- Email: [email protected]
Purification of mouse haptoglobin by antibody affinity chromatography and development of an ELISA to measure serum haptoglobin levels
Journal of Immunological Methods, 108 (1988) 53-59 Elsevier
53
JIM 04677
Purification of mouse haptoglobin by antibody affinity chromatography and development of an ELISA to measure serum haptoglobin levels C.H. Marsden and R.G. Simmonds Lilly Research Centre Ltd., Eli Lilly & Company, Erl Wood Manor, Windlesham, Surrey GU20 6PH, U.K.
(Received 15 July 1987, revised received 12 October 1987, accepted 27 October 1987)
Mouse haptoglobin was isolated from acute-phase serum initially by affinity chromatography on haemoglobin-Sepharose. This proved inefficient, but sufficient material was obtained for use as an immunogen. Rabbit anti-haptoglobin antibodies were used as immunoabsorbents to isolate larger quantities of haptoglobin. Subsequently, specific anti-haptoglobin antibodies were prepared by affinity chromatography on haptoglobin-Sepharose. A direct sandwich ELISA for mouse serum haptoglobin was developed, using affinity purified reagents. The working range of the haptoglobin standard curve was 0.02-0.5 ~tg/ml. The reagents did not cross-react with albumin or haemoglobin and the antibody also recognised rat haptoglobin. Key words: Haptoglobin; Anti-haptoglobin antibody; Affinity chromatography; ELISA, sandwich
Introduction
Haptoglobin is a serum a2-glycoprotein of approximately 100 kDa. It exists as a tetramer, comprising two smaller identical a chains and two larger identical fl chains. The a chains are linked to each other by a disulphide bond and each fl chain is similarly linked to an a chain. Human
Correspondence to: C.H. Marsden, Lilly Research Centre Limited, Erl Wood Manor, Windlesham, Surrey GU20 6PH, U.K. Abbreoiations: Hp, haptoglobin; M, mouse, Hu, human; R, rabbit; G, goat; MSA, mouse serum albumin; BSA, bovine serum albumin; IgG, immunoglobulin G; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; FCA, Freund's complete adjuvant; ELISA, enzyme-linked immunosorbent assay; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; IEP, immunoelectrophoresis; -AP, alkaline phosphatase conjugated to protein; RID, radial immunodiffusion; pNPP, p-nitrophenylphosphate.
haptoglobin is usually pol~cmeric b.ut it is thought that animal haptoglobins are rardy so (Putnam, 1984). The most characteristic property of haptoglobin is its ability to form stable complexes with extra-corpuscular haemoglobin and is thus thought to prevent iron loss through urinary excretion. As the haemoglobin binding property is unique, it has become a functional definition for haptoglobin and the basis for isolation and assay procedures (Javid and Liang, 1973; Baseler and Burrell, 1981). Haptoglobin is also one of that group of plasma proteins known as acute phase reactants, the serum levels of which show a marked increase after trauma and during inflammatory disease. Measurement of haptoglobin levels may therefore be used to monitor the progress of inflammatory reactions and assess the efficacy of test drugs. Serum haptoglobin may be measured by single radial diffusion (Mancini et al., 1965) using ex-
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
54
perimentally produced antisera or commercially available kits designed for the evaluation of clinical samples (e.g., M-Partigen, Calbiochem-Behring, La Jolla, CA). The conventional single radial diffusion test is time consuming, requiring several days to obtain a result and is difficult to quantitate. ELISAs are designed to handle large numbers of samples, results are usually available within the working day and, by using purified standardised reagents, will yield reproducibly quantitative results. The aims of this study were to prepare purified mouse haptoglobin for use as immunogen and assay standard, and subsequently to develop a simple ELISA to measure serum haptoglobin levels.
Materials and methods
Immunoabsorbents The following ligands were coupled to CNBractivated Sepharose 4B (Pharmacia, Milton Keynes, U.K.) according to the manufacturer's instructions: mouse haemoglobin (Sigma, Poole, U.K., H-5633), mouse serum albumin (Sigma, A-
3139), rabbit anti-mouse haptoglobin, IgG fraction, rabbit anti-mouse haptoglobin antibody, mouse haptoglobin. Routinely, 3 g of Sepharose were used for each preparation. The imrnunoabsorbents were resuspended in PBS and packed into 1.5 × 10 cm Econocolumns (Bio-Rad, Watford, U.K.). Chromatography was monitored by absorbance at 280 nm using a Uvicord 2 system (LKB, Croydon, U.K.).
Immunogen Haptoglobin was isolated from acute-phase mouse serum (taken 24 h after injection with 1/~g LPS) by affinity chromatography on immobilised mouse haemoglobin, using a modification of the method of Javid and Liang (1973). The clarified serum was absorbed on to the column, washed in with PBS, and bound material eluted using 2.5 M guanidine in 0.2 M sodium acetate pH 5.0. Because of low column capacity, the serum was repeatedly recycled. The pooled neutralised eluates were quantitated by absorbance at 280 nm using E1lcm mg/r,d 1.232, determined for human haptoglobin (Sigma, H-1511). The material was also characterised by SDS-PAGE and reactivity with goat
TABLE I SUMMARY OF ELISA METHODS USED ELISA method
Assay for
Plate coating
Analyte
Detection
Substrate
1
Antibody
Hp (M or Hu)
R or G anti-MHp
G anti-RIgG-AP or R anti-GIgG-AP
pNPP
2
Hp identity
R anti-MHp
MHp
R anti-MHp-AP
pNPP
(IgG) 3 4
5
Hp function
Mouse haemoglobin
MHp
R anti-MHp-AP
pNPP
R anti-MHp-AP
pNPP
G-anti-MHp-AP
pNPP
Recognition
Anti-Hp
MHp
of serum proteins
antibody
HuHp MSA Haemoglobin Transferrin al-Acid glycoprotein
Test samples
G anti-MHp antibody
MHp (standards) Test samples (replicates at 1/10 000)
55 anti-mouse haptoglobin in a single radial diffusion test.
