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Anal. Chem. 1993, 65, 1779-1704
Enzyme Immunoassay Using a Rat Prolactin-Alkaline Phosphatase Recombinant ,Tracer Daniel Gillet,’J Eric Ezan? Fr6d6ric DucancelJ Carole Gaillard,’ Thierry Ardouin? Michel Istin3 Andre MbnezJ Jean-Claude BoulainJ and Jean-Marc Grognet: DBpartement d’IngBnierie et d’Etude des ProtBines, (DIEP), and Service de Pharmacologie et d’lmmunologie, Commissariat h 1’Energie Atomique, CEISaclay F-91191 GiflYuette Cedex, France
This paper describes a competitive enzyme immunoassay of rat prolactin (rPrl) using a recombinant conjugate as a colorimetric tracer. rPrl was inserted into the N-terminal end of Escherichia coli alkaline phosphatase (AP), using a n expression vector which allows insertion of foreign DNA sequences between codons +6 and +7 of the phoA gene. The assay was performed in 96-well microtiter plates coated with a mouse monoclonal antibody raised against rabbit immunoglobulin G. Each component (recombinant tracer, rabbit antiserum against rPrl, and rPrl standard) was added in a volume of 50 pL. The sensitivity of the assay was sufficiently high to allow titration of rPrl in plasma. The detection threshold was 15 pg (0.3 ng/mL) and the B/Bo 50% value was 150 pg (3 ng/ mL). The intraassay coefficient of variation was less than 10% over a wide range of rPrl concentrations (2.9-50 ng/mL). The interassay coefficient of variation was less than 15% for rat plasma samples in the concentration range of 4-40 ng/ mL. The good parallelism observed between the standard curve and sample dilution curves showed that the immunoreactivity in rat plasma behaves like standard rPrl. Together with recovery experiments, these results indicated that assay without extraction is possible. A single immunoreactive peak that comigrates with standard rPrl is observed after molecular sieve fractionation of plasma samples. The reliability of the assay was confirmed by good correlation with conventional radioimmunoassay ( r = 0.996, slope 0.978).
INTRODUCTION Enzyme immunoassays (EIAs)often constitute a well-suited alternative to radioimmunoassays (RIAs) for the detection and measurement of peptide hormones.192 However, the conventional way to produce enzymatic tracers by chemical coupling of a peptide antigen to an enzyme suffers inherent disadvantages, including the initial purification of both the antigen and the enzyme, the use of sophisticated chemistry, and the final purification of the conjugate. In addition, stoichiometry of coupling is difficult to control, and yields are usually low.
* Author to whom correspondence should be addreeaed. DBpartement d’Ing6nierie et *Etude des ProtBines.
t Service de Pharmacologie et d’Immunologie. (1) Grasei, J.; Maclouf, J.; Pradelles, P. InHandbook ojExperimentaZ
Pharmacology;Patrono, C., Peskar, B. A., Eds.; Springer Verlag: Berlin, 1987; Vol. 82, pp 91-142. (2) Shall,R. F.; Teneso, M. J. Clin. Chem. 1981,27, 1157-1173. 0003-2700/03/0385-1779$04.0010
Attempts to use conjugates prepared in bacteria by recombinant DNA technology were previously reported.g-6 However, none of these attempts has been expanded due to their lack of versatility. Moreover, the validation of such systems to measure proteins in biological fluids was never assessed. Recently, Gillet et al. described a system which has the potential to be applied to a wide variety of proteins6 and to overcome the drawbacks of chemical conjugation. The authors developed a cloning and expression vector which allows insertion of foreign peptide sequences in the N-terminus of Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1). This system was used successfully by inserting a small toxic protein of 62 amino acids cross-linked by four disulfide bonds. The toxin-AP hybrid was exported to the bacterial periplasm from where it was easily extracted by osmotic shock. It exhibited enzymatic activity, contained a toxic domain properly folded, and was resistant to proteolytic degradation in the bacteria. The present paper describes the extension of the principle to a larger protein, rat prolactin (rPrl). rPrl is a pituitary hormone involved in the control of lactation’ and possibly also of immune response.8 Ita detection is important in evaluation of the possible effecta of pharmacological products on pituitary function. We show that the recombinant rPrlAP hybrid is an appropriate tracer for enzyme immunoassay. Validation of the assay to quantify hormone plasma levels is emphasized in this paper.
