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Microparticle‐enhanced nephelometricimmunoassay of plasminogen in bovinemilkE. Marchal a , S. Haïssat a , P. Montagne b , M. L. Cuilliere b , M.C. Bene b , G. Faure b , G. Humbert a & G. Linden aa Faculty of Science , Laboratory of Food Bioscience, Associatedwith INRA , BP 239, Vandoeuvre les Nancy Cedex, F‐54506,Franceb Faculty of Medicine , Immunology Laboratory, Jeune EquipeDRED 251 , BP 184, Vandoeuvre les Nancy Cedex, F‐54505,FrancePublished online: 16 Sep 2008.

To cite this article: E. Marchal , S. Haïssat , P. Montagne , M. L. Cuilliere , M. C. Bene , G.Faure , G. Humbert & G. Linden (1995) Microparticle‐enhanced nephelometric immunoassayof plasminogen in bovine milk, Food and Agricultural Immunology, 7:4, 323-331, DOI:10.1080/09540109509354892

To link to this article: http://dx.doi.org/10.1080/09540109509354892

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Food & Agricultural Immunology (1995) 7, 323-331

Microparticle-enhanced Nephelometric Immunoassay ofPlasminogen in Bovine Milk

E. MARCHAL,1 S. HAÏSSAT,1 P. MONTAGNE,2 M. L. CUILLIERE,2

M. C. BENE,2 G. FAURE,2 G. HUMBERT1 AND G. LINDEN1

1Laboratory of Food Bioscience, Associated with INRA, Faculty of Science, BP239, F-54506 Vandoeuvre les Nancy Cedex, France; 2Immunology Laboratory,Jeune Equipe DRED 251, Faculty of Medicine, BP 184, F-54505 Vandoeuvre

les Nancy Cedex, France

(Received for publication 7 July 1995)

A sensitive microparticle-enhanced nephelometric immunoassay of bovine plasminogen(detection limit in reaction mixture, 4 fig l-1) is described. It is based on nephelometricmeasurement of the light scattered by microparticle clusters formed during a sandwichassay that uses microparticle-anti-plasminogen conjugate and free antibodies from anti-plasminogen rabbit antiserum. This immunoassay was performed in 200-fold diluted bovinemilk, after release of plasminogen from caseins by ε-amino-n-caproic acid treatment andelimination of the caseins. It allowed the quantification of plasminogen in milk for a widerange of concentrations (0.9-14.4 mg l-1), with accuracy (linear recovery in overloadingassays) and precision (within- and between-run coefficients of variation lower than 5%).When it was applied to 106 herd milk samples, this microparticle-enhanced nephelometricimmunoassay yielded milk plasminogen concentrations ranging between 1.1 and4.9 mg l-1, with a mean of 2.0 mg l-1, and disclosed possible seasonal variations.

Keywords: Particle immunoassay, immunonephelometry, bovine plasminogen, bovine milk

INTRODUCTION

Bovine plasmin (bPl) and plasminogen (bPlg), its native zymogen, constitute the majorendogenous proteolytic system of milk (Humbert & Alais, 1979; Grufferty & Fox, 1988).Associated with casein (CN) micelles (De Rham & Andrews, 1982; Korycka-Dahl et al,1983; Richardson & Elston, 1984), this proteolytic system is responsible for the hydrolysisof a-, ß- and K-CN (Andrews & Alichanidis, 1983), and alters the quality of dairy products.Fragments of CN (Okigbo et ah, 1985) can induce gelation in sterilized and pasteurizedmilk (Driessen et al, 1981), can lead to the development of off-flavours (Mac Kellar, 1981),and can modify milk coagulation and cheese-making yields (Grufferty & Fox, 1988).

Numerous methods have been described for bPlg quantification in milk. Several are

0954-0105/95/040323-09 ©1995 Journals Oxford Ltd

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324 E. MARCHAL ETAL.

based on the measurement of bPl activity, after activation of bPlg by urokinase (Rollemaet al, 1983) and dissociation of CN micelles (Richardson & Pearce, 1981), after the releaseof CN-bound bPlg (Korycka-Dahl et al, 1983) by e-amino-n-caproic acid (e-ACA)treatment or elimination of whey proteins (Politis et al, 1993). Le Bars and Gripon (1989)used high-pressure liquid chromatography (HPLC) to follow the hydrolysis of aS2-CN bybPl. Immunological methods (ELISA), using polyclonal anti-bPlg antiserum (Collin et al,1988) or bPlg-specific monoclonal antibodies (Dupont et al, 1994, 1995), were alsodescribed.

