A competitive inhibition enzyme-linked immunosorbant assay for frog calcitonin

GENERAL

AND

COMPARATIVE

A Competitive

ENDOCRINOLOGY

Inhibition

45,

12-20

(1981)

Enzyme-Linked for Frog Calcitoninl

Immunosorbant

Assay

DOUGLAS R. ROBERTSON Department

oj’ Anatomy,

St&e University of New York, Syrucuse, Nr\ra York I.3210 Accepted

December

upstutr

Me&d

Center,

23, 1980

A heterologous enzyme-linked immunosorbant assay (ELISA) for frog (Runu pipirns) calcitonin has been developed with a sensitivity of 17 pg. In this competitive immunoassay, synthetic salmon calcitonin is used to coat polystyrene microtiter plates. Antibody to salmon calcitonin was produced in rabbits and was diluted 1: 10,000 to 1:40,000 depending upon the amount of solid-phase antigen used to sensitize the plates. Increasing amounts of soluble calcitonin added results in a decrease in the optical density of the subsequent color reaction product. The reaction is linear over the log range of 12.5 to 400 pgiwell and diluted frog plasma samples are parallel to the standard assay curve. Intraassay coefficient of variation was 10% and interassay coefficient of variation was 15%. Normal values of frog plasma calcitonin ranged from 4.1 to 27.4 rig/ml reflecting seasonal variations in circulating calcitonin in this amphibian.

says (ELISA) adsorb large molecular weight antigens to the solid phase; with some assays utilizing haptens (see review of O’Beirne and Cooper, 1979). Such assays have been found to be rapid and similar in their sensitivity and reproducibility to conventional RIA procedures. The following account describes a heterologous competitive inhibition assay of frog calcitonin using synthetic salmon calcitonin as the solid-phase antigen. The procedure is relatively simple and is as sensitive as current RIA procedures. In addition, the use of a highly purified synthetic polypeptide provides an opportunity to examine some of the steric and kinetic properties in a solidphase antigen system.

Quantitative assays for the presence of an immunoreactive hormone, calcitonin in peripheral blood have employed the use of the radioiodinated polypeptide for radioimmunoassay (RIA) (Habener et al., 1972; Deftos et al., 1972, 1974; Cutler et al., 1974). Antisera to the salmon calcitonin molecule, a 32 amino acid polypeptide, cross-react with the hormone found in several classes of vertebrates, as in eel (Orimo et al., 1977), chicken (Cutler et al., 1974), and among_rmammals with porcine calcitonin (Deftos et al., 1972). Further, antiporcine calcitonin has been shown to cross-react with frog (Rana pipiens) calcitonin (van Noorden and Pearse, 1971). This property plus the presence of several identical amino acid sequences at the single immunoreactive C terminus (Potts et al., 1972; Dietrich and Rittel, 1970; Otani et al., 1975) would suggest that antisera to salmon calcitonin may cross-react with the frog polypeptide. Most enzyme-linked immunosorbant as-

MATERIALS

Synthetic salmon calcitonin (sp act, 4700 MRC Uimg powder; Lot K7 13-081, Armour Pharmaceutical Co.) was used for antibody (Ab) production, as the solid-phase antigen (Ag) and in all assays as the standard. This preparation was about 87% peptide by weight of which 95% was salmon calcitonin sequence. Antibody to sulmon culcitonin. The immunogen was prepared by a glutaraldehyde conjugation of 100 pg calcitonin powder (87 pg peptide dissolved in 0.1 ml 0.1 M HCI) to 630 pg Bovine serum albumin (BSA,

’ Presented in part at the VIIth International Conference on Calcium Regulating Hormones, September 1980. Estes Park. Colorado.

