Protein a as an Immunoadsorbent in Radioimmunoassay: Usefulness in a Rapid Assay for Urinary Luteinizing Hormone*

FERTILITY AND STERILITY Copyright ~ 1980 The American Fertility Society

Vol. 33, No.2, February 1980 Printed in U.SA.

PROTEIN A AS AN IMMUNOADSORBENT IN RADIOIMMUNOASSAY: USEFULNESS IN A RAPID ASSAY FOR URINARY LUTEINIZING HORMONE*

ROBERT W. REBAR, M.D.t JILL AURAND, B.A. MICHAEL UNGER, PH.D.:j: AARON J. W. HSUEH, PH.D. Department of Reproductive Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093

A staphylococcal cell wall protein, designated protein A, is known specifically and rapidly to bind to immunoglobulin G. We have utilized this protein as an immunoadsorbent to separate antigen-antibody complexes from free antigen in a rapid 6-hour radioimmunoassay for urinary human luteinizing hormone (hLH). We have further documented the efficacy of this system and the optimal conditions for this assay. The ability of this assay to provide rapid and reliable measurements of hLH in timed urinary specimens provides a useful tool for determination of the preovulatory LH surge and prediction of the time of ovulation in women. Fertil Steril33:151, 1980

free antigen in RIA systems. 4 - 7 In addition, because of the rapid and specific binding of protein A to immunoglobulin G (lgG) of various species, this immunoadsorbent can be used in the development of rapid RIA systems. This report documents the utility of a rapid (6-hour) radioimmunoassay using protein A as an immunoadsorbent for the measurement of urinary gonadotropins. The applicability of this method for prediction of the time of ovulation and its possible use in different RIA systems are demonstrated.

The development of sensitive and specific radioimmunoassays (RIAs) has permitted delineation of the patterns of circulating and urinary gonadotropins. 1 Such determinations, however, have generally proved to be of more retrospective than predictive value because of the length oftime generally required for RIA. With regard to detection of the preovulatory luteinizing hormone (LH) surge, the need for sequential blood sampling frequently meets with poor patient acceptance, whereas the time and effort required to measure urinary gonadotropin excretion have generally limited widespread use of these procedures. 2 , 3 Recently, immunologic studies have demonstrated the presence in Staphylococcus aureus of a bacterial cell wall protein, termed protein A, which can serve as a convenient immunoadsorbent for separating antigen-antibody complexes from

MATERIALS AND METHODS

Preparation ofStaphylococcal Protein A. The immunoadsorbent was prepared according to the procedure of Kessler. 5 Briefly stated, S. aureus type Cowan I was grown at 37° C in trypticase soy broth. Six 4-liter flasks, each containing 2 liters of media, were inoculated with 15 ml of an overnight culture of S. aureus. The bacteria were allowed to grow for 30 hours with vigorous shaking. The cultures were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS, 0.15 M NaCI and 0.04 M NaHP0 4 , pH 7.2), and fixed with formalin

Received August 2, 1979; accepted August 16, 1979. *Supported by Rockefeller Foundation Grant RF-75029 and National Institutes of Health Research Grant HD-12303. tClinical Associate Physician of the University of California, San Diego Clinical Research Center. To whom reprint requests should be addressed. :j:Salk Institute, San Diego, Calif.