Microparticle-enhanced nephelometric immunoassays have been described previously asappropriate methods for the measurement of various human serum proteins (Montagne etal, 1992a) and bovine milk components (Collard-Bovy et al. 1991; El Bari et al, 1991;Marchai et al, 1991; Montagne et al, 1991; Prin et al, 1996). Measurement of the lightscattered by microparticle (MS) clusters, formed during a sandwich assay (Montagne et al,1992b) using MS-anti-bPlg conjugate and free antibodies from anti-bPlg rabbit antiserum,allowed determination of the concentration of bPlg. Such a bPlg assay was recently usedfor the quantitative characterization of bPlg binding to CN (Haïssat et al, 1994). Thedevelopment of a microparticle-enhanced nephelometric immunoassay of milk bPlg and itsapplication to herd milks is reported here.

MATERIALS AND METHODS

Biochemical ReagentsAffinity chromatography on lysine-agarose (lysine-Sepharose 4B, Pharmacia, Uppsala,Sweden) was used to prepare bPlg from fresh, frozen bovine plasma, following the methodof Deutsch and Mertz (1970) with some modifications previously reported (Haïssat et al,1994). The bPlg solution was stored at - 20°C at a concentration of 2 g 1~ ' in 10 mM-Tris-HC1 buffer, pH 8.0, with 3 mM-NaN3. Its purity was assessed by polyacrylamide gelelectrophoresis according to Laemmli and Favre (1973).

Anti-bPlg antisera, obtained by immunizing rabbits with purified bPlg, were collected aspreviously reported (Haïssat et al, 1994). The reactivity and specificity of anti-bPlg antiserawere studied by double immunodiffusion (Ouchterlony, 1949). The IgG fraction ofanti-bPlg antisera was obtained by chromatography on Protein A-agarose (Affi-gel ProteinA, Econo-column, Bio-Rad, Richmond, CA, USA) following the method recommended bythe manufacturer.

Alpha-lactalbumin (a-La) was prepared by precipitating fresh skimmed milk withammonium sulphate, then purifying it according to Armstrong et al (1967). Beta-lactoglob-ulin (/?-Lg) was obtained by ion-exchange chromatography (lung et al, 1987). Bovineserum albumin (bSA) and bovine gamma globulins (bGG) were purchased from SigmaChemical Company (St Louis, MO, USA). The purity of these whey proteins was controlledby polyacrylamide gel electrophoresis according to Hillier (1976).

The milk samples used for bPlg determination were fresh raw milks (two milkings),collected between June and October 1993, from 14 herds {Normande and FrançaiseFrisonne Pie Noire breeds) from Normandy, France. They were maintained at 4°C until thebPlg assay was performed on the day following their collection.

Preparation of MS-Anti-bPIg ConjugatePolyfunctional and hydrophilic MS (dry diameter = 300 ± 12 nm), produced as previouslyreported (Marchand et al, 1992), were kindly supplied by Sanofi Diagnostics Pasteur(Marnes la Coquette, France). The IgG fraction (1 g 1"1) of anti-bPlg rabbit antiserum wasbound for 18 h at 4°C, in 0.1 M-borate buffer, pH9.0, 0.3 M-NaCl) on to these MS(10 g 1 ~ '), through the formation of covalent imine bonds between MS aldehyde groups andimmunoglobulin primary amino groups (Montagne et al, 1992b). After blocking the

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PLASMINOGEN NEPHELOMETRIC IMMUNOASSAY 325

TABLE 1. Procedure for immunoassay of milk bPlg

Step

(1)

(2)

Reagent

MS-anti-bPlg conjugateFive serial dilutions of

standard or treated milksample

Nephelometry buffer

Anti-bPlg antiserumNephelometry buffer

Reactionvolume

(pl)

4030

100

Reagent dilutionor concentration

in reaction cuvette

80 mg 1 " '0.0720-O.0045 mg 1"1

1/200

Incubation for 30 min30

1001/400

Nephelometric measurement after 60 min

unreacted aldehyde groups on MS with a 0.12M-2-amino-ethanol solution at pH9.0 (for2 h, at room temperature), the MS-anti-bPIg conjugate was recovered by centrifugation(10 000 X g for 1 h at 4°C) on a discontinuous sucrose gradient (200/800 g 1" ') and storedin 0.1 M-borate buffer, pH 9.0, 0.03 M-NaCl.