00 16-6480/8

110900 I 2-09$0 1.0010

Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

12

ELISA

FOR

FROG

Fraction V, United States Biochemical Co.) dissolved in 1.0 ml phosphate buffer (pH 7.4) as described by Reichlein et ul. (1968). The volume was adjusted to 3.3 ml with phosphate buffer and mixed with 3.3 ml complete Freunds adjuvent (Sigma Chemical Co.) for a total volume of 6.6 ml. A volume of 1.1 ml (122 pg protein/rabbit) of the above suspension was injected into the neck and sides of six female white New Zealand rabbits (2 kg body wt initially). Significant antibody in the serum of four rabbits was produced 98 days after an initial injection followed by four identical booster injections at 18 to 21-day intervals. Animals were bled and the globular fraction of the antisera was isolated by ammonium sulfate precipitation (l/3 saturation; Garvey et ul., 1977) and stored in phosphate-buffered saline (PBS; pH 7.2). Antibody in this study was RA/CT-78-4 with a total protein content of 11.035 mg/ml. Rrugents f’r ELISA. Horseradish peroxidase (Type VI, Sigma Chemical Co.) was conjugated to the IgG fraction of goat anti-rabbit IgG (8 mg/ml antibody protein, United States Biochemical) by the periodate method of Nakane and Kawaoi (1974) with rabbit serum albumin substituted for bovine serum albumin for storage in PBS. Substrate for the calorimetric reaction with peroxidase was 11 mM 5aminosalicyclic acid (5AS; Aldrich Chemical Co.) plus 0.001 M hydrogen peroxide adjusted to pH 6.0 with 0.1 N sodium hydroxide. Frog plusmu sumplcs. Leopard frogs (Runu pipiens pipiens) with a body weight of lo-25 g and of mixed sexes (sexually immature) were obtained during the months of January to June from Oshkosh, Wisconsin. Upon arrival they were transferred to an environmental chamber in which the lighting schedule was 14L: 10D and the temperature range at 20-25”. After at least 8 days under these conditions blood was obtained from a leg artery in Natelson heparinized glass tubes, centrifuged, and the plasma separated and stored frozen at -30” or used in the assay within 30 min after collection. In some animals, the calcitonin producing organs, the ultimobranchial glands, were removed by surgical extirpation by methods previously described (Robertson, 1969) and blood collected at least 3 days postoperative. Assays were performed on undiluted or diluted (1:2.5 to 1:40) plasma; with assays repeated on samples stored at -30” after 2-3 months. Specificity. Rabbit antibody to immunoreactive salmon calcitonin (iSCT) was assayed against bovine parathyroid hormone (180 USP Units/mg, Lot 28C9610, Sigma Chemical Co.); somatostatin (Lot 48C01292, Sigma Chemical Co.); insulin (25.7 IU/mg; Lot 69C-0417, Sigma Chemical Co.); and glucogen (Lot 128C-0108, Sigma Chemical Co.) at concentrations from 5 x lo2 to 5 x 10” pg/well. To assess nonspecific interference or binding of antibody to proteins other than calcitonin, charcoal (Norit A; Baker Chem.) was

