Pertanika J. Trap. Agric. Sci. 19(2/3): 95-102 (1996)ISSN: 0126-6128

© Penerbit Universiti Pertanian Malaysia

A Competitive ELISA for Quantification of Protein A in Culture Mediunt

ABDUL MANAF ALII· SHARIFAH TAHIR,I BAHARUDDIN ABDUL GHANI,!UNGKU CHULAN2 and ISMAIL B. AHMAD3

1Department of Biotechnology2Department of Veterinary Pathology and Microbiology

Universiti Pertanian Malaysia43400 Serdang, Selangor, Malaysia

3Department of MicrobiologyUniversiti Kebangsaan Malaysia

43600 Bangi, Selangor, Malaysia

Keywords: protein A, cODlpetitive ELISA, IgG, Staphylococcus aureus

ABSTRAK

Satu asai imunterjerap enzim berangkai secara bertanding untuk pengukuran protin A yang dihasilkan olehStaphylococcus aureus A676 (rintang terhadap metisilin) adalah berasaskan kepada pertandingan untuk terikatkepada molikul IgG amab antara protin A yang ditandakan dengan enzim fosfatase beralkali dan protin A yangtidak bertanda. Kepekatan IgG yang optima bagi tujuan adalah diantara 2 dan 4 f..lgfml. Masa pengeraman yangoptima untuk pembentukan wama dengan penggunaan substrat p-nitrofenolfostat adalah diantara 20 dan 30 minit.Kepekatan protin A'yang terendah dapat diukur dengan menggunakan asai imunterjarap enzim berangkai secarabertandingyang telah dioptimakan adalah 20 ngfml dan kepekatan maxima adalah 2 f..lgfml. Jumlah protin Ayangdihasilkan didalam medium infusi otak-hati telah bertambah secara eksponen ketika fasa log pertumbuhan sel danmencecah kepekatan maksima pada of 22.5 f..lgfml selepas pengkulturan selama 15 jam.

ABSTRACT

A competztwe en{Yme-linked immunosorbent assay (ELISA) for quantification of protein A produced byStaphylococcus aureus A676 (a methicillin-resistant strain) was based on competitive binding to rabbit IgGmolecules between alkaline phosphatase-labelled protein A and unlabelled protein A. The optimum IgG concentrationrequired for coating was 2-4 f..lgfml. The optimum incubation time for colour development using (p-nitrophenolphosphate substrate was 20-30 min. The lowest protein A concentration that could be measured using the optimizedcompetitive ELISA was 20 ngfml, and the maximum 2 f..lgfml. The amount ofprotein A produced in brain-heartinfusion medium increased exponentially during log phase ofcell growth, reaching a maximum concentration of22.5f..lgfml after 15 h cultivation.

INTRODUCTION

Protein A is a 42 kDA polypeptide proteinproduced by most Staphylococcus aureusstrains (Lind et al. 1970; Hjelm et al.1972). This protein is capable of bindingto human immunoglobulin and of formingprecipitin lines in gel diffusion serologicaltest (Lofkvist and Sjoquist 1962). Protein Ahas four homologous binding sites for Fc

*author to whom correspondence should be addressed

receptors; each site consists of approxi­mately 60 amino acid residues. Thesebinding sites are present at the N-termi­nus, which does not bind to the peptido­glycan of the S. aureus cell wall (Sjodahl1977). However, due to the structuralconfiguration only two binding sites canbind simultaneously to the complementaryreceptor sites (Langone et al. 1978).