Immunonephelometric Study of MS-Anti-bPlg ConjugateAll immunonephelometric studies were performed in milk nephelometry buffer: 0.05 M-bo-rate buffer, pH 8.0, containing 1.5 niM-ethylene diamine tetraacetic acid (EDTA)-Na2,30mMNaN3, Triton X-100 (2g I"1) and polyethylene glycol 6000 (30g 1"1). Lightscattering measurements were carried out in disposable microcuvettes with the SanofiDiagnostics Pasteur nephelometer (Nephelia N600) after 1 h of incubation at room tempera-ture.

The immunonephelometric study of MS-anti-bPlg conjugate (80 mg 1 " ') was performedin a sandwich reaction involving free antibodies from anti-bPlg antiserum (400-folddiluted), with or without known concentrations (2.3-72.0 fig 1 ~ ') of purified bPlg and of themain whey proteins (a-La, /?-Lg, bSA and bGG).

The fidelity of the reaction observed with bPlg was studied by measuring high, middleand low concentrations (59, 31 and 15 /ig I"1) of bPlg 10 times in the same assay(within-run precision).

Measurement of bPlg in MilkBefore measuring bPlg, milk samples were treated to release bPlg from CN and to eliminateCN. The dissociation of CN-bound bPlg was performed by e-ACA treatment (Politis et al,1993) as follows. First, 1 ml of fresh, raw milk was mixed with 25 //I of a 2 M-e-ACAsolution, then incubated for 2 h at room temperature. Then, 30 /d of 5.8 M-acetic acid wereadded. After 10 min at 37°C, 30 /il of 3.3 M-sodium acetate and 0.5 ml of trichloro-trifluoroethane were added. After 3 min at 37°C with vigorous shaking, the precipitated CNwere discarded by centrifugation at 2000 X g and the supernatant containing free bPlg wasrecovered.

The residual concentrations of a-, ß- and K-CN in treated milk samples were measuredby microparticle-enhanced nephelometric immunoassays as previously reported (Collard-Bovy et al, 1991; El Bari et al, 1991).

The microparticle-enhanced nephelometric immunoassay of bPlg in treated milk sampleswas carried out according to the procedure described in Table 1. All dilutions anddispensations were performed in milk nephelometry buffer with a Hamilton (Bonaduz,Switzerland) dilutor. Serial dilutions of a frozen standard preparation, containing purifiedbPlg (3.60mg I"1), a-La (1.20g T 1 ) , J?-Lg (2.70g I"1) and bSA (0.25 g 1"1) in theJenness and Koops (1962) salt solution, were used instead of milk samples to establish thecalibration curve.

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326 E. MARCHAL ET AL

Evaluation of the microparticle-enhanced nephelometric immunoassay of bPlg in milk,including the evaluation of the treatment of milk sample by e-ACA, was performed usingreproducibility and recovery studies. The reproducibility of the calibration curve wasassessed in five successive assays with different serial dilutions (1/50-1/800) of standardpreparation. The analytical recovery of bPlg added to standard preparation was performedby measuring bPlg from the standard preparation diluted four-fold (0.9 mg 1 ~ ') andoverloaded by five known amounts of purified bPlg (0.4, 0.8, 1.6, 3.2 and 6.4 mg I"1), thenassayed as milk samples. For the parameters obtained by linear regression analysis, the nullhypothesis Ho (intercept = 0 and slope =1) versus the alternative hypothesis Hi (inter-cept = 0 and slope = 1) were tested by F-(Fisher) and /-(Student) tests, respectively.

The reproducibility of the treatment of milk by 8-ACA was assessed by measuring bPlglevels in 10 treated samples issued from the same milk (between-run precision). Assessmentof the analytical recovery of bPlg during e-ACA treatment was performed by comparing theresults of bPlg determination in a fresh raw milk sample overloaded with four knownamounts of purified bPlg (2.6, 4.1. 5.6 and 8.6 mg 1" '), then treated by e-ACA, with thoseof bPlg determination in the treated milk sample overloaded with the same amounts of bPlg.The slopes obtained by linear regression analysis of the two overloading assays werecompared by using Student's /-test. For total recovery, coupling all results obtained in thesetwo assays, the slope and intercept were tested by t- and F-tests as reported above.

The microparticle-enhanced nephelometric immunoassay was applied to the determi-nation of the concentration of bPlg in 106 herd milk samples following the procedure givenin Table 1. Comparisons of bPlg milk levels between cow breeds and according to themonth of gathering were performed using Student's /-tests.