CALCITONIN

13

added to frog plasma (0.05 g/l ml plasma), incubated for 24 hr at 4” and the response compared to nontreated plasma samples. Since Norit A will bind 50 ng calcitoninlg charcoal (Deftos et ul., 1972) changes in A,,,, after treatment would be due to non-calcitoninantibody activity. Principle of method. Salmon calcitonin is initially adsorbed to wells of polystyrene microtiter plates. In separate glass tubes antibody to calcitonin is preincubated with soluble antigen (nonequilibrium conditions). Subsequently the coated plates are washed and the wells filled with the incubation mixture. Under these conditions unreacted antibody will bind to solid-phase antigen with proportionally less antibody reacting with the solid phase with every increasing amounts of soluble antigen. Following this incubation, the soluble antibody-antigen complexes are washed from the wells and the wells reincubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (goat-HRP). The enzyme conjugate reacts with the available solid-phase antibody-antigen complexes. After incubation, the plates are washed and incubated with a peroxidase substrate with the amount of color product developed inversely proportional to the amount of original soluble antigen added in the initial antigen-antibody incubation. Procedure. The basic protocol of Engvall and Perlman (1971, 1972) was used with synthetic salmon calcitonin as the solid-phase antigen. To ensure an even distribution of iSCT to wells, the peptide was diluted in a carbonate/bicarbonate buffer (0.1 M, pH 9.5 at 4”) at a concentration of 1.0 to 8.0 ng iSCT/ml. Aliquots of 100 ~1 (100-800 pg iSCT/well) were quickly distributed to the 96 wells of Cooke microtiter (Dynatech, 1220-24A) polystyrene U plates or Linbro (Flow Laboratories, 76-302-05) flat-bottom plates. Transfer was accomplished in less than 2 set per well to reduce adsorption to the plastic tip of the pipet. Plates were sealed and incubated at 30-33” for 18-24 hr. The concentration used for coating can determine the sensitivity of the assay and was based upon an appropriate optical density of the final c&r product with a given antibody dilution. After the coating incubation, the coated plates were then stored frozen to minimize deadsorption or used immediately by washing three times for 3 min each with PBS with 0.05% Tween 20 (United States Biochemical). The final incubation medium was adjusted to a volume of 500 ,ul in 15 x 85-mm glass tubes which contained serial dilutions of soluble antigen which were made up in 1:40 calcitonin-free frog plasma in PBS, pH 7.2. Antibody was appropriately diluted, depending upon the amount of solid-phase antigen, from l:lO,OOO to 1:40,000. Highest sensitivity was achieved under nonequilibrium conditions where the mixture was preincubated for 3-7 days at 4” prior to incubation in coated wells. After this period, lOO-~1 aliquots (quadruplicate samples of standard or of each un-

14

DOUGLAS

known dilution) were transferred to coated plates with the incubation at 4” for 12 to 27 hr. The specific time period is dependent upon the amount of solid-phase antigen. Controls, with no soluble antigen added, and plasma blanks which contained frog plasma alone completed the assay set. After incubation the plates were carefully washed to avoid cross contamination of adjacent wells, with PBS and Tween 20 as above. Aliquots of 100 PI of goat-HRP conjugate in excess (1:lOO) were added and incubated in wells with constant agitation for 40 min at room temperature (23-26”). The goat -HRP was removed and the plates washed as above. Fresh peroxidase substrate was added (100 ~1) and color development allowed to preteed for 60 min at which time 25 /..d of 0.1 N sodium hydroxide was added to stop the reaction. Optical density of the reaction product in Cooke U plates was measured at A,,,, in a Spectronic 70 (Bausch & Lomb) in microcuvettes while Linbro flat-bottom plates were read with a Multiskan Plate Reader (Flow Laboratories) against substrate blanks. The A,,,, of frog plasma blanks to assess nonspecific binding was typically co.01 when compared to that of substrate blanks. Culculation of unknown srrmplrs. The A,,,, at each sample dilution (1:2.5 to 1:40) minus the plasma blank (x) is calculated from the linear portion of the response

2.0

LINBRO-24

hrs

L.

R

ROBERTSON in the assay as a linear function. is calculated when the dilution mined from the equation:

Y14,,,= rn log l/X + h., The correlation coefficient I’ is also calculated. value of h,, is substituted in the equation: (h,,,,

- h,,.)lm

= log CI,

(1) The

cv

which is the linear expression of the standard assay curve where LI is picograms iSCT/well. This procedure utilizes all data points in the single Eq. (1). Sensitivity crr~d prrcisiolz ctf the immunousscry. With Cooke plates coated with 200 pg iSCT/well and incubated under appropriate conditions a decrease in A,,,, of 2 SD below the A,,,, of controls was equilivalant to I7 pg iSCT added/well. In Linbro plates coated with 200 and up to 800 pg iSCT/well, the lower limit of iSCT detected (greater than 2 SD) ranged from 25 to 17 pg iSCT/well. Regression analysis of the linear portion of the standard assay curves have consistently revealed correlation coefficients (1.) of ~0.985 (P < 0.001). For this immunoassay, the within-assay coefficient of variation (CV) over the range of the assay and the plasma sample dilutions was lo%, and the between-assay coefficient of variation was 15%.

