Research Plus Inc Antibodies to Steroids and their Application


Antibodies to Steroids and their Application

Antisera to steroid hormones have found their greatest application in the assay of hormones. There have been, as well, physiologic studies using such antisera to neutralize the effects of indogenous and exogenous steroids. Antisera labelled with fluorescein have been used to localize steroids in tissues.

The immunologic assay procedure most commonly used for steroid hormones is termed radioimmunoassay. In this procedure, a radioactively-labelled steroid is bound by antibody. A calibration curve is then constructed correlating the ability of varying amounts of cold hormone to compete with the labelled steroid for antibody combining sites. In other words, less of the labelled steroid is bound as more of the unlabelled steroid is added. The concentration of a steroid in a biologic fluid can be determined by comparing its ability to interfere with binding of the labelled steroid by the antibody with the calibration curve previously constructed using the same antibody.

DEVELOPMENT OF THE STANDARD CURVE

Competitive protein binding assays depend on the more or less specific binding, not involving the formation or breaking of covalent bond, of a small molecule (ligand)to a protein according to the following equilibrium, where A is concentration of ligand and PR is concentration of protein and A.PR is concentration of complex. The assay is carried out using a tracer quantity of radioactive A. The quantity of radioactivity in the system is constant, and the quantity of A is variable. Under these conditions the quantity of radioactive A.PR is a function of the fraction of the total A bound to the protein. This fraction varies with the concentration of A shown below: This curve is known as the standard curve and is used as such to determine unknown amounts of ligand.

SPECIFICITY:
A number of controls must be run, and in particular, the effect of any cross-reacting steroids that may be present must be determined. (R Plus does not routinely run tests for specificity). It should be noted however, that numerous investigators have found that it is possible to develop specific, sensitive, reproducible radioimmunoassays of these steroid hormones. The range is usually in the nanogram or pilogram area. Among the steroids that have been assayed in this manner are estrogens, progesterone, testosterone and aldosterone.

Radioimmunoassays depend on the ability to separate free steroid from antibody bound steroid. Many procedures have been developed to do this. Those most frequently used in steroid radioimmunoassays involve antibody-coated polystyrene tubes, polymerized antibody, and the use of a second antibody to precipitate the anti-steroid antibody together with any bound hormone (double antibody method).

There have been very few investigators using fluorescein-labelled antibodies to localize the hormone in tissues. However, success in the localization of estrogens and testosterone has been reported.

PHYSIOLOGIC USE:
Some interesting physiologic studies using antibodies to steroids have been reported. Most of these were concerned with reproductive physiology, and it was shown that the biologic activity of estrogens, testosterone and progesterone can be neutralized by appropriate antisera. Aldosterone can also be neutralized by antisera.

Caldwell et al., in the general reference cited below, emphasize that antibodies that neutralize steroids might be useful not only in reproductive physiology, but might also serve to control some steroid-dependent cancers. In speaking of such antibodies, these authors state "....a potentially valuable tool has become available to the investigator in bio-medical sciences, the real value of which depends upon the ultimate imagination of the individual and his application of the techniques in developing new methology in the examination of physiologic processes".

For further details on teh uses of anti-steroid antibodies, see "Immunologic Methods in Steroid Determination", F.G.Peron and B.V.Caldwell, eds., Appleton-Century-Crofts, Meredith Corp., N.Y., 1970.

REFERENCES:

PREPARATION OF ANTIGENS, ANTIBODIES AGAINST STEROIDS:

	1. Erlanger,B.F., Borek, F.,Beiser,S.M. and Lieberman,S., JBC 228:713 (1957).
	2 Lieberman,S.,Erlanger,B.F.,Beiser,S.M. and Agate,F.J.,Jr. Rec.Prog.Hormone Res.,15:165 (1959).
	3. Zimmering,P.E.,Lieberman,S., and Erlanger, B.F., Biochem., 6:154 (1967).
	4. Grons,S.J., Campbell, D.H. and Weetall,H.H., Immunochem. 5:55 (1968).
	5. Smith,T.W., Butler,V.P.,Jr. and Haber,E., Biochem. 9:331 (1970).
ANTIHORMONAL EFFECTS OF ADMINISTERED ANTIBODIES AGAINST STEROIDS:

