375

Clinica Chimica Acta, 61 (1975) 375-379 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed in The Netherlands

CCA 7127

EFFECT OF EXTRACTION METHODS ON TWO PREGNANCY RELATED, ANTIGENIC COMPONENTS OF HUMAN CHORIONIC GONADOTROPIN PREPARATIONS

JOHN M. YODER and PATRICIA

K. JOHNSON

Ames Research Laboratory, Ames Company, Division of Miles Laboratories, Inc., Elkhart, Ind. 46514 (U.S.A.) (Received

December

31, 1974)

Summary One of the pregnancy related, antigenic components of HCG preparations is not readily detected in kaolin extracts of pregnancy urines; it is, however, readily detected in these urines when concentrated by lyophilization or in their benzoic acid extracts.

Introduction It is important to identify immunologically active components of an immunoassay system and to identify factors that alter their representation in test materials. At least two pregnancy related components have been immunologically detected by precipitation methods in various urinary human chorionic gonadotropin (HCG) preparations [l-4]. These two components are also readily detected in urines from patients with trophoblastic tumors but have never been detected in normal, non-pregnancy urines concentrated to various degrees [4]. Antibodies to these components cannot be readily removed from antiserum by absorption with normal, non-pregnancy human serum or urine antigens [ 1,2] . Immunodiffusion patterns indicate that antigenic determinants of both components are present on the biologically active HCG molecule [2]. This report describes a relative loss of one of these components during kaolin extraction. It is well-known that immunological levels of HCG or of a closely related gonadotropin, luteinizing hormone (LH), in kaolin extracts are much lower than those in urine concentrated by other methods or dialyzed [5-lo]. Methods Pregnancy urines Urine samples were collected

from five women during early pregnancy.

A

376

small portion (control) of each urine was dialyzed extensively (until no material absorbing at 215 nm could be detected in the dialysate) and lyophilized. Another portion of each urine was extracted by the kaolin method of Albert et al. [ 111. In three cases, a third portion was extracted by the benzoic acid method of Butt 1121.

Extraction methods In the method of Albert et al. [II] gonadotropins (acidified) were absorbed on kaolin, eluted with ammonium hydroxide, and precipitated with acetone to yield Fraction A [ll] . The method was modified slightly by the addition of 6 gm of kaolin/l (large samples) or 20 gm of kaolin/l (small samples) and allowing the kaolin mixture to settle overnight at 4°C. In the method of Butt [12], the crude gonadotropins were adsorbed on a tungstic and benzoic acid precipitate and then eluted with an ammonium acetate solution. We modified this method slightly by precipitating the crude gonadotropins with acetone (2 volumes acetone and refrigerating overnight). The wet acetone precipitates from both extraction methods were dissolved in water, desalted (Sephadex’ G-25, 21 mm X 300 mm column), and lyophilized. The volume of urine extracted varied from 260 ml to 1500 ml.

Antisera Antisera were produced to HCG as previously described (41. The rabbit antisera to semipu~fied HCG were absorbed with non-prelacy urine solids [l] to remove precipitating antibodies to one or two contaminating antigens. Precipitating antibodies to the two pregnancy related components were not removed by absorbing even with a 3-fold excess of urine solids.

Immunoelectrophoresis

(IEP)

Immunoelectrophoretic procedures were carried out using a previously described procedure [4] except that 1.0% Noble agar was used and the electrophoresis time was one hour. Experimental

Results

Examples of IEP results for the lyophilized kaolin and benzoic acid extracts, and controls are in Fig. 1. The kaolin extracts are deficient in pregnancy related component I relative to component F. Patterns of benzoic acid extracts are very similar to those obtained with the control. The extracts were each studied by IEP, using absorbed anti-HCG, at increasing extract concentrations (Table I). It was found that component I was usually still undetectable in the kaolin extracts even when they were concentrated 50-fold more than those concent~tions in which component F was just detected. In sharp contrast, in control and benzoic acid extracts, component I could usually be detected after concentrating only 2-3-fold over that level where component F was just detected. Results from unadsorbed anti-HCG that contained precipitating antibody to both components F and I were similar to those of Table I. No consistent increases of component I concentration relative to component F concentration

377

Fig. 1. Pregnancy related components of kaolin and benzoic acid extracts and controls. Antiserum in A and B is our absorbed rabbit anti-HCG (No. ZOO). A. Top well: control 5%; bottom well: benzoio acid extract 1%. 8. Top well: control 10%; bottom weR: kaolin extract 1%. The controls and extracts are all from the same individual (XI). Component designations are arbitrary. Component F (the more negatively charged component) is probably the same as “5” of Hamsshige. et al. 1131 and component I is probably the same as “6”. F is the predominating component of HCG since antibodies to it are present in all of our antisera to semipurified and HCG highly purified to such an extent that it gives a single precipitate line with unabsorbed antisera to crude HCG. Antiserum to the higbIy purified HCG routinely has no precipitating antibody to normal non-pregnancy urine components [41. Anti-F is also present in various absorbed commercial anti-HCG. Our antisera containing only precipitating anti-F readily neutralized HCG ventral prostate biological activity. Precipitating antibody to component I is present in some of our antisera to semipurified and highly purified HCG. Others [21 have reported antibodies to this second pregnancy component, in antisera to purified HCG having biological activities more than 10 000 LUJmg.

were detected in the original urines after kaolin extraction. This indicates destruction of component I in the kaolin procedure or failure of component I to elute from the kaolin.