L I NBRO - 48 hrs.

b.

3.4 3.7 4.0 4.3 46 4.9 AB +,x

AB +

AB y&x

The Y intercept (h,,.), factor is I, as deter-

pg iSCT

COATING

BUFFER

FIG. 1. Antibody titration curves as expressed by the change in A,,,, in Linbro various amounts of calcitonin and incubated (a) for 24 hr and (b) for 48 hr with dilutions. (c) The change in A,,,, in Cooke plates coated with various amounts incubated 24 hr at 4”. The responses were virtually identical after 48 hr. (d) The plot in A,,,, in antibody excess (1:500) with various concentrations of antigen coating the and Linbro (A) plates.

plates coated with different antibody of calcitonin and shows the change wells of Cooke (0)

ELISA

FOR

FROG

CALCITONIN

15

RESULTS

Adsorption Characteristics of Microtiter Plates The standard procedures described under Materials and Methods were used except 70 that plasma samples were omitted and sesi a rial dilutions of anti-salmon calcitonin were z 60 m used in Linbro and Cooke plates coated r with various amounts of calcitonin. Figures la, b, c illustrate the change in Ads,-, with various antibody dilutions when incubated 24 and 48 hr at 4”. In Linbro plates coated with 25 to 200 pg iSCT, maximal binding occurred after 24 hr of incubation while 20 antibody in 400- and 800-pg-coated wells was maximally bound after 48 hr (Figs. la, IO b). In Cooke plates coated with 25 to 800 pg : I I 1 1 I I I iSCT, maximal binding of antibody oc04/ 12.5 25 50 100 200 400 800 curred after 24 hr of incubation (Fig. lc). ADDED pg iSCT/WELL Maximal binding capacity in Linbro plates FIG. 2. A series of standard assay curves in Linbro was only about 10% of that in Cooke plates plates with varying amounts of solid-phase antigen inwhen sensitized with 25 pg iSCT in the cubated with different antibody dilutions. The dilucoating buffer; but progressively ap- tions were established to correspond to a point which was equivalent to 60-70% of the maximum A,,,, at proached that of Cooke plates when sen- antibody excess (100 pg at I:500 (Cl), 200 pg at 1: 10,000 sitized with 400 pg or more of calcitonin (A), 400 pg at 1:20,000 (0), 800 pg at 1:40,000 (0). In (Fig. Id). Analysis of the original coating the competitive inhibition assay, the addition of added buffer containing calcitonin revealed no soluble antigen results in a linear response, and is detectable iSCT after an incubation of 24 hr plotted as a percentage of the maximal A,,,, of controls without added antigen. at 33” in either plate. There are two basic procedures in which the sensitivity of the assay can be altered. antigen which will produce a 50% decrease In the first, the amount of solid-phase in AJSO of 50% inhibition (I>,,), is achieved antigen is increased and the amount of with high antibody dilutions and increased antibody decreased. Based upon the data of amounts of antigen used to coat the wells. Fig. 1, the antibody was diluted to a point In the second situation the amount of which corresponded to 60-70% of max- solid-phase antigen is held constant; for imum AdSO at antibody excess in Linbro example, at 800 pg iSCT/well. The antibody plates. This proportion was held constant dilutions are then adjusted to 75, 50, and and the various competitive inhibition 30% of the control Ah5,, of antibody excess. assay responses in a series where the Under these conditions the responses are amount of solid-phase antigen was varied is linear with a decrease in the I,, at higher shown in Fig. 2. In each instance the re- antibody dilutions which also results in sponse is linear over the log range of 25 to higher sensitivity (Table 1). However, the 800 pg in 100 and 200 pg iSCT-sensitized A,,, of the assay control is also decreased, plates and linear over the range of 12.5 to and thus the differential response is re400 pg in 400 and 800 pg iSCT-sensitized duced, which limits the range of the assay. plates. Highest sensitivity (15 pg), which is With the progressive increments in the a function of the point of added soluble time of incubation with various amounts of

