar-A R50109)

TSH (ovine) (Simpson Prep.)6

2.0

Pergonal-23 (Human post-menopausal

7.6

0

0.33

0

2.0

0

0.1

0

1000 IU/ml

(not tested)

500 IU/ml

0 0

4 male pit./ml

0

(not tested)

Hog pituitary

lipotropin

urine gonadotropin)c

(Rudman-Fr.

H)d

Human chorionic gonadotropin (Ayerst APL 29075) Pregnant

mare serum (Equinex

Rat pituitary extract (proven gonadotropin

25207)

activity)

500 IU/ml

0

The preparations indicated were kindly supplied as follows: 5 Dr. Robert Bates, Bethesda. * Dr. Miriam Simpson, Berkeley. c Dr. Pierro Donini, Rome. d Dr. Daniel Rudman! N.T.C.

serum (500 IU per ml), human chorionic gonadotropin (1000 IU per ml), and human post-menopausal urine gonadotropin (Pergonal, 7.6 mg/ml) all fail to react with the antiserum in agar-diffusion plates (Table 2). Confirming this finding, no hemagglutination reactions are observed with the above-listed preparations, as far as they have been tested (Table 2). However, contrary to the result obtained when sheep pituitary LH-Sl is tested in

undiluted sheep serum protein (Fig. 2). The sheep serum reactions may be removed by absorption and the absorbed antiserum interacts with FSH-Sl to give 2 precipitin lines. In the hemagglutination test, sheep-serum-modified erythrocytes agglutinate with a 1: 20,480 dilution of FSHSl antiserum (Table 1). Sheep serum absorbed FSH-Sl antiserum gives a titer which does not differ from that obtained with unabsorbed antiserum. The relat.ion-

16

SEGAL, LAURENCE,

PERLBACHS,

ship of this interaction between sheep serum protein and FSH-Sl antiserum to the cross-reaction obtained with LH is not clarified by the aforementioned experiments. SEROLOGIC TESTS WITH

LH-Sl

ANTISERUM

When LH-Sl antiserum is tested with LH-Sl at a concentration of 2.0 mg/ml, there appear at least 5 precipitin bands which distribute so that several adjacent bands create a peripheral group and one distinct group of bands forms with a faster moving antigen in a more medial position (Fig. 4). With diminishing concentrations TABLE3 ANTIBODY

TITER OF LH-Sl HEMAGGLUTINATION

ANTISERUM TEST

COll~~;~tiOtion Antigen

LH-Sl LH-Sl LH-Sl LH-Sl

0.33 0.2 0.02 0.002 0.0002 0.00002 14.0 2.8

LH-Sl LH-Sl Sheep Sheep

serum serum

protein protein

AB Titer

640 640 1,280 5,120 10,240 2,560 1,280 1,280

of antigen, as low as 0.2 mg/ml, the latter precipitin is preferentially retained. At optimal concentration, LH-Sl gives a hemagglutination titer of 1: 1280 (Table 3). In quantitative precipitin tests, this antigen and its antiserum prove to have an opTABLE OPTIMAL

COMBINING

LH-Sl

vs

LH-Sl

4 RATIO

AS

AG Dilution AS Dilution

1 1:2 1:4 1:8

1:4

1:s

1:10

1:32

1:l34

1:128

ago ag ag ag

OR” ag ag ag

ab OR ag ag

ab ab OR ag

ab ab ab OR

ab ab ab ab

D ag refers to antigen b OR refers to optimal c ah refers to antibody

excess. ratio. excess.

AND HAKIM

timal combining ratio of 1 mg antigen to 8 ml antiserum (Table 4). This is equivalent to 5.8 mg antibody protein: 1 mg antigen protein. CROSS-REACTION WITH SHEEP SERUM PROTEIN

