Animal Reproduction Science, 16 (1988) 225-235 225 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Sensitive Enzyme Immunoassay of Progesterone in Skim Milk Using Second-Antibody Technique

B.S. PRAKASH, H.H.D. MEYER and D.F.M. VAN DE WIEL 1

Institut [i~r Physiologie der Si~ddeutschen Versuchs- und Forschungsanstalt [i~r Milchwirtschaft, Technische Universittit Mgtnchen, 805 Freising- Weihenstephan (Federal Republic of Germany) 'Research Institute for Animal Production "SCHOONOORD", Driebergseweg 10 D, Zeist (The Netherlands)

{Accepted 16 October 1987)

ABSTRACT

Prakash, B.S., Meyer, H.H.D. and Van de Wiel, D.F.M., 1988. Sensitive enzyme immunoassay of progesterone in skim milk using second-antibody technique. Anim. Reprod. Sci., 16: 225-235.

Several methods have been described for obtaining precise physiological progesterone data from lactating cows. In certain situations it is advantageous to use skim milk for progesterone estima- tion for which we have developed a sensitive EIA. Progesterone was estimated in skim milk by EIA using progesterone-6fl-OH-hemisuccinate-horse-radish peroxidase as the enzyme label. The antiserum used had been raised against progesterone-7a-carboxyethylthioether-BSA in rabbits. The microtitration plates used in the assay were first coated with affinity purified sheep IgG developed against rabbit IgG. The immune reaction was performed by incubating a mixture of 2 /11 of skim milk ( diluted to 20 zl with assay buffer), 100 ]21 of enzyme label and 100 ~l of antiserum. After washing the plates, 150/~l of the substrate solution were added. The mixture was incubated in the dark for 40 min before the reaction was stopped and the optical density was measured. The calibration curve was sensitive in the range 0.4-25 pg/well, corresponding to 0.2-12.5 ng/ml (50% relative binding at 3 pg/well). The intra- and interassay coefficients of variation were less than 11%. Milk samples from non-pregnant dairy cows were tested for progesterone concentration twice weekly by running parallel RIA and EIA. A good correlation of 0.98 was obtained and the estimated values were similar in both techniques. In conclusion besides proving to be a very reli- able assay, with a sensitivity 10 times better than the RIA, the double-antibody technique also reduces the use of valuable antiserum to one quarter in comparison to direct coating.

INTRODUCTION

Progesterone is a good marker for determining the functional status of the corpus luteum in cattle. Its measurement in milk serves as a valuable diagnos- tic tool in identifying ovarian conditions such as silent heat, lack of cyclicity, cystic follicles, retained corpora lutea and irregular cycles (Hoffmann et al.,

0378-4320/88/$03.50 © 1988 Elsevier Science Publishers B.V.

226

1976; Bulman and Lamming, 1978; Foote et al., 1979; Claus et al., 1983 ). Most of the early progesterone measurements in milk were carried out by radioim- munoassay which restricted its practical use to specialized laboratories with the capacity to use radioisotopes.

Enzyme immunoassay (EIA) offers a better alternative due to its low cost and its non-radioactive nature. In recent years considerable attention has been focussed on developing a sensitive enzyme immunoassay for determining pro- gesterone in milk. Earlier methods involved extraction of whole milk (Van de Wiel et al., 1982), defatted milk (Nakao et al., 1983) and milk fat (Arnstadt and Cleere, 1981). EIA procedures for direct progesterone measurement in whole milk (Arnstadt and Schmidt-Adamopoulou, 1982; Sauer et al., 1982; Sauer et al., 1986; Van de Wiel and Koops, 1986) have also been developed. The apparent concentration of progesterone in milk is dependent upon the fat content, with the ratio of progesterone distribution being 50-100 times greater in fat compared to the aqueous phase of whole milk (Schwalm and Tucker, 1978). Hence, to obtain more precise physiological progesterone values, it is preferable to estimate its concentration in fat-free skim milk (Pope et al., 1976) or in milk fat (Hoffmann et al., 1976). This may be advantageous in avoiding possible errors of its estimation in either whole milk samples or strippings where milk fat content may vary considerably. In order to measure the low progesterone levels in skim milk by a direct EIA, the method employed must be very sensitive. A direct homologous EIA in skim milk has recently been demonstrated at this laboratory (Meyer et al., 1986). However, the method was no better than the progesterone RIA already in regular use.

Most of the earlier EIA methods using microtiter plates have been carried out by initially coating the plate wells directly with the progesterone-specific antibody. This often resulted in a "time dependent drift", as shown by Munro and Stabenfeldt (1984), or increased variability. It is possible to avoid this effect, using the second-antibody technique (Meyer, 1986; Meyer and Giiven, 1986) with different basic pre-conditions of the binding kinetics (Fig. 1 ). Es- sentially, this consists of coating the wells initially with a large excess of the

H I H H ?