16

DOUGLAS TABLE IN THE I,,, WITH

CHANGE LEVELS

IN A LINBRO

R. ROBERTSON

1

VARIOUS

PLATE

CONTROL

SENSITIZED

BINDING

800 pg

WITH

iSCT Control binding level (%)

Ab dilution

A,,,, of controls

I,,, (pg)

Sensitivity”

75 50 30

1:27,000 1:48,000 1:80,000

0.95 0.65 0.40

90 77 42

17.7 15.6 6.7

” Sensitivity is equal to 2 SD of control expressed as pg of added antigen.

binding

level

solid-phase antigen, the optimal time could be determined which produced maximal Ads,, in controls and a linear resopnse with increasing amounts of soluble antigen. The incubation period in coated wells is critical for any given antigen coat and ranged from 12 to 27 hr for wells coated with 100 to 800 pg iSCT, respectively. Incubation periods longer than optimal become curvilinear and stabilize to a biphasic response (Fig. 3). With increasing added antigen there is no change in A,,, from that of controls until a level is achieved which is equal to or less than the amount of solid-phase antigen. The second portion of the response is also linear, but the Iso is shifted to the right. Sensitivity is decreased from 5- to 60-fold depending upon the amount of solid-phase

‘\

s

a

‘\ 80

1

x AAiO (control),

l/I,,

(3)

where Iso is the value determined under optimal conditions and Ads,, is the absorbance of controls at various specific binding levels. The maximal value for a series with a given antigen coat established the optimal antibody dilution. For Linbro plates with a lower binding capacity, the control antibody dilutions were at 60-70% of antibody excess, while for the higher binding capacity of Cooke plates, the control binding levels could be reduced to 45% of antibody excess. Standard

Figure for the salmon petitive citonin

Curve for the Calcitonin

Assay

4 shows a typical response curve enzyme-linked immunoassay for calcitonin as a heterologous cominhibition assay for amphibian calin frog plasma. A Linbro plate FROG PLASMA DILUTIONS 140 15 l-2 I5 I

I.20

\

‘\

0

antigen. Inadequate preincubation will also result in a nonlinear response. Assessment of the various optimal conditions which produced linear responses with highest sensitivity and maximal differential response for a given antigen coat could be derived by calculating the product of

\ \\

‘. ‘\

\

\ O\

\

0\

Y=-04210gx+ll

11 ’ I I 1 I ‘AI 25

50

ADDED

100 200 pg iSCT/WELL

400

800

FIG. 3. Assay responses in Linbro plates coated with 100 pg (0) or 200 pg (0) iSCT/well and incubated 6 hr beyond the optimal time period. This results in biphasic patterns with the I,,, shifted to the right. For comparison the dotted response is the optimal standard curve for a 100 pg iSCT-coated plate.

ADDED pg ,SCT/ WELL

4. Standard competitive inhibition assay response for frog calcitonin in a Linbro plate coated with 200 pg iSCT and antibody dilution of l:lO,OOO. The response is linear over the log range of 25 to 400 pg iSCT added/well (O), while the frog plasma dilution series (A) is parallel at the lower dilutions. Calculated value for frog sample is 3.2 ng CT/ml. FIG.

ELISA

FOR

FROG

coated with 200 pg iSCT demonstrates a linear response over the log range of 25 to 800 pg iSCT added/well with the &,-, at 82 pg iSCT and a sensitivity of 25 pg iSCT. Frog plasma samples diluted over the range of 1:2.5 to 1:20 with PBS showed changes in A 450 which were both linear and parallel to the standard curve in the dilution range of 1:2.5 to 1:lO. Sensitivity of this assay can be improved by increasing the amount of solid-phase antigen to 800 pg iSCT/well and an antibody dilution of 1:40,000. Figure 5 depicts such an assay with a lower useful range from 12.5 to 400 pg where the sensitivity is 17 pg and Iso is 65 pg. Frog plasma samples were also linear and parallel to the inhibition response. A standard response curve in a Cooke plate sensitized with 200 pg iSCT and antibody dilution of 1:20,000 (Fig. 6) resulted in a linear response from 25 to 400 pg added iSCT with the Is0 equal to 108 pg and a sensitivity of 17 pg iSCT. Specificity