RESULTS

Characterization of bPlg and Anti-bPIg AntiserumAs previously reported (Haïssat et al, 1994), sodium dodecyl sulphate polyacrylamide gelelectrophoresis of bPlg preparations showed a single band at about 90kDa. Doubleimmunodiffusion confirmed the reactivity of rabbit anti-bPlg antiserum with bPlg, across-reactivity with bPl and the absence of reaction against whole CN and the main wheyproteins.

Immunonephclometric Study of MS-Anti-bPlg Conjugate AgglutinationFor bPlg concentrations ranging from 2.3 to 72 fig I"1 in the reaction mixture (Figure 1),the sandwich reaction of bPlg between MS-anti-bPlg conjugate and free antibodies fromanti-bPlg antiserum produced microparticle clusters scattering light, and allowed thenephelometric measurement of bPlg. In the same concentration range, no reaction wasdetected with a-La, ß-Lg, bSA and bGG.

A bPlg concentration of 4 /ig 1 " ' was significantly measurable in the reaction mixture,yielding an intensity of light scattered 3 X SD higher than the mean of 30 determinationsof the intensity of light scattered in the absence of bPlg. The fidelity of the measurementof bPlg was assessed by within-run precision studies at three concentrations of bPlg: n = 10;mean = 59.3/ig I"1, SD = 2.1/ig I"1, coefficient of variation (CV) = 3.5%; mean =31.0/ig I"1, SD = 0.9/Jg I"1, CV = 2.9%; and mean=14.6/jg I"1, SD = 0.6/ig P 1 ,CV = 4.1%.

Measurement of bPlg in MilkThe calibration range for the bPlg assay in milk (Figure 2) extended from 0.9 to14.4 mg I"1, with a good reproducibility (CV ranging from 1 to 4% in five consecutiveassays) for treated milk samples diluted 200-fold in the reaction cuvette. A linear profile(recovered bPlg = 0.16mg I " 1 + 0.94 added bPlg, n = 5, r = 0.999) was obtained for

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PLASMINOGEN NEPHELOMETRIC IMMUNOASSAY 327

400 -r

350 - •

300 -

£250

| 200(DÜ

CO

£ 150O)

100 - •

50 -

-I—I—I I I I I I H 1—I M i l l

10

Protein Concentration (pg/l)

10C

FIG. 1. Variation of light scattering in sandwich assays, using MS-anti-bPlg conjugate (80 mg 1 ') and400-fold diluted anti-bPlg antiserum, as a function of the concentration in the reaction mixture ofpurified bPlg (•) and of various whey proteins: D, bGG; O, bSA, ; A, a-La; O, /?-Lg; and • , lightscattered by the MS-anti-bPlg conjugate alone. The reaction time was 1 h.

analytical recovery of bPlg added in the standard preparation, with a mean percentage ofrecovery of 101% (SD = 5%); the slope and the intercept of the recovery curve were notsignificantly different (P > 0.05) from 1 and 0 respectively.

After e-ACA treatment of milk to release bPlg from CN and elimination of CN, theresidual concentration of whole CN in treated samples was lower than 0.25 g 1"1, withoutinterference on the bPlg assay. The reproducibility of this milk treatment was assessed bybPlg determination in a between-run precision study: «=10 , mean = 3.1 mg I"1,SD = 0.1 mg 1" ', CV = 1.9%. The analytical recovery of bPlg after milk treatment (Figure3) was linear for concentrations ranging from 2.6 to 8.6 mg I"1, (n = 8, mean percentageof recovery = 95%, SD = 6%, r = 0.994). The slopes of the two overloading curves (0.91for overloading of fresh raw milk and 1.00 for overloading of treated milk) were notsignificantly different (P>0.05). The slope (0.96) and the intercept ( - 0.09 mg I"1) of thetotal recovery including results of the two overloading assays were not significantlydifferent (P > 0.05) from 1 and 0 respectively.

Application of the microparticle-enhanced nephelometric immunoassay of bPlg to106 herd milk samples gave the following global results: mean = 2.0mg I"1, mini-mum =1.1 mg I"1, maximum = 4.9 mg I"1. No significant difference at P = 0.05 wasobserved between milks from Normande and Française Frisonne Pie Noire breeds. Generalstatistics of bPlg concentrations measured between June and October 1993 are given inTable 2. The levels obtained in June, July and August were significantly higher (P< 0.001)than those measured in September and October.

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328 E. MARCHAL ETAL.