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(1.5% formalin with stirring at 23° C for 1.5 hours). The bacteria were washed to remove the formalin and then heated to 80° C for 5 minutes. After two more washes, the bacteria were resuspended to a concentration of 10% (w/v) in the PBS plus 0.02% sodi urn azide and frozen in amounts sufficient for 1 week of assays. The working stock was kept at 4° C after thawing. Just before use, the bacteria were gently homogenized with a Dounce homogenizer to disrupt clumps. Typical yields ranged from 6 to 8 gm of bacteria/liter of culture media. Radioimmunoassays. Hormone concentrations were measured simultaneously by the routine RIA systems utilized in our laboratory, employing either second antibody (anti-rabbit -V-globulin) or protein A as the separating agent. RIAs for hLH and follicle-stimulating hormone (hFSH) were conducted utilizing materials obtained from the National Pituitary Agency, National Institute of Arthritis, Metabolism and Digestive Diseases, as previously described,S, 9 with the following modifications. In the standard non-equilibrium double-antibody RIA, 100 ....1 of standard or unknown sample were added to 200 ....1 of 0.1% gelatin-PBS, pH 7.4, containing 0.05 M ethylenediaminetetraacetic acid. A portion (100 ....1) of the appropriate dilution of antiserum in 0.5% normal rabbit serum was then added and the assay mixture was incubated for 24 hours at 4° C. 125I-Labeled hormone (100 ....1) (approximately 10,000 to 15,000 cpm) was added and the assay mixture was again incubated for 24 hours at 4° C. The appropriate dilution of second antibody (100 ....1) was then added, and each assay tube was incubated for an additional 24 hours at 4° C. After the addition of3 ml of cold PBS to stop the reaction, each tube was then centrifuged for 20 minutes at 3000 rpm (1000 x g) to separate antigen-antibody complex from free antigen, the supernatant was decanted, and the precipitate containing bound hormone was counted in a -V counter (Searle 1185). Separation of antigen-antibody complex from free antigen with protein A was carried out in assay mixtures incubated as described above for the first 48 hours. Protein A (usually 50 ....1) was added on the 3rd day, the assay mixture was incubated for 15 minutes at room temperature, and 3 ml of cold PBS were then added. After centrifugation at 3000 rpm for 20 minutes, the supernatant was decanted and the precipitate was counted. "Rapid" assays were also performed in which the unknown sample or standard, labeled hormone, and first antibody were added simultaneously and

February 1980 incubated in a water bath at 37° C for 4 hours. Protein A (50 ....1) was then added, bound hormone was separated from free hormone by centrifugation after an additional 15-minute incubation, and the precipitate was counted. Results of gonadotropin assays are express in terms ofthe Second International Reference Preparation for human menopausal gonadotropin (2nd IRP-hMG). Extraction of Gonadotropins from Urine Samples. A 50-ml aliquot from either a timed or first morning voided urine specimen was extracted as reported by Hansen and Ross.3 The pH ofthe sample was corrected to 4.5 with concentrated acetic acid. The gonadotropins were then precipitated by the addition of 100 ml of cold acetone, with the mixture being allowed to stand at 4° C for 30 minutes. The mixture was then centrifuged for 20 minutes at 2000 rpm, the supernatant was decanted, and the precipitate was dried under air. The dried precipitate was then redissolved in 10 ml of 0.1 % gelatin-PBS before assay, therefore providing a 5-fold concentration of the original sample. The concentrated samples were then assayed as described and the results expressed as milliinternational units per milligram of creatinine in the urine. The creatinine concentration was determined in an aliquot of urine by the alkaline picrate colorimetric method (Jaffe reaction). RESULTS

Effects ofConcentration, Time, and Temperature on Binding ofProtein A to Antigen-Antibody Complex. Various quantities of protein A were added to assay tubes incubated with buffer, labeled hormone, and antiserum (maximal binding tubes) and to others containing only buffer, labeled hormone, and normal rabbit serum (nonspecific binding tubes). Following a 15-minute incubation at room temperature, bound hormone was separated from free hormone by centrifugation. Typical results of such a test using the hLH RIA system are shown in Figure 1. Maximal precipitation of bound hormone was generally observed when 25 ....1 of protein A were added. To ensure complete precipitation, twice the amount of protein A required to achieve maximal precipitation was used in subsequent experiments. Nonspecific binding did not increase dramatically as increasing concentrations of protein A were added. From a practical standpoint, we found it technically difficult to add more than 200 ....1 of protein A to any assay tube because the bulk of the precipitate was then too large to permit