17

CALCITONIN

parathyroid hormone, somatostatin, glucagon, or insulin over the range of 5 x lo2 to 5 X lo6 pg/well. A nonspecific interference was observed in some diluted samples at 1:2.5 and 1:5 dilutions, but was not apparent at higher dilutions. Preadsorbed plasma samples treated with Norit A failed to remove the interfering substance at 1:2.5 and 1:5 plasma dilutions but the A450 at higher dilutions was increased indicating removal of an immunoreactive material with the charcoal treatment. Endogenous Levels Frog Plasma

The amount of immunoreactive calcitonin in the peripheral blood of frogs is summarized in Table 2 and shows higher levels in the plasma of animals obtained during summer compared with the plasma of spring frogs. Removal of the ultimobranchial glands, the endocrine source of calcitonin, reduced the detectable immunoreactive calcitonin below detectable levels 3 days postoperative.

Antisera to synthetic salmon calcitonin did not cross #-react w ith either bovine FROG PLASMA DILUTION

I:20 I

b/

Ii5

25’

I 50

I.10 1

100

15 I

200

I25

I

of iCT in

DISCUSSION

Success of this type of assay utilizing a small polypeptide with the immunoreactive site confined to the lo-32 region of the calcitonin molecule (Deftos et al., 1974) re-

1

400

ADDED pg iSCT/WELL

FIG. 5. Standard competitive inhibition assay curve for frog calcitonin in a Linbro plate coated with 800 pg iSCT and antibody dilution of 1:40.000. Response is linear over the range of 12.5, to 400 pg iSCT added/well (O), while the frog plasma dilutions (A) are parallel over the dilution series. Calculated value for frog sample is 7.3 t&ml.

Lf

25

510 ADDED

100 ’ pg

I

I

I

200

400

800

iSCT/WELL

6. Standard assay curve for calcitonin in a Cooke plate coated with 200 pg iSCT/well and antibody dilution of 1:20,000. Response is linear over the log range of 25 to 400 pg iSCT/well with a I,,, of 108 pg and a sensitivity if 17 pg. FIG.

18

DOUGLAS TABLE

ENDOGENOUS

2

LEVELS OF CIRCULATING IN THE FROG (Runa pipiens)”

Group 1. February -April Males (10) Females (8) 2. June-July Males (6) Females (10) 3. June-July Light period (14 hr) Dark period (10 hr) 4. February-July UBX

R. ROBERTSON

CALCITONIN

Circulating calcitonin (ng iCT/ml ? SD) 4.7 + 2.2 4.1 + 1.8 21.2 * 9.3 22.8 + 10.8 16.8 2 6.3 27.4 k 10.7” >0.3

” All animals kept on a 14L:lOD lighting schedule. * P = 0.05 from light period.

quired that binding to the polystyrene wells did not interfere with the subsequent antigen-antibody reaction. The characteristics of the antibody titration curves of the two plates tested would suggest that calcitonin adsorbed to Linbro plates was sterically different from that with Cooke plates. This apparent decrease in available antigenic sites in the Linbro plates is pronounced at low coating concentrations, suggesting that two types of binding sites are present. There is a relatively scarce binding site with a high binding affinity which does not permit binding of the antibody and a second more prevalent type which allows exposure of the antigenic site but which is near saturation at 800 pg iSCT/well. The Cooke plate appears to have a single binding type which has both high affinity and maintains an accessible antigenic site. Sensitivity of the assay is directly related to the amount of solid-phase antigen since an increase in the amount necessitates a higher antibody dilution and decreases the [Ab]/[Ag] ratio. Under these conditions there is a decrease in the Iso with increasing amounts of solid-phase antigen while maintaining an antibody dilution which is at a constant proportion to antibody excess. Potts et af. (1957) have concluded that

