Enzyme-labelled antibody-avidin conjugates: New flexible and sensitive immunochemical reagents

JOURMLOF

Enzyme-labelled antibody-avidin conjugates: new flexible and sensitive immunochemical reagents R.P.M. van Gijlswijk a.*, D.J. van Gijlswijk-Janssen b, AK. Raap ‘, M.R. Daha b, H.J. Tanke a

-Abstract We have pmpaed avidin-labelled antibodies (‘shuttles’) with the aim of increasing the sensitivity of detecting mouse IgG ad human complement facton in ELISA tests and of detecting monoclonal antibodies and digoxigenin haptens (DIG) in bvbridizarion and immuwblot tnocedures. Avidin-D was coniugated to goat IgG anti-mouse IgG or to antidigoxigenin &bodies by tbiol/maIeimide fhemisuy. Conjugates of diff&&n molec& weight were obtined by Superdex 200 gel filtration. The avidin-D-IabeIIe.3 a.nt!+&ie~ were then incubated with biotinylated horseradish peroxidax or with biotinylated alkaline phosphatase. Such preformed enzyme-labelled complexes were sudequendy used in thevarious assays. A 5-g-fold imrez in sensitivity was found when the prefomxd enzyme-labellcd antibody-avidin-D complexes were cornpad to directly enzyme-labelled antibodies or antibody fragments.. Furthermore it was shown that EIJSA procedures employing digoxigenin-labelled poIyclonal antibodies detected by shuttle conjugates were approximately tive times more sensitive than biotinylated antibodies detected by avidin-biotin complexes (ABC methal). The greatest sensirivily was obtained using antibody-avidin comptexes which consisted of two IgG molecules and 4-6 z&din-D molecules.

1. 1ntroductiin Enzyme-labelled antibodies are frequently used for the auantitative detection of a&ens and swcific nucleic ;cids in macroscopic appIic&ns suchas the enzyme-linked immunosorbent assay @LISA) and different blotting procedures (King and Kochoumian, 1979; Lang et d., 1986). as well as in immunohistochemistry (Avmmeas, 1969; Heydemnn, 1979) and in situ hybridization for microscopic investigations of cells and tissues (Dirks et al., 198% Rasp et al.. 1995). The most commonly used enzymes are horseradish peroxidase (HRF’) and calf intestinal al-

kaline phosphatase (AP) which arr: detected quaotitatively by chromogenic. fluomgenic M chemihtminescent substtates

(Wilchek

h:any methods antibodies

and Bayer,

1990).

to prepare srable enzyme-labelled

employ

biftmctiontd

linkers

(King

antibody-avidin

and

Kochoumiao. 1979). Some methods use randomly intmdttced linkers in both enzyme and antibudy to form conjugates linked by a disulphide bond (King and Kochoomian. 1979) or by a thioester bond (Doncan

et ai., 1983). King and Kochoomian

and lmigawa were

(I9791

et al. (198.2) found that these methods

superior

to monofunctional

linkers

such

3s

sodium periodate or glotarakkhyde, although many commetcially available. high qtmlity conjugates are still prepared using the latter methods. Others make use of reagents fragments

that specifically

at the hinge

region.

hydrolyse

F(ab’),

The restthing

tbiol

grwp is then allowed to react with maleimidc or disulphide activated enzymes (Ishikawa, 1983). The advantage of the latter method is that the atigen binding site of the antibody is not altered. The methods

employing

maleimitk

activation

to form

tbioether bonds are considered favwrable (Ishikawa, 1983). Although the hinge method generally results in lower enzyme to antibody ratios it is appropriate for in ELISA prwedtues taattse of its lower backgmttnd colwr (Ishikawa, 1983). Avidin is a 66 kDa protein that contains four hindiie sites with a ven hieh tinitv for biotin

(K=

_ IO-" M-'

zyme-labelled

*..

.