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FIG. 3. Effect of incubation temperature on maximal and nonspecific binding in the hLH RIA system. All tubes were incubated with 50 ILl of protein A for 15 minutes at various temperatures prior to addition of cold PBS and centrifugation.

precise, reproducible decanting ofthe supernatant fluid. Maximal binding was invariably greater following use of protein A than of second antibody for separation of bound hormone from free, indicating more complete precipitation of antigen-antibody complexes by protein A. Initial tests were also carried out to determine the time needed for the binding of antigenantibody complexes to protein A to achieve optimal separation of bound hormone from free. Results from one such study using the hLH RIA system are depicted in Figure 2. The separation of antigen-antibody complexes from free antigen could be accomplished extremely rapidly, with more than 90% of the complex bound to protein A in less than 30 seconds. To allow the addition of protein A to large numbers of assay tubes, protein A was routinely incubated for 15 minutes and then antigen-antibody-protein A complexes were separated by centrifugation after the addition of 3 ml of cold PBS to stop the reaction.

The effect of temperature on the binding of protein A to first antibody was also investigated (Fig. 3). Maximal binding was generally greatest at room temperature (25° C) and decreased with increasing temperature. At 37° C, only about 83% of antigen-antibody complex was precipitated by protein A in comparison with the quantity precipitated at 25° C. Measurement of Urinary LH Employing Protein A as Immunoadsorbent. Comparison of standard curves of hLH and hFSH RIA employing either second antibody or protein A as the separating agent is shown in Figure 4. Although the curves were not always identical, there was always good agreement between known and unknown quantities of hormone measured by each method. We were interested in applying this rapid method of separation to the measurement of LH concentrations in urine. The value for hMG added in varying quantities to a urinary aliquot containing approximately 50 mIU/ml of hLH was linear and almost identical by either method of separation (Fig. 5). Urinary LH concentrations in first morning voidings obtained daily from a patient with premature ovarian failure were measured using either second antibody or protein A as the separating agent (Fig. 6). The results were normalized by expressing them as mill i-international units ofLH per milligram of creatinine. The patterns of urinary excretion ofLH by either separation method are virtually identical except for values at very high levels. The differences in the values were not statistically significant (P > 0.2, paired t-test). To shorten the RIA procedure, antigen and antibody were incubated for 4 hours at 37° C. Protein A was then added, the tubes were incubated for an

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additional 15 minutes and centifuged, and the precipitated IgG-protein A complexes were counted as before. As shown in Figure 7, the standard curves . from this "rapid" assay were compared with standard curves for LH obtained in routine assay employing either second antibody or protein A. Although the rapid assay is less sensitive, control samples measured similarly in comparison with the determinations obtained using the two other methods and were not significantly different (P > 0.1, analysis of variance). Comparison ofthe urinary excretion ofLH measured by all three methods and serum LH levels measured by double-antibody RIA in a normally cycling woman is depicted in Figure 8. Aliquots of urine from 8-hour timed specimens were measured prior to and during the LH surge. Similar results were obtained by all three urinary RIA methods. Increased urinary excretion ofLH was observed in the first 8-hour collection during which serum concentrations of LH were elevated.

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tremely rapid, with the separation technique not dependent on any particular physiochemical characteristics of the particlar antigen. Nonspecific binding can be kept to a minimum and more antigen-antibody is usually precipitated than with second antibody. Clinically, the rapid, quantitative measurement of gonadotropin in timed or first morning voided specimens is now assuming increasing importance. The more frequent use of artificial insemination, clomiphene citrate, and· exogenous gonadotropins to induce ovulation, and in vitro fertilization require knowledge of the precise timing of

ovulation. Since patient acceptance of frequent blood sampling has always been limited, the finding that the first morning urine voiding provides an accurate index of the pattern of gonadotropin secretion and excretion3 once more turned attention to the measurement of urinary gonadotropin in timed specimens. Furthermore, despite the inability of relatively insensitive serum gonadotropin measurements to discern any patterns present in prepubertal children and individuals with diminished gonadotropin secretion, collection and measurement of gonadotropins in first morning voidings have permitted the first delineation of definite patterns of gonadotropin excretion (and secretion) in young children. 1, 13, 14 The advantage of being able to concentrate specimens containing small quantities of gonadotropins prior to measurement is obvious. The accuracy and rapidity of the current urinary LH RIA