ABDUL MANAF ALI, SHARIFAH TAHIR, BAHARUDDIN ABDUL GHANI, UNGKU CHULAN, ISMAIL B. AHMAD

Protein A reacts mainly with the Fcreceptor of IgG (Forsgen' and Sjoquist1969). Among IgG subclasses, IgGt, IgG2

and IgG4 have a high affinity to protein A,but IgG3 does not (Kronvall and Frommel1970; Arkerst et al. 1974). Other immuno­globulin classes to which protein A bindsare IgA, IgM and polyclonal IgE (Here­mans 1974; Harboe and FoIling 1974;Brunda et al. 1977). Due to their highbinding affinity towards the Fc region ofhuman, rabbit and guinea pig immunoglo­bulins, protein A and staphylococci-bearingprotein A have been used in a wide varietyof immunoassays such as agglutination,radioimmunoassay (O'Keefe and Bennett1980; Richman et al. 1982) and enzyme­linked-immunosorbent assay (ELISA)(Engvall 1976; Buchanan et al. 1981;Ahmad et al. 1988; Zainal-Abidin et al.1992). Protein A has also been used onagarose as ligand in affinity chromatogra­phy for purification of immunoglobulins(Goding 1978; Jaton et al. 1979; Gentile etal. 1984).

Langone (1982) reported that morethan 95% of S. aureus strains produceprotein A in varying amounts. Thisprotein was reported as a cell wallconstituent, which is covalently linked tothe peptidoglycan (Sjoquist et al. 1972).Some S. aureus strains are capable ofsecreting protein A into culture medium(Forsgen and Sjoquist 1969; Masuda et al.1975). For example, the strain Cowan Isecretes 30% of protein A, and methicillir:t­resistant strains secrete almost all protein Asynthesized by the cell into the culturemedium (Forsgen and Sjoquist 1969;Masuda et al. 1975). The-cell wall boundprotein A can be isolated by using enzymessuch as lysozyme, DNAase and lysostaphin(Yoshida et al. 1963; Sjoquist et al. 1972).However, the yields produced by lysozymeand DNAase are variable and heterogeneouscompared to lysostaphin (Bjork et al. 1972).

This paper describes a competitiveELISA technique for quantification ofextracellular protein A in culture mediumproduced by a methicillin-resistant strain ofStaphylococcus aureus, strain A676. Competi­tive ELISA is a heterogeneous enzymeimmunoassay where the antigen-antibodycomplexes physically sep'arate an antibodyfrom free antigen using a solid phase system(Engvall 1976). This assay system is veryspecific and sensitive, and can be used formeasuring either ant-igen or antibody. Acompetitive ELISA system, which is basedon a competitive binding between enzyme­labelled protein A and unlabelled protein Awith human IgG, was first described byGoding (1978). Human or rabbit IgG wasselected because of its high binding affinityto the Fc region by protein A throughextensive hydrophorbic interaction to thebinding sites CH2 and CH3 at the constantregions of heavy-chain (Endresen and Grov1978; Zikan 1980; Gentile et al. 1984). ThisELISA alkaline phosphatase system waschosen to be conjugated with protein Abecause it is stable and the activity has alinear relationship with substrate concen­trate compared with horseradish peroxidaseenzyme (Voller et al. 1976).

MATERIALS AND METHODS

Cultivation of Bacteria and Source of Protein A

Staphylococcus aureus strain A676, a methicil­lin-resistant strain, was provided by Prof.C. Brown of Heriot-Watt University,Edinburgh, UK. The bacterium, isolatedfrom a single colony, was sub-cultured onnutrient agar containing 0.2% methicillin(Sigma, St. Louis, MO) at 37°C for 24 h,and then transferred to 10 ml of nutrientbroth in a 100-ml flask for 10 h at 37°Cwith continuous shaking at 150 rpm(Centromat, B. Braun, Germany). Theculture was centrifuged at 3000 rpm for10 min and the pellet was resuspended inphosphate-buffered saline (pH 7.4) and

96 PERTANIKA J. TRap. AGRIC. SCI. VOL. 19 NO. 2/3, 1996

A COMPETITIVE ELISA FOR QUANTIFICATION OF PROTEIN A IN CULTURE MEDIUM

optical density (OD) adjusted to 0.6 at 600nm. The brain-heart infusion medium (pH7.4) was inoculated with the bacterialsuspension at a final concentration of 1%(v/v) and incubated at 37°C in a shakingwater bath at 150 rpm. A 5-ml sample wasremoved at 2-h intervals and centrifuged at10,000 xg for 5 min.