EXPERIMENTAL SECTION Chemicals. rPrl standard R.P.3 was a gift from the National Institute of Arthritis, Diabetes, Digestive Diseases and Kidney (NIADDK). rPrland other rat pituitary hormones were supplied by UCB (Brussels, Belgium). Microtiter plates (96F immune plates I, with certificate) were from Nunc. Biogel A 0.5 M was supplied by Bio Rad (Richmond, CA). Unless otherwise stated, all reagenta were from Sigma (St. Louis, MO) or Prolabo (Paris, France). Monoclonal antibody (mAb) VIAPl against E. coli AP was purchased from Caltag. 4-Nitrophenyl phosphate (pNPP), restriction enzymes, and T4 DNA-ligase were from BoehriigerMannheim (Mannheim,Germany). PurifiedE. coli AP obtained from Sigma has a specific activity of 55 unita/mg of protein. One unit is defined as the amount of enzyme that hydrolyzes 1 pmol of substrate in 1min at 37 O C according to the manufacturer’s specifications. Animals. Sprague-Dawley male and femalerata (Iffa-Credo, Lea Oncins, France) were housed under controlled temperature (3) Offensperger, W.; Wahl,S.; Neurath, R. A.; Price, P.; Strick, N.; Kent, S. B.; Christman, J. K.; Ace, G. Proc.Natl. Acad. Sci. U.S.A.1981, 82,7540-7544. (4) Schenk, D. B.; Spratt, S. K. Intemationl patent WO 86/06742,1988. (5) Lindbladh, C.; Pereeon, M.; Billow, L.; Stahl,5.;Mosbach, K. Biochem. Biophys. Res. Commun. 1987,149,607-614. (6)Gillet, D.; Ducancel,F.;Pradel,E.; Wnetti, M.; MBnez, A.; Boulain, J. C. Protein Eng. 1992,6, 273-278. (7) Jaffe, R. B. In Prolactin; Jaffe, R. B., Ed.; Eleevier: New York, 1981; pp 181-217. (8) Gala, R. R. h o c . SOC.Exp. Biol. hied. 1991,198,513-527.
0 I003 Armxican Chemlcel Society
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ANALYTICAL CHEMISTRY, VOL. 05, NO. 13, JULY 1, 1993
conditions (22 f 1 "C) in an artificially illuminated room (12 h on and 12 h off). Food and water were available ad libitum. Apparatus. EIAs were performed using specializedTitertek microtitration equipment, including an automatic plate washer (Microplate Washer S 8/12),an automatic dispenser (Autodrop), and a spectrophotometer (Multiskan MC) from Flow Laboratories (Helsinki, Finland). Radioactive counting was performed with an LKB Multigamma solid scintillation counter (Helsinki, Finland). Plasmids. Plasmid pJC2431 carrying the E. coli phoA gene has been described.0 Plasmid pLIPl6 carries the phoA gene in which a SmaI restriction site has been inserted, overlapping codons +6 and +7, leading to a two-base shift of the reading frame. The cDNA of rPrl in pBR32210 was the kind gift of Dr. A. Enjalbert, (INSERM U 159, Paris). Genetic and Bacteriological Procedures. rPrl DNA amplification by the polymerase chain reaction (PCR) was performed using a Perkin-Elmer Cetus DNA thermal cycler 480 (Norwalk, CT) as previously described." DNA fragment isolation and ligation with T4 ligase have been described in detail elsewhere." The phoA- E. coli strain CC118 [PA(ara-Leu)7679 araD139SlacX74 galE galKSphoA20 thi spc 'riPArgE(Am) recAl appR] was used for recombinant plasmid transformation according to the procedure described by Hanahan.12 The screening of transformed CC118 cells for the expression of AP activity was performed as previously described.6 Briefly,clones were isolated on a Petri dish with low phosphate concentration medium to induce the phoA promotor, and with 5-bromo-4-chloro-3-indolyl phosphate, a substrate of AP giving a blue color upon hydrolysis. Production of the rPrl-AP hybrid protein was performed by growing the transformed CCll8 clone in liquid medium with low phosphate concentration to induce the phoA promotor as previously described.6 The protein was then extracted from the bacterial periplasm by cold osmotic shock as previously described.13 Shock fluids containing the periplasmic proteins were kept frozen at -20 OC. Metabolic Radiolabeling and Immunoprecipitation Analysis. Metabolic radiolabeling and immunoprecipitation analysis were performed as previouslydescribed? Briefly, celh were grown in medium with low phosphate concentration to induce the phoA promotor. At the beginning of induction, 370 kBq (10 pCi) of [WSImethionine (Amersham, London, UK) were added per milliliter of culture medium. One-hundred microliters of periplasmic extract corresponding to l mL of bacterial culture was used for each sample. Initialy,nonspecific immunoprecipitation was performed to remove any radioactive material capable of binding nonspecifically to mouse immunoglobulins. Then, specific immunoprecipitation was done with either 2 pL of VIAPl ascitic fluid (anti AP mAb) or 10 pL of a 100-fold diluted antirPrl rabbit antiserum. Precipitated labeled proteins were analyzed by electrophoresis on a 12.5% SDS-polyacrylamidegel using a Mini-protean I1 apparatus (Bio Rad) followed by fluorography using Amplify fluorographic reagent (Amersham). 