Analytaca Chmuca Acta, 275 (1993) 183-187 Elsevler science Pubbshers B V , Amsterdam

183

Development of an enzyme immunoassay for the determination of porcine growth hormone in plasma

B Serpek and F Elsaesser

Immt j2.r lkmcht und 7Lmerhaiten (FL), Manemee, 3057 Neustadt I @emany)

H HD Meyer

Inshhdt jiu Physwkqpe, Technrche Uwemtat Munchen, 8050 Fmsmg- Wedmstephan (Germany)

(Recewed 22nd June 1992, rewsed manuscnpt received 12th October 1992)

A competltwe enzyme unmunoassay for the determmatlon of porcme growth hormone (pGH) was developed The assay IS based on (ant+rabbit IgQ sheep IgG-coated nucrot&e plates, antl-pGH from rabbits, blotmyl-pGH and streptamdm-horseradrsh peroxtdase The assay IS done dx&ly pvlth 40 ~1 of plasma and the cahbration graph (90% relative bmdmg at 0.36 ng ml-’ and 50% relative bmdmg at 4 4 ng ml-‘) 1 prepared m plasma mthout measurable endogenous pGH Recovery of pGH added to dtfferent plasma samples amounted to 102-119%, and all vanab&es were < 8 6% The aa~y shows 50% cro~reaction with recombmant pGH, but no s&uuficant bmdmg of ponxne pro&m, folhcle stnnulatmg hormone and h~teunzmg hormone The assay detects htgh and low plasma pGH levels wtthm the pbyrnologtcal vanatton as well as changes m plasma pGH after s~ulatxm with gmwth hormone releasmg factor or treatment wrth recombmant pGH Hence It offers a rehable altematwe to radmmunoassay However, a reference method prowdmg wrdence on the Identity of exogenous pGH anal be. reqmred for provmg tiegal treatment wttb pGH and for forensrc purposes

&ywvrd9 Enzymatuz methods, Immunoassay Plasma, Porcme growth hormone

In addition to the clas~cal sex hormones (estrogens and androgens) and the &agomsts, growth hormone has also gamed scientific and pramcal importance for growth promotlon [1,21 Porcme and bovme growth hormone (pGH, bGH) are 191 or 190 ammo acid proteins, they share a Hugh degree of homology (90%) and they are produced 111 the antenor pltu~tary Today both protems can be synthesued by transformed bac- tena [3] Liver, skeleton, adipose trssue and mus- cle are the major target tissues There are clear metabohc effects of GH and the reduced ducose

Correspandenee to F Elsaesser, Insmut fur Tlexzucht und Tterverhalten (FAL), Manensee, 3057 Neustadt 1 (Germany)

ut&atlon by adipose tissue seems to be most nnportant In addition, ammo acid degradation m the hver 1s reduced In consequence, more glu- case and ammo acids are avslllable for muscle and skeleton growth [4,5] Induced hepatlc IGF-1 may also be of importance for mcreased growth rates [6]

To study the control of GH secretlon or to monitor the apphcatron of GH, a sensitive assay 1s reqmred Recently a competrtlve enzyme un- munoassay (EIA) for lutemlzmg hormone was described that uses the double antibody tech- nique and blotm-streptavldm-horseradlsh perox- ldase amphfication 171. In order to avoid any