Preparations of IgG and Purification of IgGIgG was purified according to the methodof Clark and Adam (1977). Blood fromwhite New Zealand rabbits was allowed toclot at room temperature for 60 min andkept overnight at 4°C. Serum was sepa­rated from blood cells by centrifugation at3000 rpm for 10 min. An equal volume of40% saturated ammonium sulphate wasadded and the mixture was again centri­fuged at 2000 rpm for 10 min. Theprecipitate was dissolved in 5 mM phos­phate buffer (pH 7.4) and dialysed withthree changes of the same buffer. Immuno­globulin G in serum was chromatographedon DEAE-cellulose which was pre-swollenin 5 mM phosphate buffer (pH 7.4) andpacked into a column (1.6 x 30 cm) andequilibrated with 1 1 of the same buffer.One m of partially purified IgG wasapplied and eluted with a gradient ofincreasing ionic strength of phosphatebuffer (5-50 mM). Fractions from the firstpeak were collected and pooled. Theconcentration was determined at 280 nmand stored at -20°C.

Competitive ELISA (CELISA)Optimization of the ELISA method wasdeveloped with respect to IgG concentra­tion and incubation time for colour devel­opment. To determine the optimumconcentration of IgG, solutions of variousconcentrations (1, 2, 4, 6 and 8 ~g/ml) incarbonate buffer (pH 9.6) were dispensedat 150 ~l per well of a 96-well microtitreplate (Nunc). The plate was incubated

overnight at 4°C and then washed threetimes with PBS containing 0.05% Tween 20(Merck) PBS-T). The wells were blockedwith 150 Jll of 3% bovine serum albumin(BSA) in PBS and incubated at 37°C for 1h after a subsequent washing with PBS-To

For the assay, 200 ~l of unlabelledprotein A (Sigma) at various concentra­tions (0.005 - 4 ~g/ml) in PBS or BHIBcomplex medium (Oxoid) with the addi­tion of 200 ~l protein A alkaline phosphate(Sigma) at a concentration of 1 mg/ml wasused. The plate was incubated at 37°C for 2h and washed three times with PBS-ToFresh para-nitrophenyl phosphate (Sigma)substrate prepared at a concentration of 1mg/ml was added to each well and kept inthe dark for 15, 20, 25 or 30 min. Thereaction was stopped by the addition of 50~l of 3 M NaOH. The OD value was readusing an ELISA reader (Bio-Tek Instru­ments, USA) at A = 405 nm. The standardcurve was established by plotting the ODvalues at Y axis and protein A at X axis.The concentration of samples was thenestimated by using the Kinetic-Calc pro­gram on a computer linked to the ELISAreader (Bio-Tek Instruments, USA). Forlogit-log plot, the logit values for Y axiswere calculated by using the formula: Logit[(oDmaximum) /oDmaximum-oDstandardor sample)], versus log standard protein Ain X axis.

RESULTS AND DISCUSSION

The optimum concentration of rabbit IgGat which unlabelled protein A effectivelycompeted with the enzyme-labelled proteinA was 2-4 ~g/ml (Fig. 1). The binding ofprotein A-alkaline phosphate to the IgGmolecules adsorbed to the wells wasreduced when the concentration of unla­belled protein A in the standard or sampleswas increased. The absorbance values ofalkaline-phosphate-protein A bound to theIgG were measured 20 min after additions

PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996 97

ABDUL MANAF ALI, SHARIFAH TAHIR, BAHARUDDIN ABDUL GHANI, UNGKU CHULAN, ISMAIL B. AHMAD

4.0

3.0 -0-- 1 Ilg/ml

-0-- 2 Ilg/ml

E-A- 4 Ilg/ml

c:I/)0 2.0:!..c0

1.0

2.01.81.51.21.00.80.50.20.0 -t------.---.,...--...-..------..-..-_.._--...--,....-.....---,--.....,.-.-I

0.0

Protein A (J.1g/ml)