1%-Labeledmoleculer mass marker proteins were purchased from Amersham. Enzyme Immunoassay. EIA was performed in the following assay buffer: phosphate buffer (0.1 M, pH 7.4) containing 0.4 M NaC1, 1 mM EDTA, 0.1% bovine serum albumin, and 0.01 % sodium azide. EIA for rPrl was performed by the procedure already described for rPrl competitive EIA using acetylcholinesterase as label." The total incubation volume was 150 pL, with each component (standard or sample, anti-rPrl antiserum, and conjugate) being added in a 50-pLvolume. A standard curve was generated using purified rPrl (NIADDK R.P.3 or rPrl from (9) Lazzaroni, J. C.; Atlan, D.; Portalier, R. C. J. Bacteriol. 1985,164, 1376-1380. (10) Cooke, N.E.; Coit, D.; Weiner, R. I.; Barrter, J. D.; Martial, J. A. J.Biol. Chem. 1980,255,6502-6510. (11) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2th ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. (12) Hanahan, D.In DNA Cloning: APractical Approach;Glover, D. M., Ed.; IRL Press: Oxford, UL, 1985; Vol. 1, pp 109-135. MBnez, A. Protein Eng. (13) Ducancel, F.; Boulain, J. C.: TrBmeau, 0.; 1989,3,139-143. (14) Duhau. L.: Grassi,J.: Grouselle, D.: Enialbert.. A.;. Grognet, J. M. J. Immunoassay 1991,12,233-250.
UCB calibrated against R.P.3 standard). Rat plasma samples are assayed without dilution. When concentrations of rPrl were above the ICm of the assay, plasma was reassayed after dilution in assay buffer in order to obtain a concentration of rPrl close to the ICm. The anti-rPrl antiserum was obtained as already described'' by immunization of rabbita using pure rPrl. It was used at a final dilution of l / g o ~ and , rPrl-AP hybrid was introduced at a dilution corresponding to an increase in optical density (OD) of 6.7 mL-l h-l cm-I. Immunoreaction was performed at 30 OC in the case of the one-step assay. With a two-step incubation procedure, the standard or plasma sample (50 pL) and diluted antiserum (50 pL) were added and left for 16 h at 30 OC. The enzymatic tracer was then added and incubated for 24 h at 20 "C. In both cases, the plates were washed after incubation with 10 mM Tris-HC1 buffer (pH 7.4) containing 0.05% (v/v) Tween 20. Two-hundred microlitersof substrate-containing buffer (0.1 M Tris-HC1 (pH 8.2), 2 M NaC1, 1mM MgCl2,O.l mM ZnCh, 3 mM triethylamine, 10mM pNPP) was added to each well. The enzymatic reaction was allowed to proceed with gentle agitation at room temperature (generally for 4-5 h), and the absorbance of each well was then measured at 405 nm. Radioimmunoassay. RIA using '%I-radiolabeled rPrl was performed as recommended by the kit manufacturer (Amersham). Cross-Reactivity Studies. The specificity of EIA was checked by comparing standard curves obtained with rPrl and various rat pituitary hormones (LH, TSH, GH). Results were expressed in terms of percent cross-reactivityusing the Abraham criterion.I6 ICWis the concentration of the hormone that induces a 50% decrease in tracer binding to antibodies. cross-reactivity is defined as the ratio of the ICs0 of rPrl to the ICm of the tested hormone. Imprecision Profiles. Imprecision profiles of EIA were established on the basis of standard curves for which eight replicates of each standard concentration were used. Resulta are expressed in terms of the coefficientof variation (CV % ) vs the logarithm of the dose.I6 Characterization of rPrl Immunoreactive Material. Three milliliters of rat plasma (pool of samples from three rata) was analyzed by molecular sieve chromatography using a Biogel A 0.5M column (100 X 3 cm). Elution was performed with 0.06 M phosphate buffer (pH 7.0). Fractions of 1mL were collected and assayed for rPrl immunoreactivity as described above. Absorbance at 278 nm was recorded for each fraction. Data Analysis. Resulta were expressed in terms of BIB0 X 100where B and BOrepresent the radioactivity or the absorbance measured on the bound fraction in the presence or absence of hormone, respectively. Fitting of the standard curves and calculations of the quantity of hormone in biological samples were performed using a microcomputer (IBM/PC) and software developed in our laboratory, using a linear log-logit transformation. Unless otherwise stated, all measurementa were made in duplicate. The detection limit was defined as the rPrl concentration inducing significant reduction (three standard deviations) in the Bo value. Animal Study. Male rata were injectedintraperitoneallywith haloperidol (1mg/kg) in 1 mL of 0.9% NaC1. Rata were killed by decapitation 1 h after injection. Heparinized blood samples were obtained and centrifuged at lsoOg for 20 min. Plasma was collected and kept at -20 OC until assayed.