	1. Neri,R.O.,Tolksdorf,S.,Beiser,S.M.,Erlanger,B.F. and Agate,F.J.,Jr.,Endocrinology 74:593 (1964).
	2. Ferin,M.,Zimmering,P.E. and VandeWiele,R.L.,Endocrinology 84:893 (1969).
	3. Ferin,M.,Zimmering,P.E.,Lieberman,S. and VandeWiele,R.L.,Endocrinology 83:565 (1968).
USE OF ANTIBODIES AGAINST STEROIDS AS REAGENTS FOR ASSAYS:

	1. "Second Karolinska Symposium on Research Methods in Reproductive Endocrinology-Steroid Assay by Protein Binding", Supplement No.147 (March, 1970) Acta Endocronologica (Kbh).
	2. Mayes,D., Furuyama,S.,Kem,D.C. and Nugent,C.A., J.Clin.Endocr. 30:682 (1970).
	3. Midgley,A.R., Niswender,G.D. and SriRam,J., Steroids 13:731 (1969).

General Notes On the Use Of This Antisera:

A. Development of titration curves should be run as follows:

	1. Mass of 3H Steroid under investigation 100 pg.
	2. Antibody diluted with 0.1 ml borate buffer plus 0.6% human gamma globulin (Estriol has also been run with 1% human male serum - see comments below).
	3. Incubation volume: 0.5 ml.
	4. Equilibrium time: 20' at 4^oC.
	5. Separation: 0.5 ml ice-cold ammonium sulphate.

B. We do not freeze our stored material. However, some investigators have done so with no apparent degradation. We recommend only diluting that amount you anticipate using in a relatively short period of time. Diluted material has a tendency to deactivate over a prolonged period of time.

C. Anti-Cortisone, Anti-DOC, Anti-Progesterone and Anti-Testosterone react with one another to some extent but react primarily with the homologous determinant groups. They do not cross react with Anti-Estrone nor does Anti-Estrone cross react with the other.

D. Anti-Estriol may be used in measuring estriol in blood or urine in late pregnancy. The amount of estriol in blood in pregnancy is related to fetal well being and clinicians should find this useful in the management of certain high risk pregnancies. The procedure to follow is noted in the following reference:
	Gurpide,E.,Giebenhain,M.E.,Tseng,L. and Kelly,W.G.,"Radioimmunoassay for estrogens in human pregnancy urine, plasma and amniotic fluid", Am.J.Obstet.Gynec., 897,109,6 (1971).

E. The buffer solution used for Anti-Estriol was 0.01M sodium phosphate in distilled water adjusted to pH 7.4. For assays, 10 microliters at at time was diluted 1:10,000 (or the titre noted) in buffer containing 1% human male serum.

F. Specificity of Aldosterone: These antibodies find aldosterone and its gamma etiolactone about equally well. However, the antibodies cross react significantly with all delta four 3-ketosteroids tested. On the other hand, cross reactivity with estrogen and 3B-ol-delta five steroids is slight. Thus, the separation of aldosterone from other steroids is necessary before an assay can be carried out.
The sensitivity of a competitive binding assay will vary with experimental conditions under which the assay is carried out. Standard curves can be constructed using our serum 141 showing significant displacement of radioactive aldosterone with 10 pg of unlabelled aldosterone. This anti-serum should provide a sensitivity of less than 50 pg per sample obtained from a body fluid.

G. Specificity of Estriol: These antibodies find estrone, estrone, estradiol and estradiol about equally well. Estriol-16-glucuronide and estriol-17-glucuronide bind as well as estriol. Estrogens conjugated with glucuronic and/or sulfuric acid at the phenolic group in ring A do not cross react. However, 3-benzoates, 3-acetates and 3-methylethers have various degrees of cross reactivity. No significant cross reactivity towards delta five 3b-ol steroids or delta four-3-keto steroids has been reflected. The specificity of these anti-sera depend on the concentration of the anti-serum used in the assay. The cross reactivity with ring D conjugates of estriol makes possible an assay for the conjugates as such.

A method for the determination of estriol in blood, urine or amniotic fluid during pregnancy has been reported (see D above). This method has a range of approximately 100 pg to about 5,000 pg. This sensitivity is more than ample for blood in pregnancy.

H. All serum contains 0.2% sodium azide as a preservative.


Disclaimer
The information contained herein is, to the best of our knowledge, true and accurate. However, all recommendations or suggestions are made without guarantee, since the conditions of use are beyond our control. We disclaim any liability incurred in connection with the use of these data or suggestions. This publication is not to be taken as a license to operate under, or a recommendation to infringe any patents. The observance of all legal regulations and patents is the responsibility of the user.