LH-Sl antiserum gives a strong crossreaction in agar diffusion plates with sheep serum protein. The bands formed correspond to the peripheral group that develop between the antiserum and LH-Sl (Fig. 5). These reactions may be removed by absorption with sheep serum, at an optimal combining ratio of 14 mg sheep serum protein to 1.0 ml antiserum. When tested with LH-Sl, the sheep serum absorbed LH-Sl antiserum gives only the medial band of precipitin lines (Fig. 6). These appear to represent the reaction with the LH component that is not associated with sheep serum proteins. That this reaction is not due to endogenous LH present in the sheep serum employed in these tests is shown by identical reactions obtained when serum from a hypophysectomized ewe is used. Reactions of identity occur between normal sheep serum protein and that of the hypophysectomized sheep serum (Fig. 7). The optimal LH-Sl concentration for modifying erythrocytes in hemagglutination tests is 2 pg/ml. With cells so modified, the hemagglutination titer reaches 1: 1280. When sheep serum protein is used as antigen in this procedure, the optimal concentration proves to be 2.8 mg/ml (Table 3). This, too, gives a titer of 1: 1280. However, preabsorpt.ion of LH-Sl antiserum with sheep serum eliminates the responsible antibodies completely without reducing the reaction to LH-Sl. Absorption of the antiserum with LH-Sl reduces significantly the antibody titer for sheep serum protein (Table 5). From these combined results, it appears that LH-Sl antiserum is comprised of distinct and separable antibodies. Some antibodies react specifically with sheep serum _ proteins and the others with the LH molecule.

I.MMUNOLOGIC

FIQ. 5. LH-Sl AS sheep serum protein Fxo. 6. LH-Sl A5 mg/ml, 0.2 mg/ml; FIQ. 7. LH-Sl AS pophysectomized ewe 14.0 mg/ml. FIG. 8. LH-Sl AS LH-Sl AS incub. with LH-Sl (0.125 m&ml)

ANALYSIS

OF

17

GONADOTROPINS

in center well tested against LH-Sl 0.2 mg/ml; FSH-Sl 2.0 mg/ml, 02 mg/ml; 1.4 mg/ml, 14.0 mg/ml; LH-Sl 2.0 mg/ml. (Ss Abs) in center well tested against LH-Sl 2.0 mg/ml, 6.2 mg/ml; FSH-Sl 2.6 sheep serum protein 1.4 mg/ml, 14.0 mg/ml. in center well tested against LH-Sl 2.0 mg/ml; sheep serum protein from hy1.4 mg/ml ; sheep serum protein 1.4 mg/ml; saline ; saline; sheep serum protein in

center LH-Sl

well tested (0 25 mg/ml)

against ; sheep

LH-Sl serum

TABLE ANTIBODY

TITER

OF LH-Sl

2.0 protein

mg/ml; saline; LH-Sl 2.0 mg/ml; 14.0 mg/ml; LH-Sl AS incub. with

5

ANTISERUM

(ABSORPTION

TESTS) AB Titer

Optimal

Antigen

LH-Sl Sheep

serum

4 Ss refers

protein to sheep

serum

concentration

LH-SI

LH-Sl (Ss Abs)a

LH-SI (LH Abs)

2 a/ml 14 mg/ml

1280 1280

1280 0

0 320

in this

and

subsequent

tables.

18

SEGAL, LAURENCE, PERLBACHS, OF LH-Sl ANTISERUM TROPIC HORMONES

CROSS-REACTIONS WITH OTHER

absorbed with sheep serum. For the experiment reported in Table 8, absorption was carried out with optimal concentrations of sheep serum and the absorbed antiserum tested by both hemagglutination and agar

The antiserum to LH-Sl does not give precipitin bands in agar-diffusion plates when tested with ovine pituitary tropic TABLE SEROLOGIC

REACTIONS

AND HAKIhI

6 LH-Sl

WITH

ANTISERUM

Agar-diiusion Con~;ce~;ion

%?z

Antigen

LH-Sl LH

(ovine)

(Fraction

C, Li)

FSH-Sl

LTH

(ovine)

4B74948

LTH

(ovine)

R10109

Pergonal-23 (Human GH

(bovine)

(Squibb) (Panlitar)

post-menopausal R50109

urine

gonadotropin)

(Somar-A)

hormones other than LH or FSH (Table 6). The cross-reaction with FSH-Sl is limited to a single precipitin band that appears to correspond to the reaction obtained with sheep serum protein (Fig. 5). An ovine pituitary LH preparation (ICSHFraction C) different from that used in immunization gives a very strong positive reaction with LH-SI antiserum (Table 6). Other gonadotropic hormones, from various heterologous sources, fail to cross-react with the LH-Sl antiserum (Table 6). OF LH-SI LH-Sl ANTISERUM As low as 2.5 pg of LH-Sl may give a positive weaver finch reaction. At a dose of 40 pug in 0.25 ml normal rabbit serum, 100% of the tested birds respond positively. If, however, this dose is mixed in vitro with LH-Sl antiserum, in the region of antibody excess and the precipitate centrifuged out, the supernatant shows a complete loss of hormonal activity (Table 7). Biologic activity is also reduced considerably when the LH-Sl is incubated with antiserum that has been previously In