RESULTS Construction of an rPrl-AP Hybrid Protein. In order to insert a DNA coding for rPrl(l98 amino acid residues, 594 base pairs (bp))in the phoA gene, vector pLIPl designed for the insertion of foreign proteins in A P 6 was modified. pLIPl is a vector which has been previously designed for a rapid visualization of a correct insertion of a foreign DNA intophod. The principle of the method consists of introducing a frame shift immediately after the SmaI restriction site (Figure 11, between codons +6 and +7, so that the empty vector will not synthesize AP. Production of AP is expected to occur only (15) Abraham, E. G. Acta Endocrinol. 1974, Suppl. 7 5 , 7 4 2 . (16) Ekins, R.P.;Newman, G. B. Acta Endocrinol. 1970,154, 11-30.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993 1781 Native phoA gene (A!? activity)
... ATG CCT QlT CTG GAA ............... AAA TAA ... met pro Val 1 e L gl,, ............... lys stop +7
+6
1
+450
pL1Pl (no AP activity)
SmaI
... ATG ccc ggg lTC 'TGG ..... TGC TGA ... m e t pro gly pbe trp ..... cys stop ... ATG ... m e t
1
+49
+6
sa11
sa11
sac1
ccc DTC TTC AQ!J' CGA CGA GCT CCC 009 gTT CTG G A A pro Val asp phe per arg arg ala pro gly Val leu glu +7
+6
SalI SacI ccc 9 T C Q& P g Q CT€ CCQ ggg TTC TGG m e t pro Val asp glu leu pro gly phe trp
... ATG ...
...
(Ap
t450
pLIP4 (no AP activity)
+49
SalI
rPrl ctg cca met pro Val asp leu pro ATG ccc g T C +6
AAA TAA
lys s t o p
..... TGC TGA ..... cys stop
+6
...
... ...
pLIP4.0 activity)
SacI
... tgc g m QCT CCC g T T CTG ... cya gly ala pro gly Val l e u glu +I Qgg
GAA
...
...
pLIP4-rPrl AAA TAA (AP activity)
lys s t o p +450
Figure 1. Clonlng strategy used to insert the rPri Into AP. Vector pLIP4, designed for the lnsertlon of the fPrlcDNA, was derived from pLIP1. A s both vectors carry a phoA gene with a reading frame shift, unable to encode an acthre enzyme, the corresponding clones cannot be dlstlngulshed one from the other uslng AP activity screenlng. Thus, we deliberately constructed an Intermediate plasmid, pLIP4.0, whlch can express an active AP, and from which pLIP4 can easily be derived. At each step of the construdon strategy,the new genetic construct can be screened out from the precedent by the presence or absence of AP activity in the bacterial clones. This can be seen by colony color when bacteria are grown on appropriate medium containing a colorimetric substrate of AP. First line: nucleotide and deduced amlno acid sequences from the native phoA gene.% Nucleotidesfrom codons +6 and +7 are in bold letters. Second ilne: insettion of a SmI restrlctlon site (lowercase letters)by sitedirected mutagenesls overlapping codons 4-6and +7 of AP. The phoA gene's reading frame Is shifted. Third ilne: insertion at the SmI site of a synthetic DNA fragment contalnlng two Sail and one SacI sites (underilned). The four nucleotides (lTCA) between both Sail sites restore the reading frame of the gene leading to synthesls of a functional AP. This TTCA sequence was chosen in order to generate a stop codon In case of lnsertbn in the reverse orlentation and thus to allow discarding of these undesired clones. The CCQ sequence terminating the DNA insert was chosen in order to insert small uncharged residues between the foreign proteln and AP In the flnai construction (Pro-oly). Fourth line: one of the Sail sites and the four nucleotides TTCA are removed by digestion with the restriction enzyme Sail followed by Ilgatlon. The readlng frame of the phoA gene Is shlfted as in the pLIPl vector. Fifth line: the PCR-ampiitied rprl DNA sequence (lower case letters), with a Sail and a SacI site added at each end. Is Introduced at the CorresDondina - sites in vector D L I P ~ .An extra guanine at the end of the rprl DNA restores the reading frame
of the phoA.sequence.