Fig. 1. Standard curves ofprotein A (Wells were coated with IgG at 1, 2 or 4 fJ.g/mland the competitive ELISA performed as described in the text)

4.0

--0-- 15 min

3.0 -.- 20 min

--0-- 25 min

E --A- 30 min

c:I/) 2.0'~c0

1.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Protein A (Ilg/ml)

Fig. 2. Determination of optimum incubation time with IgG coated at 2 fJ.g/ml (TheIgG at 2 fJ.g/ml was coated on the microtitre plate and the competitive ELISA

performed; absorbance was measured every 5 min)

of the substrate. The absorbance at zeroconcentration of protein A was increasedwith increasing concentrations of IgGcoated. The significant reduction ofabsorbance values was observed with theincrease in unlabelled protein A for thewells coated with 2 and 4 Ilg/ml IgG. Athigher IgG concentrations (4 Ilg/ml or

above), no competition occurred becausethe binding sites for protein A were notlimited. Conversely, at IgG concentrationslower than 1 Ilg/ml the number ofprotein Areceptors was too small to allow anycompetition to occur between alkalinephosphatase-protein A and the unlabelledprotein A molecules (Goding 1978).

98 PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996

A COMPETITIVE ELISA FOR QUANTIFICATION OF PROTEIN A IN CULTURE MEDIUM

o-4 -3 -2 -1

In (Protein A concentration)-5

-5 L-_--&__-I....__...L.-_---IIo.--_--"'- ..&...-__•

-6

4----------------------,3

:0 2c:::Jo.0

~ 0 I---------"~~------------o:e -1c:::- -20

0o..J

Fig. 3. Logit-log plot of standard curve using IgG coating at 2 Jlg/ml with a 25-minsubstrate incubation time

Fig. 2 shows the standard curve, whichwas plotted using absorbance values takenat time intervals of 15, 20 and 30 min afterthe addition of substrate. At 20 and 30 minthe absorbance values were inverselyproportional to a wide range of protein Aconcentrations. Fig. 3 shows a linearstandard curve using IgG coating at 2 J.1g/ml with incubation time of 25 mintransformed into 10git-log plot. This plotallows approximation of sample concentra­tion in the region which is approachingsaturation (Peterman and Butler 1989).The minimum detectable limit of proteinA in this competitive ELISA system wasdetermined by constructing the standardcurve with a concentration range of 0-5 ng/ml using 4 J.1g/ml IgG coating. Fig. 4 showsthat the minimum detectable limit was 20ng/m!.

The production of protein A frommethicillin-resistant S. aureus A676 cul­tured in brain-heart infusion medium inthe shake flask was measured using thecompetitive ELISA as described above.The supernatant of samples was obtainedevery 2 h for the first 10 h and subsequently

every 10 h until 48 h of total incubationtime. The sample was then diluted 10 x inPBS before measuring the protein Aconcentration. Using this competitive ELI­SA measurement, absorbance of the proteinA standard and samples was performed inthe same 96-well plate. Fig. 5 showsconcentration of protein A in the mediummeasured for a period of 48 h usingcompetitive ELISA technique. The con­centration of protein A produced in thisexperimentwas correlated with increase incell number. The production increasedexponentially during log phase andreached maximum concentration of 22.5J.1g/ml after 15 h cultivation. Thus, thistechniques is successful in measuring pro­tein A concentration in S. aureus cultures.

ACKNOWLEDGEMENTS

This project was partly funded by IRPAGrant No. 1-07-05-003 from the Ministry ofScience, Technology and EnvironmentMalaysia and Japan International Co­operation Agency UICA) for the develop­ment of the Department of Biotechnology,Faculty of Food Science and Biotechnology,

PERTANIKA Jo TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996 99

ABDUL MANAF ALI, SHARIFAH TAHIR, BAHARUDDIN ABDUL GHANI, UNGKU CHULAN, ISMAIL B. AHMAD

4.00

Ec:It)0~

C-O

3.00

60.050.040.030.020.010.02.00 .t---..,...--....--.....--.....-..-.......-......-~

0.0

Protein A (ng/ml)

Fig. 4. Standard curve ofprotein A with a concentration range of 0-50 ng/ml usingIgG coating at 4 J1.g/ml. (The OD was measured 25 min after substrate was added)

. 30

-~25

. 20 ~Cl.:!:

15 «c:

'CD(5...