radiolabel a sumlar assay was developed for pGH

Elsevler Saence Pubbshers B V

184 B Serpek et OL /Anal. Chm. Actn 275 (1993) 183487

EXPERIMENTAL

Preparatwn of bwtuayl-rpGH label A 200~mg amount of blotmyl-e-ammocaproic

acid N-hydroxysuccmnmdeester (Blotm-X-NHS, Sigma, Delsenhofen, Germany) mlved m 20 ml of N,N-dnnethylformanude (Aldrich, Stemheun, Germany) was used for couplmg vvlth 500 pg of recombinant porcme growth hormone (rpGH) (kmdly provided by Pltman-Moore, Mundelem, IL, lot number 148-215) dissolved 111500 ~1 of 50 mM carbonate buffer (pH 9 98) The reagents were rmxed and mcubated at room temperature wth gentle stlrrmg for 4 h The reactlon was stopped by adding 0 4 mg of glycme (Serva, Hel- delberg, Germany) m 1 ml of carbonate buffer After overmght mcubatlon at 4”C, 2 mg of bovme serum albumin (BSA) (lot number 12031, Serva) m 1 ml of carbonate buffer were added to the mxture and dlalysed three tnnes agamst carbon- ate buffer at 0°C ovemlght After dralysls the coqugate was diluted with assay buffer to achieve a final concentration of 50 pg ml-l, plpetted mto ahquots and stored at -20°C unttl used

pGH antibody A GH-specific antibody riused m rabbits (code

K-14) was kmdly supphed by Dr Schams (In- stitute of Physiology, Techmcal Umverslty of Mu- nich, Welhenstephan, Germany) The cross-reac- tlvltles of rpGH (Pitman-Moore, lot number 148- 2151, prolactm (porcine PRL, USDA pPRL I-2, AFPJOO), porcine lutemmng hormone (LH) (Bloproducts, Brussels, lot number 004/l) and porcme folhcle smulatmg hormone (FSH) (Blo- products, Brussels, lot number 008/2) were de- termmed at 50% bmdmg of pltmtary GH

Preparatwn of affinity-punjied sheep IgG an- tlrabbrt IgG

A 15-20-ml volume of plasma from a sheep lmmunlzed agamst rabbit IgG, contammg 6 mM EDTA (Merck, Darmstadt, Germany) was ap- phed to a small column prepared with 5 g of rabbit IgG agarose gel &gma) Low affimty bound protems were eluted with 10 ml of 0 5 M NaSCN (pH 8 0) followed by 10 ml of 0 1 M glycme-HCl (pH 3 5) Protems bound specd?cally

were eluted with 15 ml of 0 1 M glycme-HCl (pH 2 0) All steps were performed at room tempera- ture To achieve an nmnedlate neutrahzatlon of the eluted product, the collectmg vials were pre- filled with 2 ml of 1 M Tns-HCl (pH 8 0) The eluted IgG was dralysed agamst 66 mM NaH,PO,-Na,HPO,, (pH 72) and determined by the bmret procedure

Enzyme unmunoassay procedure Arst coatang The first coatmg was achieved by

addmg 1 pg of sheep-anti-rabbit IgG dissolved m 100 ~1 of coatmg buffer (15 mM Na,CO,, 35 mM NaHCO,, pH 9 6) per well of the mlcrotltre plate (Nunc, Roskdde, Denmark, No 439454) The plates were subsequently mcubated for el- ther 2 h at room temperature or overnight at 0°C with shght shakmg After the incubation the plates were decanted

Second coatrng To achieve saturation of the remammg bmdmg sites, 350 ~1 of assay buffer (40 mM Na,HPO,, 150 mM NaCl, pH 7 2, contam- mg 0 1% BSA) were added per well and mcu- bated for 15-45 mm at room temperature before subsequent decantatlon Non-specfic bmdmg (NSB) m the absence of antibody was checked m duphcate on each plate Optical density values for NSB ranged between 0 045 and 0 135 Coated plates can be stored at - 20°C for up to 6 months

Wizshmg Prior to use m the assay, the coated plates were washed twice with 375 ~1 of 0 05% Tween 80 per well