E E H I~ H H A H

I I

Fig. 1. Bonds after completed immunoreactions. Y = Sheep IgG antirabbit IgG; Y = hormone- E

specific rabbit IgG; H =unlabelled hormone; I = enzyme-labelled hormone. H

227

sheep IgG antirabbit IgG ( 1 ttg/well) for total binding of the hormone specific rabbit antibody, and minor variations of coating become negligible.

Bearing in mind the above considerations, the present study was planned to develop a sensitive heterologous progesterone enzyme immunoassay using the second-antibody coating technique for determining the hormone in bovine skim milk.

MATERIALS AND METHODS

Collection and initial processing of milk samples

The monitored cycling post-partum Brown Swiss Cows belonged to the In- stitute's Dairy Cattle Farm. For progesterone estimation, milk samples from the final strippings were collected on a routine basis twice weekly, until con- firmation of pregnancy by rectal palpation. The samples were placed in stop- pered polypropylene tubes, initially stored in a refrigerator for about 2 h and later frozen at - 30 ° C until further analysis.

Skim milk was prepared as follows. The frozen milk samples were thawed at 45 °C and incubated at this temperature for about 1 h. The samples were then centrifuged at 4500 rpm for 20 min under refrigerated conditions in an inverted position to facilitate easy decantation of the skim milk after centrifugation.

Progesterone antibody

The anti-progesterone-7~-carboxyethylthioether-BSA used in the present study was specific for estimating progesterone. Its specificity for EIA and RIA has been discussed elsewhere (Van de Wiel et al., 1978; Van de Wiel and Koops, 1986). The cross-reactivities are briefly summarized: progesterone 100%; es- tradiol, dihydrotestosterone, testosterone and 17~-hydroxy-progesterone <0.1%.

Preparation of enzyme label

Horse-radish peroxidase (Serva, Federal Republic of Germany) was used for coupling to progesterone-6fl-OH-hemisuccinate (Steraloids, U.S.A. ) using the mixed anhydride method (Liebermann et al., 1959) modified in this labo- ratory (Meyer et al., 1986). Briefly, the coupling procedure was carried out as follows. Solution A was prepared by freeze-drying a mixture of 2 mg of proges- terone-6fl-hydroxy-hemisuccinate dissolved in 50 ttl of methanol and 20/A of 1 N HC1 at 50/lm for 2 h. After dissolving the residue in 500 ttl of N, N-di- methyl-formamide (Merck, Federal Republic of Germany), 6.25 ]A of 4-meth- ylmorpholine (Merck, Federal Republic of Germany) were added and the solution cooled to - 15 ° C.

228

Solution B was prepared by slowly adding 375/A of dimethyl-formamide to 500/~l of horse-radish peroxidase (4.75 mg/ml water) and the solution cooled to 0°C.

The coupling reaction was performed by first adding 6.25/A of isobutyl- chloroformate (Sigma, U.S.A. ) to solution A with continuous stirring for 3 min at - 15 ° C. This was followed by the addition of solution B to solution A under continuous stirring at - 15 ° C and the pH of the resulting mixture was imme- diately corrected to pH 8.0 with 1 N NaOH. The mixture was initially incu- bated for 1 h at - 15°C followed by 2 h at 0°C. After the addition of 10 mg of NaHCO~, the product was dialysed overnight and further purified by gel chro- matography { Sephadex G 25 fine; column: 1.6 cm × 50 cm). The enzyme label so obtained was immediately tested for its titer and stored at - 60 ° C.

Preparation of affinity purified sheep IgG antirabbit IgG

Fifty ml of plasma containing 6 mM EDTA from a sheep immunized against rabbit IgG were mixed with 2 g of rabbit IgG-linked agarose gel ( Sigma, U.S.A. ). The resulting mixture was incubated overnight at 0 ° C under constant mixing. Following centrifugation at 4000 rpm, the gel was recovered and transferred to a small column with a filter at the bottom. It was then washed with 7 ml of phosphate buffer (43 mM NaH2PO4, pH 7.2 ) followed by 10 ml of 0.5 M NaC1 in phosphate buffer. The sheep IgG bound to the agarose gel was then eluted with 10 ml of 2 M NaSCN (pH 8.0) at 0°C. The eluate was dialysed overnight against the phosphate buffer. The affinity purified sheep IgG so obtained was determined by protein estimation (Biuret method).

EIA procedure

(1) First coating. This was performed by dispensing 100 pl of affinity purified sheep IgG antirabbit IgG in coating buffer (50 m M NaHCO3, pH 9.6) at a dilution to provide 1 zg/well of the microtiter plate (No. 439, 454; Nunc, DK- 4000 Roskilde ) followed by overnight incubation (20 h) at 0 ° C on a slow me- chanical horizontal shaker.