Vitro WITH

INACTIVATION

Hemagglutination AB Titer

2.0

6

0.02

1,230

2.0

1

0.2

5,120

2.0

1

1.0 0.33 0.2

4.0

0

0.33

0

4.0

0

0.33

0

0.36

0

0.33

0

4.0

0

2.0

0

0

diffusion to assure that there was complete removal of the sheep serum antibodies. The absorbed antiserum was incubated under various conditions with proportional amounts of LH-Sl. At all concentrations used, the amount of LH-Sl present in the TABLE ABSORPTION

OF LH-Sl

7 WITH

LH-Sl

ANTISERUM

Weaver finch reactions (positive/total number) YE1 PI3

NRSa

LB-AS

40.0 20.0 10.0 5.0 2.5

15/15 10/15 6/15 2/15 l/15

015 o/10 o/10

o NRS subsequent

refers to normal tables,

O/5 O/5 rabbit

serum

in this

and

final solution is adequate to give 100% response in all birds. When the LH preparation is incubated with sheep serum absorbed antiserum at a ratio of 5: 1 (antiserum: antigen) or less, the preparation is not inactivated sufficiently to eliminate the biologic activity of the LH. This

IMMUNOLOGIC

ANALYSIS

indicates that insu$cient antibody was present in relation to the amount of LH. When the ratio is increased to 7 ml antiserum to 1 mg antigen only one of five birds gives a positive response (Table 8). TABLE SS-ASS.

8

OF LH-~1

INCUBATION

LH-Sl

WITH

ANTISERUM

immature female mice. In normal rabbit serum, FSH-Sl gives a maximal uterine stimulation at doses of 1 mg and 0.5 mg. The activity of the material is significantly reduced following incubation with LH-Sl antiserum (Fig. 9). In this experiment, antiserum was added to the point of completion of visible precipitation. It, was

LH-Sl

dose

Weaver finch reaction (positive/tots1 number)

ratio AS/-W bl/ms)

rr/~;,total

28/.25 45/.25 50/.25

LH-AS

515 5/5 5/5

l/5 5/5 5/5

to antiserum;

Ag refers

to antigen.

It should be recalled that serologic tests, reported above, show that the optimal combining ratio for the LH antiserumantigen system is 8: 1 (Table 4, Fig. 8). Presumably, inactivation at this ratio would reduce further the biologic activity. OF FSH-SI LH-Sl ANTISERUM From the results above, it should be possible to remove the LH molecules that are present in FSH-Sl by selectively absorbing with LH-Sl antiserum. Normally FSH-Sl gives a positive weaver finch reaction at the dose level of 375 pg. Following absorption with LH-Sl antiserum, more than 5 times this dose is negative (Table 9). The effect of this selective LH In.

Vitro

INACTIVATION

WITH

LH-Sl

of FSH-SI Antiserum

9 OF FSH-Sl

50 i

4o

5 z jj 3

30

a m

1

Normal rabblt serum LH-SI antiserum

20 10

1.0

a5 Total

FIG.

9. Absorption

dose FSH-St

of FSH-Sl

0.1

0.0 I (mg)

with

LH-Sl

anti-

serum.

proven to be an antibody excess by agar diffusion tests. After centrifugation the supernatant was injected into the mice. At a dose of 0.1 mg, which normally is above the minimal effective dose for doubling the uterine weight, pre-absorbed FSH-Sl fails to increase uterine weight significantly above the control level. DISCUSSION

TABLE ABSORPTION

Absorption with LH-SI 60

7/l 5/l 4.5/l

5 AS refers

NRS

19

OF GONADOTROPINS

WITH

ANTISERUM Weaver finch reactions (positive/total number)

FSH-Sl dose La

NRS

LH-AS

2ooo 1000 500 375 250

3/3 4/4 4/8 l/4 o/5

O/5 O/5 O/5 O/5 o/5

removal on the total gonadotropic potency of FSH-Sl has been further analyzed by means of the uterine weight response of