when the frame shift is abrogated, a phenomenonwhich should take place when a DNA fragment containing 3n + 1 bp is correctly inserted. In order to increase the cloning efficiency of PCR-amplified DNA fragmenta at the insertion site, we have constructed a new vector, pLIP4. Two restriction sites giving cohesive ends upon cleavage, Sal1 and SacI, absent from pLIP1, have been inserted at the SmaI site of pLIP1. This was done using a strategy which is described in Figure 1. A DNA fragment correspondingto the rPrl sequence was produced from the rPrZ cDNA1O by PCR amplification. Synthetic oligonucleotides for PCR were designed in order to introduce a SalI site at the 5' end and a guanine and a SacI site at the 3' end. The guanine was added to restore the reading frame of the phoA gene after insertion in pLIP4. The amplified fragment and pLIP4 were both digested by SacI and Sal1 and ligated together. One out of 103 colonies expressed AP activity after transformation of AphoA cells CC118. The plasmid extracted from the positive colony corresponded to the desired DNA construct as shown by restriction analysis and DNA sequencing. The hybrid rPrZ-phoA gene was expected to encode an rPrl-AP hybrid protein with the following organization: the 21 amino acids of the AP leader sequence (MKQSTIALALLPLLFTPVTKA), the first 6 amino acids of mature AP (RTPEMP), a VD dipeptide, the 198 amino acids corresponding to the total rPrl sequence, a GAPG tetrapeptide, and the remaining 444 C-terminal amino acids of AP. This product was presumably localized in the bacterial periplasm
since AP is active in this compartment and not in the cytoplasm.17 Detection of an rPrl-AP Hybrid Protein. Expression of a hybrid rPrl-AP protein was investigated by immunoprecipitation followed by SDS-PAGE. Cells transformed with plasmid pJC2431 (native phoA gene), pLIP4 (frameshifted phoA gene), or pLIP4-rPrl (rPrZ-phoA hybrid gene) were grown under conditions appropriate for phoA promotor induction, and proteins were metabolically radiolabeledwith [SSImethionine. Periplasmicproteins were extracted by cold osmotic shock and subjected to immunoprecipitation with either anti AP mAb VIAPl or rabbit polyclonal anti-rPrl antibodies. The fluorography of the electrophoresis of immunoprecipitated material is shown in Figure 2. No radioactive material was immunoprecipitated from pLIP4 transformant using VIAPl mAb (Figure 2, lane D), while a 47-kDa component, corresponding to AP, was precipitated from pJC2431 transformant using mAb VIAPl (Figure 2, lane A). As expected, AP waa not precipitated by the antirPrl antibodies (Figure 2, lane E). However, a molecule with an apparent molecular mass of 80 kDa was detected from pLIP4-rPrl transformant using either AP-specific or rPrlspecific antibodies (Figure 2, lanes B and C). These results clearly indicate that the hybrid rPrZ-phoA gene led to synthesis of a hybrid rPrl-AP protein retaining both AP and rPrl antigenicity. This protein is exported to the periplasm. Given the imprecision of SDS-PAGE molecular weight ~~
(17) Derman, A. I.; Beckwith, J. J. Bacterial. 1991, 173, 7714-7722.
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ANALYTICAL CHEMISTRY. VOL. 85, NO. 13. JULY 1. 1993
-- M
200 92,5 69
46 30
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-
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C
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E
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-
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70 60
0 50
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.-
--
Flgure 2. Immunopreclpltatbn and SDS-PAOE analysis of Um rRIAP hybrid protein. Perlplasmlc protehsfrcinCCll8straintranslwmed wlth pJC2431 (natlvephoA gene), p L I P k R l (rW-pMA hybrid gene). or pLIP4 (frame-shined phoA gene) were Immunoprecipitated after [36S]methloninemetabolic radlolabellng. Lanes: (A) AP precipitated with VlAPl mAb; (Band C)rPri-AP preclpitated wlth V l A P l and antirPrl antibodles. respectively; (D) immunoprecipitatlon from pLIP4 hansformants using VIAP1; (E) Immunoprecipitation from pJC2431 hansforrnantsusing ant+rPrlantibodies;(M)molecular massstandards: valves given in kllodanons. The gel was loaded with &fold less AP sample than rPrl-AP samples.