(Green, 1990)). This m&es enavidin suitable as an immonochemical

reagent to detect biotin haptens in applications similar to those of enzyme-labebelled antibodies (Wilchek and Bayer. 1990). Usually saepfavidin ot chemically modified avidit-D is used to minimize backgmtmd staining of the avidin. because of the fact that both molecules have a lower isaelecttic point and do oat contain carbohydrate moieties (Hiller et al.. 1990). We have used &din-D as a link between the antibody and the enzyme of choice to prepare pre formed enzyme-avidin-emtituly complexes. First, the antibody

was labelled with multiple

body-avidin complexes suitable for lahelling with a variety of biothtylated reagents. We have optimized the coupling of &din-D to IgG and the lahelling of

avidin molecules

conjugates

with biotinylated

HRP or

AP. Tltese new shuttle conjugates were tested in ELISA and immunoblot omcedutes. Detection sensitivity was improved by a factor of 5-8 compared to the rest& obtained with antibodies directly labelled with etuymes. Futthetmore ELISA pmcedores employing

digoxigenin-labelled

polyclonal

antibodies

detected by shuttle conjugates were approximately live times more sensitive than h~otinylated antibodies detected by avidin-biotin cample~es (ABC method).

2.Mataiakattdmethods

Reagents used for the preparatioit of antibodyavidin-D and antibody-pemxidase complexes were: Hydmxyl

amint.

hydrochloride

(HONH,

HCl),

SMCC. SATA HRP, N-ethyhnakimide, Ellmano’s reagent, BCA tugem (all Pierce) and avidi-D (Vex-

tori Antibodies used for conjugation wete aftinity pttritied goat IgG ant-mouse I&i (H +L, Pierce). Affinity putitied sheep IgG anlidigoxigenin (DIG)

..

was kindlv movided hv Boetineer Mannheim. The goat IgG anti-DIG an&diis were obtained by immunizing a goat with digoxigenir-labelkd chicken albumin. Goat IgG was isolated using ammonium sulphate precipitation and DEAE chromato~y (cnmd, 1993). The total Igc fraction war used for conjugation

Goat IgG anti-DIG

war dialysed

for 16 h against

PBS md adjusted to a concentration of 9 mg/tnl. To I ml IgG 6. IO M 25 $30 tnM SMCC in DMSO

by randomly intmduced thioether bonds. The complexes were sobsqoently labelled by means of aftinity chemirtty with biotinyla!ed enzymes. Since avidin has foot biotin binding sites one avidin can be labelled with multiple enzymes. Futtbemtore. an eas-

were added and the t-e-ction allowed to ptuceed for 4 b at 4’C. Umeacted SMCC was removed using Centricon filten hmicod with 50 mM sodium ohosuhate OH 6.8. 0.1 M MaC1 ami 5 mM EDTA r. (buffer pH’6.8) as at eloettt. following the iMmtc-

ily petfomted

tions of the manufaetorer.

tiity

chemistry

step makes the anti-

Makimide

activated

IgG

was adjusted to a concentration of 3 mg/ml in buffer pH 6.8. To 10 mg HRP in 1 ml 50 mM sodium phosphate pH 8.0. 0.1 M N&l and 5 mM EDTA (buffer pH 8.0). 7 pl SATA (50 mg/ml in DMSO) were added and the reaction allowed 80 proceed for 90 min at 3O’C. Umeacted SATA was removed using Cent% con 30 filters with 50 mM sodium phosphare pH 7.5. 0. I M NaCl and 5 mM EDTA (buffer pH 7.5) as an eluenr SATA-aeated HRP was adjusted to a coneent&on of 4 mg/ml in buffer pH 7.5. I h prior to incubation with maleimide activated IgG the SATAtreated HRP was iocobated at 3PC with l/IO volume 35 g/l NH,OH.HCl in buffer pH 7.5. The thiol: HRP mtio was 1.3 as determined by tifration with ElImann’s reagent to calculate thiol concenuations and BCA reagent with untreated HRP as a standard lo calculate HRP concenbations (Riddles et al., 1983). For each mg maleimide activated IgG (0.33 ml). 0.67 mg SATA/HONH,-mated HRP (0.18 ml) was added. The reaction was allowed u) prowd for 16 h at 4°C and subsequently quenched by adding l/100 vol. of 0.1 M N-ethyhnaleimide in DMSO.