400 T

300 -

Ien

I 200(Suw£

100 -

-4- -4- +0.9 1.8 3.6 7.2

bPIg Concentration in milk (mg/1)

14.4

FIG. 2. Calibration curve for the microparticle-enhanced nephelometric immunoassay of milk bPIg. Lightscattered is shown as a function of the bPIg concentration in standard preparation. ±, mean ± 2 SDin five successive assays; • , light scattered in the absence of bPIg.

DISCUSSION

The microparticle-enhanced nephelometric immunoassay of bPIg developed in this studyfollowed a two-step procedure: first, capture of bPIg by MS-anti-bPlg conjugate, thenreaction of free antibodies from an anti-bPlg antiserum with the bPIg trapped by theconjugate (Montagne et al, 1992b). Such a sandwich reaction produced conjugate clustersthat were able to scatter light, and allowed the nephelometric quantification of bPIg. Theassay was found to be sensitive (4 pg 1 " ' of bPIg significantly detected), accurate (linearrecovery of bPIg in the overloading assay) and reproducible (CV in the precision studylower than 5%). The main whey proteins (oe-La, ß-hg, bSA and bGG) did not interfere inthe reaction.

Because bPIg is bound to CN (De Rham & Andrews, 1982; Korycka-Dahl et al, 1983;Richardson & Elston, 1984), milk samples must be treated to release bPIg from CN and todiscard CN before the assay of bPIg. Dissociation of bPIg from CN was performed bye-ACA treatment, and CN were precipitated at low pH. Such a milk treatment appeared tobe reproducible (CV in between-run assay lower than 2%); it allowed a nearly totalelimination of CN (higher than 99%) and an accurate release of bPIg (similar recoveries ofbPIg in overloading assays performed before and after milk treatment). A standardpreparation, containing whey proteins in addition of bPIg and the salt solution of Jennessand Koops (1962), was used to build a calibration curve under the same physico-chemicaland biochemical conditions to those of the treated milk samples assayed.

The anti-bPlg antiserum used in the sandwich assay of bPIg, to prepare MS-anti-bPlgconjugate and as reagent containing free anti-bPlg antibodies, reacted against bPIg and bPl.The immunoassay described therefore measures the concentration of both bPIg and activebPl, and thus allows an evaluation of the active and potential endogenous proteolytic system

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PLASMINOGEN NEPHELOMETRIC IMMUNOASSAY 329

10 -r

8 -

o

t 6ooU

Q. 4.a

13V)

S 2

4- + +2 4 6 8

Theoretical bPIg Concentration (mg/l)

10

FIG. 3. Analytical recovery of bPIg. D and —, milk overloaded with purified bPIg then treated by e-ACA;O and milk sample treated by e-ACA then overloaded with purified bPIg; [—], linear regressionfor total recovery

of bovine milk. The concentrations assayed in herd milks with this microparticle-enhancednephelometric immunoassay ranged from 1.1 to 4.9 mg I"1. They were close to thosepreviously reported using another immunological method (2.6-4.8 mg I"1 by Collin et al,1988) but were often slightly higher than the levels obtained by the spectrophotometricmethods based on the measurement of bPl activity (0.2-2.8 mg I"1; Richardson & Pearce,1981; Korycka-Dahl et al, 1983; Rollema et al, 1983; Benslimane et al, 1990). Month-by-month comparison of the results of this study indicated a possible seasonal variation of thebPIg concentration in milk. Such a variation has been already observed, with an increaseof bPl activity in the summer (Benslimane et al, 1990). However, this seasonal variationcould also be the consequence of the stage of lactation, milk bPIg levels reaching 12 mg 1~ 'and even 30 mg I"1 (Kaartinen & Pyörälä, 1989; Dupont et al, 1995) during early lactation.

Already described as a method of quantification of the main milk proteins, thismicroparticle-enhanced nephelometric immunoassay also appears to be an appropriate

TABLE 2. Statistics: bPIg concentration (mg 1 ') measured in 106 herdmilks between June and October 1993

Month

JuneJulyAugustSeptemberOctober

n

1225132927

Minimum

2.11.51.91.21.1

Maximum

4.93.42.42.42.1

Mean

2.92.32.21.8°1.6"

SD

0.70.60.20.20.2

"Significantly different (P< 0.001) compared with the three othermonths.

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330 E. MARCHAL ETAL.

method to measure milk bPlg levels, thus allowing evaluation of potential endogenousproteolysis in bovine milk.

ACKNOWLEDGEMENTS

P. Montagne is a research engineer at the Institut National de la Santé et de la RechercheMédicale. M. L. Cuillière is a study engineer at the Centre National de la RechercheScientifique.

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