TABLE I COMPARISON OF IEP THRESHOLD CONCENTRATIONS COMPONENTS IN DIFFERENT URINE EXTRACTS

FOR THE TWO PREGNANCY

RELATED

The figures denote ratios of extract concentrations of I/F where each of the two components was just faintly detected by IEP. For example: if component I were just faintly detected by IEP at 1% extract concentration and F at 0.3%. the ratio would be 3. For some kaolin extracts. component I was not detected even in extract concentrations of 30%. In some cases, a short I spur was visible in the concentrated kaolin extracts. Arbitrarily, it was not considered as detectable unless its length was over one-ha@ the length of the F precipitin arc. Component F was always detectable at lower extract concentrations than was component I. Absorbed anti-HCG was used. Treatment

Urine

LW Month of gestation: 3.5 Antisera: Control Benzoic Kaolin

143

200

2 3

1 3

>66

10

MA 3.0

VR 1.5

JC 1.6 143 3 5

200 2 lOO >lOO

145 2 3 >60

200

145

2 60

1

>lOO

BM 2.5 200 -

1

100

246 -

2

200 lOO >so

318

Discussion This deficiency of pregnancy related component I in kaolin extracts probably, in some specific assays, contributes to the reported overall loss in immunological activity of kaolin extracts. The kaolin extraction method recovers only part of the immunologic gonadotropin activity from pregnancy urines [5,9]. Kaolin extracts of non-pregnancy urines also have much less immunological LH activity than do the dialyzed, unconcentrated urines [6,8]. Since component I has very low rat accessory gland activity [13,14], its depletion could reduce the immunological activity of kaolin extracts relatively more than the biological activity. Thus, the biological/immunological activity ratio for kaolin extracts is greater than that for unextracted urine [ 51. This can occur when pregnancy specific immunologic assays are used that partly react with the pregnancy immunogens having low biological activity such as component I or other immunogens. On the basis of molecular size, Ryan [15] has described a biologically inactive, immunologically active LH in ultrafiltered urine that also is depleted in kaolin extracts relative to the biologically active LH. Other pregnancy related immunogens or forms of HCG reacting with the assay but having reduced biological activity (such as partially desialated HCG [16] or free subunits of HCG [17-191 also may be relatively depleted in kaolin extracts and, therefore, contribute to this altered biological/immunological activity ratio. Pregnancy related component I is not simply desialated HCG, since removal of sialic acid from HCG results in an IEP pattern showing slower migrating material [20] with no spur. This pattern does not match the IEP patterns previously reported [1,4] for unextracted, concentrated urinary HCG. The relationship of component I to free subunits is not known. When comparing the antigenic determinants and biological activities of comparable extraction or preparation various LH or HCG preparations, methods should be used. In immunoassays, for optimal results, the standard should be prepared in a similar manner to the unknown [20,21]. Acknowledgement We thank HCG biological

Dr S. Hamashige for carrying activity by antisera.

out studies on neutralization

of

References 1 S. Hamashige and E.R. ArquiUa. J. Clin. Invest., 43 (1964) 1163 2 C.E. Wilde and K.D. Bag&awe, in G.E.W. Wolstenholme and J. Knight (eds), Gonadotropins: Physico-Chemical and Immunological Properties, Little, Brown, Boston. 1965, p. 46 3 C.L. Robyn, J.M. Limbosch and P.O. Hubinont, Rev. Franc. Etud. Clin. Biol. 10 (1965) 101 4 J.M. Yoder, E.C. Adams, Jr, K.W. Chambliss and B.E. Loughman, J. Clin. Endocrinol., 27 (1967) 509 5 B. Hobson and L. Wide, Acta Endocrinol. (Kobenhavn), 46 (1964) 632 6 V.C. Stevens J. Clin. Endocrinol.. 29 (1969) 904 7 H. Schmidt-Elmendorff and E. Kaiser, Acta Endocrinol. (Kobenhavn). Suppl. 141 (1970) 39 8 H.E. KuIin. A.B. Rifkind and G.T. Ross, J. Clin. Endocrinol., 28 (1968) 543 9 T. Tamada. M.L. Taymor and J. Stark, Am. J. Obstet. Gynec., 95 (1966) 249 10 A. Baghdassarian, S. Fisher, H. Guyda, A. Johanson, T.P. Foley, Jr. R. Penny and R.M. BIizzard, Am. J. Obstet. Gynec., 108 (1970) 1178

379 11 12 13 14 15 16 17 18 19 20 21

A. Albert, S. Kelly, L. Silver and J. Kobi, J. Clin. Endocrinol., 18 (1958) 600 W.R. Butt J. Endocrinol.. 17 (1958) 143 S. Hamashige. M.A. Astor, E.R. ArquiBa and D.H. van Thiel. J. Clin. Endocrinol., 27 (1967) 1690 C.E. Wilde. A.H. Orr and K.D. Bag&awe, J. Endocrinol., 37 (1967) 23 R.J. Ryan, Acta Endocrinol. (Kobenbavn). Suppl. 142 (1969) 300 H. Van HeII. R. Matthijsen and J.D.H. Homan. Acta Endocrinol. (Kobenhavn). 59 (1968) 89 P. Franchimont. in Report 2nd Int. Symp. Protein Polypeptide Hormones (Liege), Nature New Biol.. 233 (1971) 256 B.D. Weintraub and S.W. Rosen, J. Clin. Invest., 52 (1973) 3135 J.L. Vaituhaitis, J. Clin. Endocrinol., 37 (1973) 505 C.E. Wilde. Acta Endocrinol. (Kobenhavn). Suppl. 142 (1969) 360 A. Albert, E. Rosemberg, G.T. Ross, C.A. Paulsen and R.J. Ryan. J. Clin. Endocrinol., 28 (1968) 1214