Goat I&i an&DIG was dialysed against PBS and adjusted to a concentration of 9 mg/ml. To I ml IgG, 4. 10 or 25 ~130 mM SMCC in DMSO were adde&thewaaiontintewas4hat4”C.Umeacted SMCC was removed using C’en~icoo30 filters with pH 6.8 buffer as an elumt Mabzimide activated IgG was adjusted to a concentration of 3 mg/ml in pH 6.8 buffer. To 10 mg avidin-D in 1 ml buffer pH 8.0, 14 pl oc 5 pl SATA (25 mg/ml in DMSO) were added and incubated for 90 min at UPC. Unreacted SATA was removed wing Centicon 30 filters with pH 7.5 buffer as an elocnL SATA-treated avid&D was adjosfedtoa wwnUation of 4 mg/ml in pH 1.5 bat%. I h prior to incobation with maleimide activated IgG the SATA-treated avidin-D was incubated a 3pC with l/IO volume 35 g/l NH,OH HCl in pH 7.5 buffer. The hiol:avidin-D ratio was either 5.; oi 2.1 wilh 14 and 5 1.r1SATA ~spmively. Avidin- L) ancentioos were measured with BCA magem using mmeated avidm-D as a stand&.

FM each mg maleimide activated IgG (0.33 ml), 1.0 mg SATAjHONH,-mated avidin-D (0.28 ml) was added. The reaction was allowed to proceed for 16 h L 4Y and subsequently quenched by adding l/100 vol. of 0.1 M N-etbyhnaleimide in DMSO. Both HRP and avidin-D conjugations were performed starting with either I, 10 or 15 mg IgG. 2.1.4. Conjugarion of &din-D to goat IgG mouse IgG and sheep IgG anti-DIG antibodies

an&

Antibodies were dissolved in or dialyzed against 10 mM phosphate, 0.25 M NaCI, pH 7.6 at 2.3 mg/ml. To 1.0 ml either 5, 10 or 20 gl 12 mM SMCC in DMSO were added and the reaction was allowed to proceed for I h at 30°C. Unreacted SMCC was removed using gel filtration over GF5 columns (Pierce) with pH 6.8 buffer as &em. Fractions containing maleimide activated IgG were pookd and adjusted 10 a concentration of 1.5 mg/ml in buffer pH 6.8. To 5 mg avidin-D in 0.5 ml pH 7.5 buffer, 7 pl SATA (25 mg/ml in DMSO) were added and incubated for 90 min af 30°C. Unreacted SATA was removed using gel filtration with pH 7.5 buffer as eluent. SATA--eated &din-D was adjusted to a concentration of 5 mg/ml in pH 7.5 buffer. 1 h prior to incubation with maleimide activated IgG the SATA-treated avidin-D was incubated at 37°C with l/lOvolume 35 g/l Nli,OH HCl in pH 7.5 buffer. Thz thiol:avidin-D ratio was 5.1. For each mg maleimide activated IgG (0.67 ml), either 1.2 mg (0.24 ml) or 2.5 mg (0.5 ml) SATA/HONH,-treated avid&D were ad&d. The reaction conkoxd for 2 h at 3PC and was stopped by adding l/SO vol. of 0.1 M Nethylmaleimide in DMSO. All conjugations were performed starting with 0.5-2 mg IgG. 2.15. PuriJicarion D-labelled IgC

and analyses

of HRP-

and ouidin-

Gel filtration by Superdex 2Otl was used to purify labelled IgGs from unlabelled IgG. avidin-D or HRP and to separate formed conjugates on the basis of their molecular weight Either a 24 ml column (Soperdex 200 HR 10/30. Pharmacia Biotech) operated at a flow 0.5 ml/min or a 320 ml column (Superdex 200 HiLoad 26/c%, Phwmacia Biotech) &ted at a flow of I ml/min was used PBS with 2 mM EDTA

was 4°C.

used as eluent and the conjugates were stored at Detection

was

performed

by

measuring

the

absorbance at 280 nm or by determining the protein concentration of the fractions obtained using the BCA reagent.

HRP-labelled

anti-DIG

antibodies

were

also tested

in a %-well plate coated with DIGlabelled rabbit IgG; HRP development was achieved by the addition of 2 mM ABTS (Sigma) in 0.1 M sodium acetate pH 4.2.0.01% H,O, and absorbance was measured at 415 not. Fractions were pooled and only the goat IgG anti-DIG

conjugates

were concen-

trated to approximately one quarter of the original volume using size exclusion filters (Amicon). Standard SDS-PAGE using a 5% gel war performed 6ambrook et al., 1989). Proteins were stained with Coomassie blue &mbrook et af.. 1989) or, in the case of avidii-D-labelled goat IgG anti-mouse IgG,

blotted

onto niuocelhdose

stained with an

and

AP-labelled rabbit IgG anti-goat IgG (Sigma. l/looO) or with an AP-labelled goat IgG antiavid&D (Vector, I/500). AP-labelled antibodies were diluted in PBS/OS% blocking reagent (Boehringer)

to reduce

Tween

20 was

were detected

non-specific used

binding.