Assay protocol Lyophdrzed pltultary porcme GH (pit pGH) (Broproducts, lot number 009/l) was dissolved m 50 mM carbonate buffer (pH 9 98) at a concentration of 100 ng ml-’ Standard solutions (0 306-40 ng ml- ’ pit pGH) were pre- pared by senal dllutlon (steps of 1 2) m plasma vvlth undetectable levels of endogenous porcme GH (< 0 3 ng ml-‘) Volumes of 40 ~1 of stan- dards and unknown plasma samples were diluted m duplicate mto 100 ~1 per well of antlbody-con- taming (1 40000) assay buffer usmg a dduter- dispenser (Ham&on Mxrolab 1000) The plates were then mcubated under constant gentle shak- mg for 48 h at 4°C and subsequently decanted After decantatlon, 2 ng per well of blotmylated rpGH m 100 ~1 of assay buffer were added and

B Setpek et eL/AnaL Chm Acta 275 (1993) 183-187

mcubated for 2 h at 4°C Followmg another de- cantatlon, 20 ng per well of streptawdm peroxl- dase (Boehrmger, Mannhelm, Germany) m 100 ~1 of assay buffer were added and mcubated for 15 mm at 4°C until decantatlon

Substrate reactwn The plates were washed four tunes wth 375 ~1 of 0 05% meen 80, after which 150 ~1 of substrates A and B (1 1) were added per well [substrate A, 10 g I- ’ hydrogen perox- lde-urea, 18 g I-’ Na,HPO, 2H,O, 103 g I-’ cltnc acid monohydrate, 0 1 ml 1-l Kathon (Rohm and Haas, Frankfurt, Germany), pH 5 0, sub- strate B, 500 mg I-’ tetramethylbenzldme, 40 ml l- ’ dlmethyl sulphoxlde, 10 3 g I- ’ citric acid monohydrate, pH 2 41 The reaction was stopped wth 50 ~1 of 2 M H,SO, per well and the extmctlon was measured at 450 nm usmg an eight-channel mlcrotltratlon plate photometer (Tltertek MultIscan MC, Flow Lab, Meck- emheun, Germany)

RESULTS

Trtratwn of porcuae bwtutyl-rpGH and anti-pGH serum

In order to determme the optimum concentra- tlon of antlbody K-14 and blotmyl-rpGH for the assay a two-dnnenslonal tltre determmatlon was performed Antibody dllutlons of 1 10 OOO- 1 1280000 and blotmyl-rpGH concentrations of 0 125-4 ng per well were tested The mmmnun concentrations of antibody and blotmyl-rpGH ac- cepted as suitable for the assay were taken as those sufficient to achieve an optlcal density (OD,,,) of ca 1 For the actual assay wrth an mcubatlon period of 2 days an antlbody dllutlon of 1 40000 and a blotmyl-rpGH concentratron of 2 ng per well were chosen

Assay vahdktwn Injluence of uuxbatwn temperature on assay

kznetrcs To determine the optlmum mcubatlon tnne and temperature, rmcrotltre plates were m- cubated for either 4 or 24 h at 22°C or for either 24 or 48 h at 4°C (Fig 1) The relatrve bmdmg was less with mcubatlon for 4 h at 22°C and 24 h at 4°C compared w&h 24 h at 22°C and 48 h at

-I

0001 0306 0612 125 25 50 10 20 40 80

Standard concentrmon of pGH (ng/ml)

Fu 1 Intluence of mcubation penod and temperature on pGH bmdmg + + -4hat22”C, A---A =24hatZZ*C, o---o=24hat4”C. +-+ = 48 h at 4°C

4°C Desprte the good relatrve bmdmg Hrlth mcu- batlon for 24 h at 22°C the slope of the callbra- tlon graph was mfetror vvlth a low repeatabrllty and higher standard dewatlons among duphcate determmatlons For this reason an mcubatlon permd of 48 h at 4°C was chosen as bemg optl- mum for the assay

Assay sen,s&vzty To determme the posstble mterference of plasma with the assay sensltlWy, vanous amounts of plasma (5, 10, 20 and 30 ~1) were added to the prt pGH standard ddutlons An mterference of added amounts of plasma with the cahbratlon graph was observed Because of this, all standard dllutlons were subsequently pre- pared m plasma, which had unmeasurable amounts of endogenous pGH (g 0 3 ng ml-‘) Samples and standards were standardized to a volume of 40 ~1 per well to guarantee sufficient assay sensltlvlty (Fig 2) Usmg standards pre- pared m plasma, recovenes rates of various con-