highest sensitivity in RIA procedures is obtained with high antibody dilutions. Ansari et al. (1978) commented on the usefulness of comparing the amount of inhibitor which would produce 50% inhibition. The study also concluded that higher antibody dilutions increased sensitivity. The present observations concur, but also indicate that for a given control binding level the amount of solid-phase antigen is an important factor in establishing maximal sensitivity in the ELISA procedure. It can be seen that, in this system, there is no advantage in utilizing a plate with high binding affinity, such as the Cooke plate, since low concentrations of solid-phase antigen are coincident with high [Ab]/[Ag] ratios. Under optimal conditions, with low [Ab]/ [Ag] ratios, the Adso of controls was about equal for both plates. Additional factors which dictate the optimal conditions include the period of preincubation and incubation in the wells. Use of low concentrations of solid-phase antigen (and lower antibody dilutions) requiring short incubation periods in the plate are not as reliable since the critical period of linearity is short and transitory. Higher antibody dilutions which requried longer incubation times in the sensitized plates provided a broader range of optimal response time and increased reliability. The nonspecific binding effect of antibody at low sample dilutions has been observed by Deftos et al. (1972) and Tashjian and Voelkel (1979) in calcitonin radioimmunoassays. Since the value of plasma calcitonin in frogs is above 1.O rig/ml the use of higher dilutions, which eliminates the interfering property, can be used in this assay. The values obtained in this assay for frog calcitonin levels varies from a mean value of 4.4 to 32 rig/ml depending upon the season of the year the plasma sample was obtained. These values are within the range observed in fish (Deftos et al., 1974). Diurnal variations in circulating calcitonin are also maximal at night in the frog as they are

ELISA

FOR

FROG

in the rat which is also a nocturnal animal (Roos et al., 1978). However, contrary to that seen in fish by Deftos et al. (1974), sexual differences were not pronounced in the frog. The fall below detectable levels of iCT in ultimobranchialectomized frogs, not only confirms the endocrine origin of calcitonin in the frog, but demonstrates the specificity of the assay and the crossreactivity of the rabbit antisera with the endogenous polypeptide in the amphibian. In conclusion, this heterologous competitive inhibition assay for calcitonin may well be applied to other animal systems, while the basic procedure may be applicable to other smaller polypeptides. However, it must be recognized that such molecules may be more sensitive to steric alterations when adsorbed to the solid phase. ACKNOWLEDGMENTS The author wishes to acknowledge the aid of the late Dr. Americo A. Marucci, Department of Microbiology, who as teacher and friend provided the inspiration and continued interest for a major portion of this project. My thanks to Dr. J. Philip Aldred, Armour Pharmaceutical Company, for the generous gift of salmon calcitonin, and Dr. Robert Dougherty, Department of Microbiology, for his comments throughout this project and for reading the manuscript. Support for this project was provided by funds from Biomedical Research Support Grants from the U.S. Public Health Service. 5S07RR0540217-540218.

REFERENCES Ansari. A. A., Bahuguna, L. H., and Malling, H. V. (1978). Comparison of double antibody and solid-phase radioimmunoassays. J. tmmunol. Methods 23, 219-226. Cutler, B. G.. Jr., Habener, J. F., Dee, P. C., and Potts, J. T., Jr. (1974). Radioimmunoassay for chicken calcitonin. FEBS Lett. 38, 209-211. Deftos, L. J., Murray, T. M., Powell, D., Habener, J. F.. Singer, F. R., Mayer, G. P., and Potts, J. T., Jr. (1972). Radioimmunoassays for parathyroid hormones and calcitonins. In “Proceedings, Fourth Parathyroid Conferences,” pp. 140- 151. Excerpta Medica. Amsterdam. Deftos. L. J., Watts, E. G.. Copp. D. H., and Potts, J. T.. Jr. (1974). Radioimmunoassay for salmon calcitonin. Eutlocrirtology 94, 155 - 160.