(2) Second coating. The wells of the microtiter plates were coated with 250 ~l of 1% BSA in phosphate buffer and incubated at room temperature for 20-40 min under constant shaking.

(3) Washing. The plates were washed twice with 300 #l/well of washing solu- tion (0.05% TWEEN 80).

(4) Immune reaction. The progesterone standards in skim milk and unknown samples were initially diluted 10 times with assay buffer (40 mM Na2HPO4,

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0.14 M NaC1, 0.1% albumin; pH 7.2 ); 100 ]A of diluted enzyme conjugate plus 20 zl of diluted s tandards/unknowns were incubated with 100 zl of diluted progesterone antiserum for 1 h at 37 ° C and then overnight at 0 ° C in the dark.

(5) Substrate reaction. The microtiter plates were washed four times, as before, with the washing solution and then incubated with 150 #l of the substrate solution/well (0.01% tetramethyl benzidine plus 0.004% H202 in 100 mM so- dium acetate, pH 5.5 with citric acid), for 40 min in the dark at room temperature.

The reaction was stopped by adding 50 #l of 4 N HeSO4 to each well and the yellow color so obtained was measured at 450 nm with an eight-channel auto- matic microtitration plate reader (Titertek Multiskan, Flow Laboratories, Federal Republic of Germany).

RIA procedure

Twenty ]A of skim milk samples were taken for analysis for progesterone by RIA method according to the procedure detailed by Meyer et al. (1986).

RESULTS

Titration of enzyme label and anti-progesterone serum

A two-dimensional titer determination for opt imum dilution of the enzyme label and progesterone antiserum was carried out. For routine use in the EIA opt imum dilutions of 1:16 000 and 1:80 000 were used for the enzyme label and the antiserum, respectively.

Assay validation

(1) Assay sensitivity. The sensitivity of progesterone estimation by EIA and RIA in terms of the lowest detectable amounts significantly different from zero concentration and mid-point relative binding of the calibration curve are pre- sented in Fig. 2. It is apparent from this figure that the EIA method was about 10 times more sensitive than the conventional RIA.

(2) Calibration of standard curve. The progesterone standard curve by EIA was calibrated by adding known amounts of tritiated progesterone {Amersham; 84 Ci /mmol) in progesterone-free skim milk samples, and run along with the rest of the unknowns in an assay. The amount of progesterone obtained after ex- trapolation from the standard curve was compared with the actual amounts added. The accuracy of the estimation is presented in Table 1.

230

100

90

80

7C

6C

~- 5C

,- L,C g_

3O

20

10

\ 50% binding

\ I2.Spg 130pg

0 0.~0.81.6326.3 25 100 400 Progesterone ( pg )

EIA RIA

0.6 pg I, pg

2.5 pg 30 pg

Fig. 2. Comparison of progesterone standard curves between EIA and RIA in skim milk. O RIA (20/~1 skim milk); • • , E I A (2/~1 skim milk).

TABLE 1

,

Calibration of EIA standard curve

H-Progesterone Progesterone Progesterone % Accuracy added (cpm) equivalent (pg) estimated

1495 5.003 4.4 88.0 2975 9.956 9.0 90.5

Counter efficiency: 50.4%. Specific activity of label: 84 Ci/mmol.

(3) Intra- and Interassay precision. Two progesterone controls from pooled skim milk samples were selected for this purpose. The "low" control contained about 0.9 ng/ml of skim milk, and the "high" control about 2.0 ng/ml. The controls were assayed in duplicate at four different points on each EIA and RIA and the mean and the intraassay coefficient of variation for each were determined. A mean duplicate determination from each quality control on each plate was randomly selected to determine the mean interassay coefficients of variation in nine different tests. The results of precision for EIA and RIA are summa- rized in Table 2.

Influence of different amounts of skim milk used on the assay sensitivity in EIA

The effect of different volumes of progesterone-free skim milk on the stan- dard curve was determined and compared with the standard curve in assay

TABLE 2

Intra- and interassay precision for EIA and RIA

231

Quality EIA (n=9) control

RIA (n=5)

Mean Intraassay Interassay Mean Intraassay Interassay CV (%) CV (%) CV (%) CV (%)

Low 0.92 6.6 10.8 0.79 High 2.28 4.0 9.7 1.93

Overall mean CV 5.3 10.25

12.1 10.8

11.45

13.9 13.1

13.5

0.8

0.7

0.6

0.~,

d 0.3 -~

0.1

oZ~ 018 116 312 6'.3 ff.s 2's Progesterone pg/wel.t

Fig. 3. Progesterone standard curves in buffer and skim milk. • skim milk; O ©, 10/A skim milk; A - - A , 20/A skim milk.