* 0

0001 0306 0612 125 25 5 0 10 20 40 80

Stlndud concentrdmn of pGH (ng/ml)

FIN 2 Intluence of volume of standard on pGH bmdmg Volume per well + + =lO, A---A =20, O---O-30, +-+=40~1

186 B Serpek et al /Anal Chun. Acta 275 (1993) 183-187

centratlons of pit pGH (0 625, 125, 2 5, 5 0 and 10 0 ng per ml) added to unknown samples were determined and acceptable results were obtamed (Table 1)

At a volume of 40 ~1 of standard per well the detection lmut of the assay (optical denstty values srgmflcantly drfferent from those at 100% B/B,) was found to be 0 361 f 0 11 ng per ml of ptt pGH CmeanfSD, n=14, RSD =21%, 90% B/B,) and the 50% relative bmdmg (B/B,) was at 4 41 f 0 43 ng ml-’ of pit pGH (mean f SD , n=14,RSD -98%)

Intra- and anter-assay vanatwn Intra- and mter-assay varratlon were deter-

mmed usmg plasma samples wrth varrous pit pGH concentrations Repeated measurements of plasma concentratrons (mean f SD ) of 4 79 f 028 (n-141, 1104fO43 (n-51, and 1722f 0 93 ng ml-’ (n = 5) of pit pGH resulted m R S D s of 5 75,3 87 and 5 4%, respectrvely The mter-assay vanatlon was 8 6% and 4 3% usmg plasma samples wrth concentrations of 120 f 0.19 (n = 14) and 4.44 f 0 19 (n = 14) ng ml-’ of pit pGH, respectwely.

Cross-reacttvrty wrth other ho- The antii K-14 that was used m this assay

showed 50% cross-reaction wrth rpGH, but no cross-reactMy (g 1%) was observed wrth either porcme LH, FSH and prolactm

Clamcal vahdatwn Plasma samples obtamed from a German

Landrace sow (DL 685) and a Gottmgen mmta-

TABLE 1

Recovenes of pltmtary-demed GH added to different plasma samples at various concentrations

Pit pGH added Replthons Recovery Vanatlon (nt3 ml-‘) (n samples) (%) (%‘c)

0 612 6 119 387 1250 12 107 4 16 2500 18 108 354 5ooo 18 104 490

loo00 12 102 14 14

50

40 1

2 30

8 20

P 10

0

-240 -180 -120 -60 0 60 120 180

Time (mm)

Fig 3 Plasma pGH levels m two mdmdual pw (+ - MINI 667, A = DL 68.5) before and after stuuulatlon wh GRF (7 bg kg-* body we&t iv)

ture prg (MINI 667) at 15 mm mtervals vra an mdwelhng jugular catheter were analysed usmg the standard EIA procedure descrrbed above Both ammals had recerved a single rv sumula- tron wrth human growth hormone-releasmg factor (hGRF l-29, 7 pg kg-’ body we&t) (Saxon Bmchermcals, Hannover, Germany) at 0 h to m- duce marked changes of plasma pGH concentra- tions (Fig 3) In the German Iandrace pig a first mcrease m pGH m plasma could be observed wrthm 5 mm after the nqectlon, reaching a peak concentratron at 30 mm, and followed by a steady declme until basal levels were reached at 90 mm after the nqeetmn In the GMngen multature ptg the pGH response to hGRF was delayed; the peak concentratron of pGH was found at 75 mm after hGRF The response curves were sLrmlar m mcrease and tlrmng to those m other expenments performed prevrously