CALCITONIN

19

Dietrich, F. M., and Rittel, W. (1970). Immunochemical analysis of antibodies against synthetic human calcitonin M. In “Proceedings, Second International Symposium. Calcitonin, 1969,” pp. 87-94. Heinemann, London. Engvall, E., and Perlman, P. (1971). Enzyme-linked immunosorbant assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8, 871-874. Engvall, E., and Perlman, P. (1972). Enzyme-linked immunosorbent assay (ELISA). III. Quantitation of specific antibodies by enzyme-labeled antiimmunoglobulin in antigen-coated tubes. J. Immunol. 109, 129- 135. Garvey, J. S., Cremer, N. E., and Sussdorf, D. H. (1977). “Methods of Immunology,” 3rd ed., pp. 218-219. W. A. Benjamin, Reading, Mass. Habener, J. F., Singer, F. R., Neer, R. M., Deftos, L. J., and Potts, J. T., Jr. (1972). Metablism of salmon and porcine calcitonin: An explanation for the increased potency of salmon calcitonin. In “Proceedings. Fourth Parathyroid Conference,” pp. 152- 156. Excerpta Medica, Amsterdam. Nakane, P. K., and Kawaoi, A. (1974). Peroxidaselabeled antibody-A new method of conjugation. J. Histochem. Cytochrm. 22, 1084- 1091. O’Beirne, A. J., and Cooper, H. R. (1979). Heterogeneous enzyme immunoassay. J. Histochem. Cytochrm. 27, 1148- 1162. Orimo, H.. Yamauchi, H., Ohyama, T., Matuo, M., and Otani, M. (1977). Radioimmunoassay and immunological properties of eel calcitonin. G‘en. Camp. Endocrinol. 31, 482-485. Otani, M., Noda, T., Yamauchi, H., Watanabe, S.. Matsuda, T., Orimo, H.. and Nartia, K. (1975). Isolation, chemical structure and biological properties of ultimobranchial calcitonin of the eel. In “Proceedings, Fifth Parathyroid Conference,” pp. 1 ll- 115. Excerpta Medica, Amsterdam. Potts, J. T., Jr., Naill, H. D.. Keutmann, H. T., and Lequin, R. M. (1972). Chemistry of the calcitonins: Species variation plus structure-activity regions, and pharmacologic implications. In Proceedings, Fourth Parathyroid Conference,” pp. 12 I- 127. Excerpta Medica. Amsterdam. Potts, J. T., Jr., Sherwood, L. M., O’Riordan. J. L. H., and Aurbach, G. D. (1967). Radioimmunoassay of polypeptide hormones. Adwn. Intern. Med. 13, 183-240. Reichlin, M., Schnure, J. J., and Vance, V. K. (1968). Induction of antibodies to porcine ACTH in rabbits with nonsteroidogenic polymer of BSA and ACTH. Proc. Jot. Exp. Med. 128, 347-350. Robertson, D. R. (1969). The ultimobranchial body of Rrrrltr pipienJ. VIII. Effects of extirpation upon calcium distribution and bone cell types. G’un. Camp. Endocritd. 12, 479-490.

20

DOUGLAS

Roos, B. A., Cooper, C. W., Frelinger, A. L., and Deftos, L. J. (1978). Acute and chronic fluctuations of immunoreactive and biologically active plasma calcitonin in the rat. Endocrinology 103, 2180-2186.

Tashjian, A. H., Jr., and Voelkel, E. F. (1979). Radioimmunoassay of human calcitonin: Application of affinity chromatography. In “Methods

R. ROBERTSON

of Hormone Radioimmunoassay” (B. M. Jaffe and H. R. Behrman, eds.), pp. 355 -385. Academic Press, New York. van Noorden, S., and Pearse, A. G. E. (1971). Immunofluorescent localization of calcitonin in the ultimobranchial gland of Runu temporuriu and Runu

pipiens.

Histochemistry

26, 95-97.