to wash

by NBT/BClP

PBS/O.Z%

the filter. reagents

2.2. ELBA and dot-blot procedures ~nribody-avidin-hi~z;nylafed enzyme

AP labels

(Promega).

with preformed complexes

Bio:Inybtted AP (I otg/mI. 2OW U/ml) and biotinylated HRP (5 mg/mI, 3500 U/ml) were purchased from Boehringer. In some cases HRP (Pierce) was labelled with biotin-LC NHS ester (Pierce) as follows: IO mg HRP (+3000 U) was dissolved in 1 ml pH 8.0 buffer and 100 ~1 freshly prepared 10 mg/ml added.

biotin-LC-NHS ester in pH 8.0 buffer were After 2 h at 30°C incubation, Centicon

filters were used for purification and the biotinylated HRP was adjusted to a concentration of 2.5 mg/mI in PBS/2 mM EDTA pH 7.4. Ail biotinylated enzymes were stored at 4°C. 2.2.2. ELISA procedures Different sandwich ELISAs

were

used

fractions

obtained.

ated

HRJ’ resultinz

in a ratio

of 4:I.

Thev

were in

incubated for at least 15 min at RT and diluted PBS/2DI,

casein/0.051

Tweeo20.

Human complement components C2, C3, C4, factor H and factor B concentrations were determined by ELISA (Timmemmn et al.. IW595). These ELlSAs were used lo evaluate avidin-D-Iab-elled anti-DIG complexes. In short, a %-well plate (Greiner) was coated with a polycIooaI antibody followed by sequential incubations with the appmpriale staodard. DIG-labelled polyclonal anti human C2, C3. C4, factor B or factor H antibodies and finally detection with the preformed anti-DIG-avidin_(bio)HRP complexes. HRP labels were. detected by ABTS. Results obtaioed using the preformed complexes were compared with directly HRP-IWled anti-DIG antibodies. AS antibodies HRI-labelled goat IgG anti-DIG, HRP-labelled Fab fragments from sheep IgG antiDIG and poIyHIWIabe1led Fab fragments from sheep IgG anti-DIG (borh Fab hagments purchased fmm Boehringer) were used. FM comparison the polyclonal anti complement factor antibodies were also biotinylated (Timmerroan et al.. 1995) and detected with various (streptkwidin-HRP reagents. A sandwich ELISA for measuring moose IgG concentrations in ascites was used to evaluate the performance of goat IgG anti-mouse IgG-avidin(bio)HRP complexes. %-we11 plates were coated with 4 .ug/mI goat IgG aai-moose IgG &nverTech). The plates were subsequently incubated with serial dilutions of mouse IgG containing ascites in PNBT (IO mM phosphate pH 7.4. 0.15 M NaCI, 0.5% blocking reagent, 0.05% Tween 20). After incubations with goat IgG anti-mouse IgG-avidin(bio)HW complexes, the HRP labels were visualized with 0.4 mg/ml OPD in 0.1 M citrate/phosphate pH 5.3 containing 0.03% H,O,. Between each step plates were washed with PBS/O.O5B Tween20. Results obtained using the preformed complexes were compared with commerciaIly available labelled antimouse IgG am&&es directly labelled with HRP, obtained from Boehringer. Sigma and Dakopatts.

to deter-

mine optimal ratios between biotinylated HRP and the antibody-avidin of antibody-avidin

amounts of biotinylated HW, e.g. 10 1~1avidin-Dlabelled goat IgG anti-moose IgG + 2.5 .rl biotinyl-

Fixed amounts

complex were mixed with various

2.2.3. Dot-blot procedures Immunodot-blot procedures and dot-blot hybridization procedures were used IO evaluate the

performance of the HRP- and AP-labelled complexes. The HRF-l&lied complexes were prepared with biotinylated HRP using ratios found to be optimal for ELISA. HRP-lab&d antibodies and complexes were used threefold mne concentrated than in the ELlSA pmcedwes. The ratios of antibcdyavidin-D complexes to biotinylated AP were determined as described for HRi’ labelling. Chromogenic detection of the membrane bound enzymes wa performed with NBT/BCIP stining of AP or with 0.05% DAB
The AP-labelled sheep IgG anti-DIG-avidii-D complexes were used in a dot-blot hybridization acisay. Plasmid DNA was spotted in serial dilutions and hybridized with a homologous plasmid labelled with digoxigcnin-IldUTP. Dettction of the DIG labels was performed with HRP-lab&d Fab fngments from sheep IgG anti-DIG (Boebringcr) or bio-HRF-labelled anti-DIG complexes. Probe labelling. hybridization and detection by cbemiluminescence were pafonned as previously described (Van Gijlswijk et al.. 1992).