deterrnination,'s the observed molecular weight in consistent with the predicted value calculated from the primary sequence of the rPrl-AP hybrid (70 kDa). No immunoreactive proteolytically degraded form of rPrl-AP was detected since only one band was observed on the gel after precipitation using antibodies directed againstrPrlor AP. This does not formally exclude the presence of other degraded forms of the fusion protein; however, such products unable to bind antibodies and devoid of AP activity would not interfere in an EIA. Characterization oftherPr1-AF' Hybrid. Twomethods wereusedtoshowthatantigenicallyactiverPrlwascovalently bound to AP. First, RIA measurements were made on the rPrl-AP hybrid. In these experiments, rPrl-AP hybrid provided dilution curves parallel to the curves obtained with standard rPrl (R.P. 3 from NIADDK). This allowed us to quantify the rPrl content of our rPrl-AP preparation (905 ng/mL). Assuming that the hybrid enzyme and the commercially available enzyme have identical specific activities, it is possible to evaluate the quantity of AP present in our preparation of tracer (1062ng/mL). We thus calculated that 85% ofAPiscovalentlyconjugatedtorPrl. Second,antibody dilution curves were constructed. Under equilibrium conditions, the maximum bound enzyme activity observed in the presence of a large excess of rPrl-specific antibodies was at least 85% of the total activity introduced in the assay. Taken together with the immunoprecipitation experiments, these results strongly suggest that the great majority of the AP present in the periplasmic extract was conjugated to rPrl and that the rPrl moiety has native-like antigenic properties. Standard Curve. The rPrl-AP hybrid allowed determination of a standard curve for rPrl assay in a one-step incubation for 16 h at 30 "C (Figure 3). A mean ICso value of 10 ng/mL of rPrl was observed and the limit of detection was lower than 0.5 ng/mL. The sensitivity of the assay could be improved by performing a two-step incubation: a 16-h incubation of antibody and rPrl standard followed by a 24-h incubation with the tracer. Under these conditions, the ICs0 value was approximately 3 nglmL and the detection limit was lower than 0.3 ng/mL (Figure 3). For both assay conditions,the sensitivity wassufficienttodetect physiological plasma levels of rPrl.? (18) See, P. Y.;Jackowski. G. In Rotein Structure: A Practical Approoeh;Ciaighton,T.E.,Eda.;IRLPress:Word,UK,1989;ppl-23.
10
- 1rPrl (ng/rnl)
0 0.1
I
LOO
10
-'O.I
IO
tm
Dose (ng/ml) or Volume (pl)
Figure 3. (Len) Callbration curve of the enzyme immunoassay of rat prolactin: omstep (0) and two-step (X) Incubations. Each point is the mean f SEM 01 SIXindependent experiments. (R1ght)Comparlson of male (') and female (0) rat plasma dilution curves and the rat prolactln standard curve (X). The logarithm of the prolactin Conwntration(~/mL)wthe!qarlthmof samp1evolume~L)ispIonedagalnst loan 816. me assay was performed using one-step incubation condnins. Slopes were determined after linear regression (slope SE): male rat, 1.238 0.075 (df = IO); female rat. 1.287 f 0.099 (df = 12); standard. 1.222 0.1239 (df = IO).
*
+
*
Validation of the Assay. None of the four rat pituitary hormones (rLH, rTSH, rFSH, rGH) was able to inhibit the binding of the rPrl-AP hybrid to the antibodies, even a t a concentration of 1 ag/mL. This indicates that no crossreactivity coefficient exceeded 1%. These values are in good agreement with those previously found for EIA using the same anti-rPrl antiserum and a chemical AChE-rPrl conjugate" or an lsI-radiolabeled rPrl (D. Grouselle,unpublished results). Thesedatashowthat theuseofarecombinantrPr1AP conjugate does not modify the specificity of the assay. Whatever thespecificityoftheantibodiesused,thevalidity of an immunoassay, as well as its applicability to biological fluids, must be checked. This was first done by comparing dilution curves of a standard antigen and plasma samples. Figure 3 shows the classical log-logit transformation of the standard curve as well as a series of sample dilution curves for male and female rat plasma. The corresponding slopes were respectively-1.222 f 0.1239for standard antigen,-1.238 f 0.075 for male plasma, -1.287 0.099 for female plasma (slope f SE). A statistical analysis based on Student's discriminant function indicated that there were no statistical differences between the standard curve and the plasma dilution curves a t a threshold of 5%. Table I presents the results of the recovery experiments performed on ratplasmaspikedwith different concentrations of standard rPrl. Good recovery was observed. Taken with the results of the dilution curve comparisons, these results strongly indicate that the assay was not significantly influenced by nonspecific components present in the plasma samples. The absence of nonspecificallyinterfering components was also supported by molecular sieve fractionation of plasmasamples. Figure 4shows the immunoreactivityprofile observed for a plasma sample chromatographed on a Biogel A 0.5M column. Only one peak of immunoreactive material was observed in an exclusion volume identical to that measured for standard rPrl under the same conditions. This contirmsthespecificityoftheassayandthelackofnonspecific interference due to the plasma. The imprecision profile obtained in the range 2500 (50 ng/ mL) to 1.9 pglwell(O.38 ng/mL) is presented in Figure 5 for both one- and two-step incubation. An intraassay coefficient of variation of less than 10% was observed above 145 pg/well (2.9 ng/mL). The coefficient of variation exceeded 20% for rPrl concentrations of less than 40 pg/well (0.8 ng/mL).