The relationship of the hormonally active moiety to the antigenic constitution of the NIH-Sl sheep pituitary gonadotropins is made complicated by the presence of non-specific sheep serum protein antigens. Immunization of laboratory animals with LH-Sl or FSH-Sl induced the formation of at least 4 antibodies, of which only 1 or 2 are directed toward the hormones themselves and the remainder towards the sheep serum contaminant. That this cross reaction with sheep serum proteins is not due to circulating endogenous LH or other pituitary protein hormones has been shown by the identity

20

SEGAL,

LAURENCE,

PERLBACHS,

of the sheep serum reaction when the serum used is taken from a hypophysectomized ewe. From this it must be concluded that the initial immunizing material contains sheep serum protein as a significant antigenic component. It is apparent, however, that the antibodies developed against the sheep serum prot,ein constituent of the NIH-Sl gonadotropins are distinct from the specific LH or FSH antibodies. If the antisera, first absorbed with sheep serum, are then tested against the original gonadotropin preparation, the hemagglutination antibody titer is not reduced. A further indication of the presence of separate and distinct antibodies was established when it was observed that the antibody(s) remaining after the sheep serum absorption still retained the capacity to inhibit biologic activity of these hormone preparations. These results clearly indicate the individuality and distinctiveness of the two antibodies present in the sera. The combining ratio at the equivalence zone of the antibody for the LH moiety would indicate that the hormone itself is weakly antigenic. It would require at least 8.0 ml of antiserum to completely neutralize the hormonal activity of 1.0 mg of the LH-Sl preparation. Ratios of antiserum: antigen less than 8: 1 failed to inhibit completely the biologic activity of the LH molecule. Analysis of the antibody: antigen protein precipitate in the zone of optimal proportions reveals a ratio of .5.8: 1. Assuming that the molecular weights of the antibody and the LH antigen are 165,000 and 28,000 (Ward, 1959) respectively, the molecular antibody : antigen rat.io at equivalence was approximately 1 to 1. This same antiserum combined much more efficiently with sheep serum proteins than it did with the purified LH preparation. On a volume: weight basis, only 1.0 ml of antiserum was required to completely precipitate 1.0 mg of sheep serum protein. This indicates that the antiserum that resulted from immunization with LH-Sl had a higher antibody content for sheep serum protein than for the LH antigen itself.

AND

HAKIM

That the antigenic and hormonal properties of the FSH molecule are independent was indicated by the absence of cross reactions when sheep FSH-Sl antiserum was tested against heterologous FSH containing preparations, Cross reaction with other tropic hormones was limited to the reaction with LH purified from sheep hypophyses. Similarly, the sheep LH-Sl antiserum failed to cross react with heterologous LH containing preparations, but did have the ability to cross react to a limited degree with the FSH-Sl antigen. This latter reaction can be attributed to the presence of sheep serum proteins common to both the FSH-Sl and LH-Sl. We do not mean to imply, however, that the same sheep serum contaminant is common to both, but only that these two hormone preparations contain sheep serum protein. These cross reactions which occur between LH-Sl and FSH-Sl were detected only by agar gel diffusion techniques. The hemagglutination test failed to reveal this cross reactivity and remains unexplained. The FSH-Sl preparation does contain a minor contamination of LH, as determined by the weaver finch reaction. The LH content in the FSH-Sl preparation is apparently below the threshold for detecting visible reactions by agar gel diffusion techniques. The LH-Sl antiserum is, however, capable of immunologicaly neutralizing the LH activity in the FSH-Sl as shown in the specific LH bio-assay. In addition, this antibody also reduces considerably the total gonadotropin activity in the mouse uterine weight test. SUMMARY

Antisera have been developed in rabbits against follicle stimulating hormone and luteinizing hormone purified from sheep hypophyses. The resulting antiserum contains separate and distinct antibodies for sheep serum protein and for the active hormone moiety. The antigenic and hormonal properties are independent as indicated by the lack of cross reactivity with heterologous FSH and LH containing substances. A cross reaction occurs between