DISCUSSION

The method described here 1s the first report of a pGH EIA The use of the second antibody for coatmg the wells mstead of hormone-specrflc antrbody is preferred as rt reduces assay varrabth- ties associated wrth uneven bmdmg of the latter antrbody to the wells and further reduces the amount of hormone-specrflc antrbody needed m the EIA [B] The amount of hormone-spectflc antrbody reqmred IS ten times less than m a radlommmnoassay and 1000 tunes less than 111 a sandwrch ELISA

B Serpek et a,! /Anal Chtm Acta 275 (1993) 183487

In our EIA an acceptable decrease m opt& density was observed when mcreasmg plasma vol- umes were used Nevertheless, It 1s essential to compensate for this effect and to use the same plasma volumes for standards and unknowns Suitable sensltlvlty and low non-speck bmdmg were obtamed over a range of up to 40 ~1 of plasma, sufflaent to measure low baseline and high pGH levels 111 the pig The sequential satu- ration of the antlbody w&h cold and labelled hormone further improved the sensltlvlty of the EIA However, this required optnmzatlon of the tracer concentration and mcubatlon period so as to produce the desirable saturation Hrlthout dls- placement of the cold hormone Lower tracer concentrations than those employed m both EIAs could be used but were less preferred as longer mcubatlon periods were needed to attam satura- tion

The vahdatlon data presented above indicate that the performance characterlstlcs of the EIA are acceptable Hrlth respect to senat&y, vanabll- ity, specificity and recovery of pGH m dtierent samples The stimulation of pGH by GRF was clearly detectable The results of a current exper- unent m which pigs were treated mth rpGH show a S-lo-fold elevation of pGH levels m porcine plasma

In conclusion, the assay detects high and low plasma pGH levels withm the physlolog& range and changes m plasma pGH after stunulatlon vvlth GRF or treatment with recombmant pGH Hence it offers a reliable alternative to radlolm- munoassay However, a reference method proved- mg evidence on the identity of exogenous pGH will be required for provmg illegal treatment wth pGH and for forensic purposes

187

The authors thank the Alexander von Hum- boldt Foundation for provldmg a fellowship for B S , Professor D Schams, Instrtut fhr Physlolo- gle, Techmsche Umversltat Munchen, Frelsmg- Welhenstephan, for provldmg the pGH-specific antibody and NHPP, Umversrty of Maryland, School of Medlcme, for provldmg pPRL

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M Kuchgessner, FX Roth, D &hams and H Karg, J Amm Physlol Amm Nutr ,58 (1987) 50 P van der Wal, G.J Nleuwhof and R D Pohtiek @ds 1, Biotechnology for Control of Growth and Product Quahty m Swme, Imphcatlons and Acceptabdity, Pudoc, Wagemn- gen, 1989 P H Seeburg, S Stas, J Adehnan, HA. de Boer, J Hayfbck, P Jhuram, D V Goeddel and J L Heyneker, DNA, 2 (1983) 37 T D Etherton, m P van der Wal, G J Nieuwhof and R D Pohtiek (Eds 1, Biotechnology for Control of Growth and Product Quahty m Swme, Imphcahons and Acceptabdlty, Pudoc, Wagemngen, 1989, p 111 HHD Meyer and H Karg, m TK. Mukhenec (Ed 1, FAO/CAAS Workshop on Biotechnology m Anmud Pro- duction and Health m Asla and Latm America, Begmg, 1989 FAO, Rome, p 49

6 HP Guler, J Zapf, K. Bmz and E R Froeach, m R B Heap, C G Presser and G E Lammmg (Eds 1, Blotechnol- ogy m Growth Regulanon, Butterworths, Gmkiford, 1989, p 119

7 B M Mutayoba, H H D Meyer, D Schams and E Schal- lenberger, Acta Endocrmol (Copenhagen), 122 (1990) 227

8 H H D Meyer, m Proceedmgs of International Symposuun on the Use of Nuclear Techmques m Studies of Ammal Prodution and Health m Dtierent Enwromnents, IAEA 292-3, FAO/Int Atormc Energy Agency, Vienna, 1986, p 255