10 Cl.

-0-- Growth--- Protein A ·5

1.5Ec:00~.r:. 1.0 .i0

~

0.5

2.0.----------------

0.0 ·...-.--,.---.--r--....-.--y-------I. 0o 10 20 30 40 50

Incubation time (h)

Fig. 5. Growth ofS. aureus A676 in BHIB medium and concentration ofprotein Aproduced when cultured in shake flask at 37'Cfor 48 hours

Universiti Pertanian Malaysia. The authorswould also like to thank Prof. C. Brown forsupplying the culture.

REFERENCES

AHMAD, LB., A.M. ALI and S. FARIDAH. 1988.Percubaan pengesanan antigen PMV dalamsap kasar pasi dengan teknik ELISA. InProceedings of Tenth Malaysian MicrobiologySymposium, ed. LB. Ismail and J.A. Kadir, p.

88-95. Kuala Lumpur.

ARKERST, J., P. CHRISTENSEN, L. KjELLER andG. KRONVALL. 1974. A routine diagnostictest for IgA and IgM antibodies to rubellavirus adsorption of IgG with Staphylococcusaureus. Journal ofInfectious Diseases 130: 268-273.

BJORK, I., B. PETERSSON and J. SJOQUIST.1972. Some physicochemical properties ofprotein A from Staphylococcus aureus. EuropeanJournal of Biochemistry 29: 579-584.

100 PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3,1996

A COMPETITIVE ELISA FOR QUANTIFICATION OF PROTEIN A IN CULTURE MEDIUM

BRUNDA, M.J., P. MINDEN, T.R. SHARPTON,J.K. MCCLATCHY and R.S. FARR. 1977.Precipitation of radio1abelled antigen-anti­body complexes with protein A-coatingStaphylococcus aureus. Journal of Immunology199: 193-198.

BUCHANAN, R.E., M. KAMARCH and N.H.RUNDDLE. 1981. Development of a proteinA enzyme immunoassay for use in screeninghybridomas. Journal of Immunological Methods42: 179-186.

CLARK, M.F. and A.N. ADAM. 1977. Character­istics of the microplate method of enzyme­linked immunosorbent assay for the detectionof plant viruses. Journal of General Virology 34:475-483.

ENDRESEN, C. and A. GROV. 1978. Isolation ofenzymatically derived fragments ofguinea pigIgG and examination of their reactivityagainst staphylococcal protein A. Acta Patho­logica Scandinavia Section C 86: 193-198.

ENGVALL, E. 1976. Determination of antibodiesDNA by ELISA. Lancet 2: 1410-1414.

FORSGEN, A. and]. SJOQUIST. 1969. Protein Afrom Staphylococcus aureus VII. Physiologicaland immunological characterization. ActaPathologica et Microbiologica Scandinavia 75:466-480.

GENTILE, T.C., S.E. DIEKS and R.M. WATT.1984. Interaction of porcine immunoglobulinM with protein A of Staphylococcus aureus.Biochimica et Biophysica Acta 791: 102-111.

GODING, J .W. 1978. Use of staphylococcalprotein A as an immunologies reagent.Journal of Immunological Methods 20: 241-253.

HARBOE, M. and I. FOLLING. 1974. Recognitionof the two distinct groups 'of human IgM andIgA based on different binding to staphylo­cocci. Scandinavian Journal ofImmunology 3: 471­482.

HEREMANS, ].F. 1974. Immunoglobulin A. InThe Antigens, ed. M. Sela, Vol II p. 365. NewYork: Academic Press.