0 , Buffer; A A, 4~1

buffer. Skim milk volumes greater than 4/A reduced the sensitivity of the flat- tened standard curve (Fig. 3). All tested volumes ~<4/~l were in good agree- ment with the s tandard buffer curve.

Comparison of skim milk samples estimated [or progesterone by EIA and RIA

A total of 175 milk samples was taken for comparison of progesterone esti- mation by EIA and RIA. Good correlation was obtained between EIA and RIA values, and the values obtained by the two methods were quite similar (y-- 1.18 x + 0.04; r-- 0.98; n-- 175; x = RIA value, y = EIA value ). This is well illustrated from the hormonal profiles in two cycling cows before and after fertile insem- ination (Fig. 4).

232

~0

3O

o

20 eJ

cL 10

IDA

/', ,

!, -10 10 20 30 ~0 50 60

Doys

LOLLA

n . . . . . . . .

-20-10 0 10 20 30 1,050 Days

Fig. 4. Progesterone levels in the skim milk of two cows as measured by RIA and EIA. ~ , Insem- ination; © . . . . . . ©, RIA; • . . . . . . @, EIA.

DISCUSSION

The method described is the first reported using a heterologous system and the second-antibody technique in a progesterone EIA. The necessity of obtain- ing an optimal fit of enzyme label and antiserum for high sensitivity requires the right grade of heterology. The present combination using progesterone-6fl- OH-hemisuccinate-horse-radish peroxidase as the enzyme label and anti-pro- gesterone-7a-carboxyethylthioether-BSA as the antiserum demonstrates the tremendous improvement in the sensitivity from earlier reports either for ho- mologous (Arnstadt and Cleere, 1981; Foulkes et al., 1982; Meyer et al., 1986) or heterologous systems (Sauer et al., 1982; Munro and Stabenfeldt, 1984; Sauer et al., 1986). The EIA method in the present study was 10 times more sensitive than the regular progesterone RIA in use, to the extent that only 2/~l of skim milk are required for a reliable, precise hormone determination of physiological progesterone levels.

In former techniques, since the hormone-specific antibody was directly coated to the wells, a time lag ensued while pipetting different samples and standards, resulting in a distinct drift (Munro and Stabenfeldt, 1984). This problem is avoided by the present technique in which the wells are initially coated with the second antibody and the hormone-specific antiserum is quickly dispensed after the addition of the sample and enzyme label, thereby keeping the incu- bation time for all samples for the immune reaction constant. This also vastly improves the reproducibility of the assay which is seen to be better than the RIA (Table 2). The application of the second-antibody technique also brought about a four-fold reduction in the use of valuable hormone-specific antiserum from that reported earlier for the direct-coating method (Van de Wiel and Koops, 1986).

233

The observation that increasing the volume of skim milk clearly decreases the sensitivity of EIA has also been recorded for whole milk (Sauer et al., 1981; Chang and Estergreen, 1984). In our studies 10/~l or 20/~l of skim milk per well result in flattened calibration curves, but 4/A or less produce parallel curves comparable to the standard buffer curve. However, due to the high sensitivity of the assay analysis, 2/~1 of skim milk are sufficient to give a clear indication of the luteal activity in the cow. The calibration curve range of 0.4-25 pg in 2 ill corresponded to 0.2-12.5 ng/ml skim milk, which covers the whole range of possible physiologic variations. In this respect 2/~l is the optimal volume to be analysed and there is no need for different dilutions. For routine classification purposes, values < 0.4 ng/ml corresponded to the early follicular phase, 0.4- 1.0 ng/ml to the early or ending luteal phase, and > 1.0 ng/ml skim milk in- dicated full luteal activity.

The very good parallelism of progesterone levels in plasma and skim milk throughout the oestrous cycle has been demonstrated (Pope, 1982). Hence, skim milk presents a good alternative to plasma because it also provides access to reliable, relevant data independent of fluctuating fat content in milk sam- ples. Skim milk can be prepared much easier than fat, but levels in skim milk are lowest and an assay with a sufficient sensitivity is required. The described assay may be applied in every laboratory. However, it must be mentioned that enzymes have a limited stability if handled inappropriately. Therefore, some experience is required prior to the successful completion of an EIA.

In conclusion, this EIA method has shown at least a 10-fold improvement in sensitivity over RIA. The use of the second-antibody technique considerably economizes on the need for the valuable progesterone antiserum besides im- proving the precision of the system. Former direct EIA procedures required purification of the hormone antiserum to obtain anti-progesterone rabbit IgG which is no longer required in the present technique.

ACKNOWLEDGEMENT

The Fellowship awarded to B.S. Prakash by the Deutscher Akademischer Austauschdienst (DAAD), Bonn, Federal Republic of Germany, for carrying out this research is gratefully acknowledged.

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