3. Results

The IgG fraction of the goat IgG anti-DIG antibody was labelled with HRP as described by Duncan et al. (1983). As shown in Table I, little or no unlabelled IgG was found and the ratio of pcmxidae to IgG could be monitored by changing the SMCC concentration during the IgG activation step. As proven by SPS-PAGE and gel filtration, the majority of the complexes consisted of on= IgG with 2-4 HRP molecules attached. Furthermore, the conju-

05 ml. min. ‘. unlabelkd IgG elmes ai 12 + 0.7 ml and unlabelkd avid&D at 13.8 + 0.8 ml. Bv cbawY

At high molar excesses of SMCC over Ifi, molecular weight compkxs

hi_eb

wets formed at avidin-D

toIgGratiosof3:1a6:1.Atamolar~~ioof6:1 over 95% of the IgCi am-4858 of the widin-D were bcxmd. The compkx was fwnd 10 have an average mokcular weight of 520 Id)a and ber.ce the conjugates fmmed clhnsisted of I IgG and 5-6 &din-D

activity which &creased strongly upon more enten-

the tmmoditied goat IgG anti-mouse IgG antibody used fix the of the wttjugates were

we

twmnak& to KU%. Signals ebtaincd by the mnju~~~~from82+4~;08+1J.The~~gates ef luw molecular weight retained the highest

conjugates of similar molecular weight but prepared km-n higher SMCC ratios were compared. Furthermore, complexes containing 2 IgG molecules

labelling with avidin-D and deceased slowly as

:IgG

Iy we11 in E!_ISA.

specirt for differem human complement factors. ranging from a factor 3 to 7. Larger complexes

vide higher sensitivity than biotinylated antibodies. Different &rept)avidin-HRP conjugates from different commercial sources were used, consisting either of direcdy HRP-labelled Istrep)avidin or preformed avidin-bioHRP

complexes (ABC

method). The besr

results were obtained with ABC complexes. although, DIG-labelled antibodies detected by commercially available HRP-labelled anti-DIG anlibodies (Fab fragments) were cm average twice as sensirive, predomiaantly because the DIG method provided much lower background values.

Similar results wem

with goat IgG antiFig. 5A shows the results obtained by aa imaunadot blot in which human IgG was detected fir. with a mouse IgG anti-human IgCi antibody. followed by an incubation with HRP- or AP-lab&d anti-mouse IgG antibodies or with preformed HRPor AKlabelled anti-mouse IgG/avidin-D complexes. The conjugate used consisted of twc IgGs and 5-6 avidins. optimal

shows tM

digoxigenin-labelled polyby shuttle conjugates pro-

bio-AP/umjagate

ratios and dilu-

dcms W~IC calculated by checkerbvard experlmmts on similar blots. For bio-HRP, the ratios use4 were those found optimal for ELISA. The ca~jugate could

a&tin-D

80 IgG

ef al. (1983) tk

using

activation

repmer mokcuk be&d

of choice.

with bioH?W

Shuttle conjugates

la-

or bioAF’ were found to improve

ths sensirivity of various immunockmical procedures compared u) procedures employing antibody directly laklkd

We intera

wish

with enzyme.

to thank R

in this

WC&

supported by Et&uittger

lands Organii

Seibl

Tnis

for his stimulating

study

Mannkim

for .%entifK

was

fnancially

and the Nether-

Research (grant no.

PGS 900-790-129~. J. HiwoFhml. Cytockm. 34.164. Rue. AK.. Gxknbt. M.P.C. “al da?. “av,,

RAW..

“An

Tnrnemun.JJ.. Van &r~k&Je. N...Van Gijtwijk-Janea. DJ, wkwey. CL. “An Es. LA. ud Mla, MR. w95)