HJELM, H., K. HJELM and]. SJOQUIST. 1972.Protein A from Staphylococcus aureus. Itsisolation by affinity chromatography and itsuse as immunosorbent for isolation of im­munoglobulin. FEBS Letters 28: 73-76.

JATON, ].C., D.C. BRANDT and P. VASSALLI.1979. The isolation and characterization ofimmunoglobulin antibodies and their consti-

tuent polypeptide chains. In ImmunologicalMethods ed. 1. Lefkovits and B. Pernis, p. 43.New York: Academic Press.

KRO VALL, G and D. FROMMEL. 1970. Defini­tion of staphylococcal protein A reactivity forhuman immunoglobulin G fragments.Immunochemistry 7: 124-127.

LANGONE, J.J. 1982. Protein A of Staphylococcusaureus and related immunoglobulin receptorsproduced by streptococci and pneumonococ­ci. Advances in Immunology 32: 157-251.

LANGONE, J.J., M.D.P. BOYLE and T. BORSOS.1978. Studies in the interaction betweenprotein A and immunoglobulin G. II.Composition and activity of complexesformed between protein A and IgG.Immunology 121: 333-339.

LIND, 1., 1. LIVE and B. MANSA. 1970. Variationin staphylococcal protein A reactivity withgamma globulin to Staphylococcus aureus. ActaPathologica et Microbiologica Scandinavia B 78:673-677 .

LOFKVIST; T. and J. SJOQUIST. 1962. Chemicaland serological analysis of antigen prepara­tion from Staphylococcus aureus. Acta Pathologicaet Microbiologica Scandinavia 56: 295-304.

MASUDA, S., S. SAKURAI and 1. KONDA. 1975.Simple and effective method for selectingprotein-deficient mutants by cosedimentationwith sensitized sheep erythrocytes. Infectionsand Immunity 12: 245-251.

O'KEEFE, E. and V. BENNETT. 1980. Use ofimmunoglobulin loaded protein A-bearingstaphylococci as a primary solid-phase im­munosorbent in radioimmunoassay. Journal ofBiological Chemistry 255: 561-568.

PETERMAN, J.H. and J.E. BUTLER. 1989.Application of theoretical considerations tothe analysis of ELISA data. Biotechniques 7:604-614.

RICHMAN, D.D., R.H. CLEVELAND, M.N.OXMAN and K.M. JOHN. 1982. Staphylo­coccal protein A from S. aureus by sera ofdifferent animal species. Journal of Immunology128: 2300-2305.

SjODAHL,]. 1977. Structural studies on the fourreceptive Fc-binding regions in protein Afrom S. aureus. European Journal of Biochemistry78: 471-490.

SjOQUIST,]., J. MOVITZ, LB. JOHANSSON andH. HJELM. 1972. Localization of protein A in

PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996 101

ABDUL MANAF ALI, SHARIFAH TAHIR, BAHARUDDIN ABDUL GHANI, UNGKU CHULAN, ISMAIL B. AHMAD

bacteria. European Journal of Biochemistry 30:190-194.

VOLLER, A., A. BARTLETT, D.E. BIDWELL,M.F. CLARK and A.N. ADA. 1976. Thedetection of viruses by enzyme-linked immu­nosorbent assay (ELISA). Journal of GeneralVirology 33: 165-167.

YOSHIDA, A., S. MUDD and N.A. LENHART.1963. The common protein agglutination ofStaphylococcus aureus. II. Purification, chemicalcharacterization and serologic comparisonwith Jensen antigen. Journal of Immunology91: 777-782.

ZAINAL-ABIDIN, B.A.H., S.R.H. IDRIS, S.DOLAH and LB. AHMAD. 1992. Teknik Pai­ELISA untuk pengesanan antibodi terhadaproden. Sains Malaysiana 21: 133-142.

ZIKAN, J. 1980. Interaction of "Pig" Fabfragment with protein A from Staphylococcusaureus. Folio Microbiology 25: 246-253.

(Received 11 March 1994)

(Accepted 20 August 1996)

102 PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996