IMMUNOLOGIC

ANALYSIS

the FSH-Sl and LH-Sl preparations and their antisera which can be attributed to the common presence of contaminating sheep serum protein in both hormone preparations. This cross reactivity could be detected by the agar gel diffusion technique, but not by the hemagglutination procedure. The biologic activity of the hormones could be neutralized only in the region of optimal proportions and in the region of antibody excess. Immunologic evidence indicates that the two gonadotropins, FSH and LH, are distinct chemical and antigenic entities. There is present in the FSH-Sl a cont,aminant with LH activity. REFERENCES BOYDEN, S. V. (1951). The adsorption of proteins on erythrocytes treated with tannic acid and subsequent hemagglutination by antiprotein sera. 3. Exptl. Med. 93, 107-120. COLLIP, J. B., AND ANDERSON, E. M. (1934). Serum inhibitory to the bhyrotropic hormone. Lancet

226, 7678. DEAN, H. R., AND WEBB, R. A. (1926). Influence of optimal proportions of antigen and antibody in serum precipitation reactions. J. Pathol. Bacterial. 29, 473492. FEINBERG, J. G. (1957). Identification, discrimination and quantification in Ouchterlony gel plates. Intern. Arch. Allergy Appl. Immunol.

2, 129-152. GESCHWIND, I. I. (1959). Species variation in protein and polypeptide hormones. In “Comparative Endocrinology” (A. Gorbman, ed.), p. 421443. Wiley, New York. HAYS, E. E., AND STEELMAN, S. L. (1955). Chemistry of the anterior pituitary hormones. In “The Hormones” (G. Pincus and K. V. Thiman, eds.), pp. 201-234. Academic Press, New York. HENRY, S., AND VAN DYKE, H. B. (1958). A study of the antibodies produced in response to purified preparations of sheep interstitial cell stimulating hormone. J. Endocrinol. 16, 310-

325. LEATHEM, J. H. (1949). The antihormone problem in endocrine therapy. In “Recent Progress in Hormone Research” (G. Pincus, ed.), Vol. IV, pp. 115-152. Academic Press, New York. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1952). Protein measurement with the Folin phenol reagents. J. Biol. Chem. 193, 265-275.

OF

GONADOTROPINS

21

MADDOCK, W. 0. (1949). Antihormone formation complicating pituitary gonadotropin therapy in infertile men. I. Properties of the antihormones. J. Clin. Endocrinol. 9, 213-233. SEGAL, S. J. (1957). Response of weaver finch to chorionic gonadotrophin and hypophysial luteinizing hormone. Science 126, 1242-1243. SEGAL, S. J., NIU, L., AND HAKIM, S. (1960). Im-munologic analysis of sheep pituitary LH. Proc. 1st Intern. Congr. Endocrinol., Copenhagen, p. 1093. THOMSON, D. L., COLLIP, J. B., AND SELYE, H. (1941). The antihormones. J. Am. Med. Assoc. 116, 132-136. V.~N DYKE, H. B., P’AN, S. Y., AND SHEDLOVSKY, T. (1950). Follicle-stimulating hormones of the anterior pituitary of the sheep and t,he dog. Endocrinology 46, 563-573. WARD, D. N., MCGREGOR, R. F., AND GRIFFIN, A. C. (1959). Chromatography of luteinizing hormone from sheep pituitary glands. Biochim. et Biophysics Acta 32, 305-314. ZONDEK, B., AND SULMAN, F. (1942). “The Antigonadotrophic Fact,or with Consideration of the Antihormone Problem,” 185 pp. Williams and Wilkins, Baltimore. DISCUSSION LEATHEM: (1) Can one absorb out the LH activity from human menopausal urine? (2) What, is the anti-LH activity when tested in animals given LH and anti-LH separately? SEC.4~: (1) Although we have established, by the weaver finch test, that human post-menopausal urine (Pergonal) contains LH, we have not yet tried to eliminate this activity with the use of antisera. (2) Using immature male and female rats, we have not been able to demonstrate the inhibition of the rat’s endogenous gonadotropins by the administration of sheep pituitary anti-FSH or anti-LH. This parallels our serologic studies which failed to give cross reactions between rat pituitary extracts and the sheep gonadotropin anti-sera. However, in ot,her experiments we have been able to achieve in vivo inactivation of exogenous gonadotropins, administered to 23-day-old male rats. In this experiment the young rats received daily subcutaneous injections of LH-Sl. Some were given, also, intraperitoneal injections of LH-Sl antiserum. Seminal vesicle and ventral prostrate weights, as well as testicular weight, were reduced in the latter group as compared to the group receiving LH without anti-serum.