*
ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993
601
Table I. Assay of Rat Plasma Spiked with Standard Prolactin prolactin concn, ng/mL rec," % added mead (n = 4) rec, ngJmL (A) One-Step Incubation Conditionsb 17 5 0 (basal level)
* * *
10 50 100 200
27 t 3 81 5 133 & 16 244 34
10 64 116 227
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50
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lo
100 128 116 114
(B) Two-step Incubation ConditionsC 0 (basal level)
llfl
10 50 100 200
24*4 67 7 111 5 238 & 10
*
13 56 100 227
130 112 100 114
"":"'ly"
5
0
Recovery index is taken as the ratio of measured to added quantities. b Linear regression: YfoUd = 1 . 1 3 Y d ~ +1.70;rz= 0.9993. c Linear regression: Yfomd = 1.12Yddd - 1.30; P = 0.9978.
.
o.......,.,,..............,....,
0
10
15
3'5
T
20
25
30
35
40
45
rPr1 (ng/ml)
0
Flgure 6. Day-tcnley varlatkm of the rPri assay on 30 rat plasma samples. Fitting was achieved using a power function.
-2 \
'"T
R2 Slope = 0.963 = 0.96 t/- 0.04
40
30
a4
52 &
20 10
0
0
10
20
30
40
50
rPrl (ng/ml)
one-step EIA
rPr1 (ng/ml) two-step EIA
Flgure 7. Correlation between enzyme immunoassay (EIA) and radioimmunoassay (RIA) of rat prolactln in 30 rat (male and female) plasma samples.
Flgws4. Elutionprofileof prolactin Immunoreactivematerial: molecular sieve chromatography (Biogei A 0.5M,100 X 3 cm) of 1-mL plasma sample from female rat. (- -) absorbance 278 nm; (-) rPrl immunoreacthrlty. I n this system, rPrl standard (NIAWK R.P.3) shows a single Immunoreactive peak at fractions 87-92 (1-mL fraction). V, (total volume of the column) was taken as the retention volume of ferricyanide sodlum.
-
70
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using a power function as described by Rodbard.'e The mean day-to-day variation was below 15% for concentrations between 4 and 12 ng/mL and below 10% for concentrations above 12 ng/mL in the case of the one-step incubation. The same range of values was obtained in the case of the two-step incubation procedure (data not shown). For a series of 30 rat plasma samples, a high correlation was observed between values obtained using EIA with the one-step or two-step incubation procedure and RIA (Figure 7a,b). One hour after intraperitoneal injection of 1 mg/kg haloperidol in rats, the change in plasma rPrl levels was measured (Figure 8). A marked increase in rPrl levels was observed after injection, as expected for a butpophenone drug.' This indicates that this assay, which uses a recombinant tracer, is well-suited to monitor effects of drugs on rPrl levels in plasma.
DISCUSSION
Flguro 5. Imprecision proflie of the rPrl assay (intraassay Variation, n = 8): (- -) onastep incubation; (-) twestep incubation.
We produced an rPrl-AP hybrid protein which is expressed in E. coli as a periplasmic protein. Both rPrl and AP domains retained antigenicity. Moreover, the hybrid exhibited AP activity. These properties prompted us to investigatewhether the bacterial periplasmic extract could be used aa a tracercontaining reagent in an rPrl EIA. When compared to other isotopic20*21 or n o n i ~ o t o p i ccompetitive ~~*~ assays, this method
To assess reproducibility of the assay, 30 rat (male and female) plasma samples ranging from 1 to 43 ng/mL were assayed in three daily experiments. Plasma was diluted to give a decrease in tracer binding close to BIB0 = 50% when possible. For each sample, the day-to-day variation was plotted against concentration (Figure6). Fitting was achieved
(19) Rcdbard, D. Quality Control for RIA. InRadioimmunoausay and Related Procedures in Medicine; International Atomic Energy Agency: ~. Vienna, 1978. (20) Neill, J. D.; Reichert, L. E. Endocrinology 1971,88, 548-666. (21) Thordareon, G.; Holekamp, K. E.; Talamantee, F.Biol. Reprod. 1987,36,1186-1190. (22) Signorella, A. P.; Hymer, W.C. Anal. Biochem. 1984,136,372381.