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procedure should make such determinations simpler and more feasible. Although we have utilized protein A for the developement of a rapid radioimmunoassay for urinary LH excretion, the possibilities for application of this separation procedure to other RIA systems are unlimited. In most RIAs it is the separation of bound antigen from free that is the most timeconsuming. Further application of protein A to such RIA systems should dramatically decrease the time in which such measurements can be made. Acknowledgments. We wish to thank Dr. Samuel S. C. Yen for his support and critical evaluation of this work and Ms. Del Austin for preparation of the manuscript. Portions ofthis study were conducted in the clinical research center of University Hospital with the aid of Dr. M. E. Quigley and J. Hoff, whom we also thank. REFERENCES 1. Rebar RW, Yen SSC: Endocrine rhythms in gonadotropins and ovarian steroids with reference to reproductive processes. In Endocrine Rhythms, Edited by DT Krieger. New York, Raven Press, 1979, p 259 2. Midgley AR Jr: Radioimmunoassay: a method for human chorionic gonadotropin and human luteinizing hormone. Endocrinology 79:10, 1966 3. Hansen JW, Ross GT: A new method simplifying collection of senal. specimens for gonadotropin determinations. J Clin Endocrinol Metab 41:241,1975 4. Kronvall G, Seal US, Finstad J, Williams RC Jr: Phylogenetic insight into evolution of mammalian Fe fragment of aG globulin using staphylococcal protein A. J Immunol 104:140, 1970

February 1980 5. Kessler SW: Rapid isolation of antigens from cells with a staphylococcal protein A-antibody absorbent: parameters of the interaction of antibody-antigen complexes with pro· tein A. J ImmunoI115:1617, 1975 6. Dubois-Dalcq M, McFarland H, McFarlin D: Protein Aperoxidase: a valuable tool for the localization of antigens. J Histochem Cytochem 25:1201, 1977 7. Jonsson S, Kronvall G: The use of protein A-containing Staphylococcus aureus as a solid phase anti-IgG reagent in radioimmunoassays as exemplified in the quantitation of a-fetoprotein in normal human adult serum. Eur J Immunol 4:29, 1974 8. Yen SSC, Llerena 0, Little B, Pearson OH: Disappearance rates of endogenous luteinizing hormone and chorionic gonadotropin in man. J Clin Endocrinol Metab 28:1763, 1968 9. Yen SSC, Llerena LA, Pearson OH, Littell AS: Disappearance rates of endogenous follicle-stimulating hormone in serum following surgical hypophysectomy in man. J Clin Endocrinol Metab 30:325, 1970 10. Frohman MA, Frohman LA, Goldman MB, Goldman IN: Use of protein A-containing Staphylococcus aureus as an immunoadsorbent in radioimmunoassays to separate antibody-bound from free antigen. J Lab Clin Med 93:614, 1979 11. Gupta RK, Morton DL: Double-antibody method and the Protein-A-bearing Staphylococcus aureus cells method compared for separating bound and free antigen in radioimmunoassay. Clin Chem 25:752, 1979 12. Ying S-Y, Guillemin R: Dried Staphylococcus aureus as a rapid immunological separating agent in radioimmunoassays. J Clin Endocrinol Metab 48:360, 1979 13. Hansen JW, Hoffman HJ, Ross GT: Monthly gonadotropin levels in premenarcheal girls. Science 190:161, 1975 14. Hayes A, Johansen A: Excretion ofFSH and LH in urine by pubertal girls. Pediat Res 6:18, 1972