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appeared slightly less sensitive when tracer, antiserum, and standard antigen were incubated simultaneously (one-step incubation procedure). Sensitivity was substantially improved using sequential incubations of first the competitor and then the tracer, with the antiserum (two-step incubation procedure). However, under both sets of incubation conditions, we have shown that the assays are well-suited to monitoring rPrl secretion in rat plasma, under both physiological and pharmacological conditions. Several attempts to use recombinant fusion proteins as tracers for EIAs have been previously reported. A pre-S2 peptide-&galactosidase fusion was used to detect antibodies against the pre-S2 region of the hepatitis B virus? but the assay was complicated by centrifugation steps. A human surfactant apoprotein peptide-@-galactosidasefusion was used for the assay of human surfactant apoprotein.* In both cases, the use of &galactosidase, which is a cytoplasmic protein, is unsuited to the expression of full-size disulfide-containing proteins. The reducing conditions present in the cytoplasm do not favor disulfide bond formati0n.~3 Moreover, purification of the peptide-&galactosidase hybrid is required. An (23) Gilbert, H. F. Advances in Enzymology; Meister, Alton: New York, 1990; pp 69-172. (24) Neuberger, M. 5.;Williams, G. T.;Fox, R. 0. Nature 1984, 312, 604-608. . . .~~ ~
(25) Casadei, J.; Powell, M. J.; Kenten, J. H. Proc. Natl. Acad. Sci. U.S.A. 1990,87,2047-2051. (26) Pugsley, A. P. Protein Targeting;Academic Press Inc.: San Diego, CA, 1989. (27) Boquet, P.; Boulain, J. C.; Ducancel, F.; Gillet, D.; MBnez, A. European Patent Application 90-401794.4, 1990. (28) Ducancel, F.; Gillet, D.; MBnez, A.; Boulain, J . 4 . C. R.Acad. Sei. Parak 1992,315, 221-224. (29) Gosling, J. P. Clin. Chem. 1990,36, 1408-1427. (30) Chang, N. C.; Kuang, W.-J.;Ellaon, Y. C. Gene 1986,44,121-125. (31) Abbreviations: AChE, acetylcholinesterase (EC 3.1.1.7.); AP, alkaline phosphatase (EC 3.1.3.1.);rPrl, rat prolactin; rLH, rat luteinizing hormone;rTSH, rat thyrotropin; rGH, rat growth hormone;EIA, enzyme immunoassay;RIA, radioimmunoassay;NIADDK, National Institute of Arthritis, Diabetes, Digestive Dieeases and Kidney; pNPP, 4-nitrophenyl phosphate.
antihapten antibody was fused successfullyto a nuclease from Staphylococcus aureus24 and to the photoprotein aequorin.26 In both cases, the hybrid was produced by hybridoma cells. Detection of the hapten by the hybrids was possible, but the assays are fairly involved and require unconventional conditions since nuclease activity is monitored by the extinction of fluorescence of ethidium bromide-labeled DNA and aequorin must be highly purified for photoactivity assay. Finally, a proinsulin-AP fusion protein using the proinsulin signal sequence was constructed6 and was used to design an insulin assay. The strategy used for exportation of this proinsulin-AP fusion protein, which consisted in using the eukaryotic protein's own signal peptide, is not generally applicable since such signal peptides may be incompatible with the prokaryotic protein export machinery.% Moreover, the assay was not validated in the case of plasma measurements. The system which is described in this paper offers many advantages. First, screening of the recombinant DNA constructs is based on colorimetric detection. Second, the insertion strategy should lead to exportation of the hybrid whatever the inserted foreign protein. Indeed, exportation was observed for over 10 different hybrids that we have constructed, including a snake neurotoxin: small peptide hormones,27 and antibodies.% Third, the system is welladapted for inserting disulfide-containing proteins. This was previously proven in the case of the neurotoxin.6 As rPrl contains three disulfide bonds, the fact that an anti-rPrl antiserum recognizes rPrl-AP without loss of affinity, as compared to purified rPrl, suggests that the conformation of the rPrl domain of the hybrid is properly folded. Fourth, the hybrid appears remarkably stable during culture and extraction procedures, as no degradation product was observed following immunoprecipitation. This is of importance because proteolysis is a frequent problem when foreign proteins are expressed in E. coli. Fifth, the hybrid is produced using a fast and inexpensive fermentation process. It is extracted by osmotic shock and may be used as such for the assay, without the need for further purification. This process allows largescale production of the conjugate. Due to ita origin from a single bacterial clone, this tracer may be qualified as monoclonal. Sixth, calf intestine AP is widely used for commercial immunoassay kits for diagnosis29 and many automated apparatuses have been developed for AP tracers. Seventh, our system makes it feasible to produce enzymatic conjugates for proteins which are not available in sufficient quantities or which are difficult to purify, especially in the case of pituitary hormones. Finally, as indicated by recent preliminary experiments,% one can now insert proteins composed of two different chains, like the Fab portion of immunoglobulins.
~
ACKNOWLEDGMENT This work was supported by the Commissariat A l'Energie Atomique (CEA). The authors are indebted to Dr. A. Enjalbert for a generous gift of cDNA for rPrl. We warmly thank Drs. J. Grassi and P. Pradelles for fruitful discussions.
RECEIVEDfor review November 9, 1992. February 22, 1993.
Accepted