Antibodies Male New Zealand White rabbits were injected twice, with a 4 week interval, with 100/~g putative haptoglobin in 0.5 ml PBS, emulsified with an equal volume of FCA. After a further 2 weeks, 100 ~tg haptoglobin in saline (0.5 ml) were given intravenously. Blood samples of up to 40 ml were taken from the central ear artery at intervals starting 7 days after the third injection. The presence of anti-haptoglobin antibodies was determined by ELISA (method 1, see Table I). The IgG fraction was obtained by chromatography on Protein A-Sepharose (Pharmacia). The serum was washed in using PBS and bound material eluted using 0.1 M citrate-phosphate pH 3.5. After dialysis against PBS, the eluate was absorbed against immobilised MSA. Specific antibodies were then isolated on mouse haptoglobinSepharose. Bound material was eluted using 1 M potassium thiocyanate/0.5 M ammonia (KCNS/ NHaOH ), neutralised, dialysed against PBS and quantitated by absorbance at 280 nm, using Elmg/ml_ 1.43. lcrn Goat anti-mouse haptoglobin serum was kindly donated by Mr. M. Smith (I.C.I. Ltd., Alderley Edge, U.K.). The IgG fraction was obtained by precipitation with 50% ammonium sulphate. The precipitate was washed with 50% ammonium sulphate solution, dissolved in and then dialysed against PBS. After absorption against MSA, the antibody was isolated on haptoglobin-Sepharose as above. Both antibody preparations were tested for reactivity with mouse haptoglobin and other serum proteins using ELISA method 4 (see Table I).
Preparation of haptoglobin Rabbit anti-mouse haptoglobin IgG fraction was absorbed against mouse serum albumin and then immobilised on CNBr-Sepharose. This immunoabsorbent was used to isolate mouse haptoglobin directly from acute-phase serum. The column was run in PBS and bound material eluted using KCNS/NH4OH. The neutralised, pooled eluates were dialysed against PBS and characterised by SDS-PAGE, single radial diffu-
sion and ELISAs for identity and functional binding (Table I, methods 2 and 3). A portion of the haptoglobin so isolated was in turn immobilised on CNBr-Sepharose for routine isolation of anti-haptoglobin specific antibodies. Finally, when sufficient antibody had been prepared, a rabbit anti-mouse haptoglobin-CNBrSepharose immunoabsorbent was made, to provide a more efficient, direct isolation procedure for haptoglobin, using the method described above.
Electrophoretic techniques All electrophoresis experiments were performed using an LKB Multiphor and associated equipment. Procedures were run according to the manufacturer's relevant application notes. SDS-PAGE was carded out under non-reducing conditions using 5% acrylarnide in 0.1 M phosphate buffer pH 7.0. Crossed IEP. Samples of mouse haptoglobin (4 /~g), normal and acute-phase mouse serum were first run in 1% agarose in Tris-barbiturate buffer pH 8.6 and then perpendicularly into 1% agarose containing rabbit or goat anti-haptoglobin antibody (10 /~g/ml), or rabbit antiserum to mouse, rat or human serum proteins (Nordic, Maidenhead, Berkshire, U.K.) at 1/200. Electrophoresis was carried out for 1.5 h in each direction at 10 V/cm. The gels were processed as described in the application notes and stained using Coomassie blue.
General ELISA protocol Flat bottom rnicrotitre plates (Nunc Immunoplate II, Gibco, U.K.) were coated with target protein, usually at 10 # g / m l in 0.1 M sodium carbonate pH 9.3, 200 #l/well, and incubated for 3 h at 37 °. Plates were stored sealed at 4°C, still containing the coating solution. Between each stage of an assay the plates were washed six times by immersion in wash buffer (0.15 M sodium chloride, 0.05% Tween 20, 0.025% sodium azide) and then emptying. Samples and reagents were diluted in 1% BSA in 0.15 M sodium chloride, 0.05% Tween 20, 0.05% azide. Sample dilutions were prepared in Eppendorf tubes, then 100/zl aliquots pipetted per well, and incubated for 2 h at room temperature.
56
Enzyme conjugates were prepared by coupling anti-haptoglobin antibodies to alkaline phosphatase (Sigma, P-5521) by glutaraldehyde (Engvail and Perlmann, 1972). 100/zl of an appropriate dilution were added per well and the plates incubated for 1 h at room temperature. p-Nitrophenylphosphate (Sigma 104 phosphatase substrate) was used at 1 mg/ml in 0.1 M sodium carbonate, 0.002 M magnesium chloride, pH 9.3. 100/~1 were added per well and incubated at 37 °C for about 1 h, until the development of maximum readable coloration. The reaction was stopped by the addition of 50 /~1 5 N sodium hydroxide per well and the absorbances read at 410 nm as sample wavelength and 630 nm as reference, using an MR600 microplate reader (Dynatech, Billingshurst, U.K.). A reagent blank value was automatically subtracted from all sample readings. The five ELISA methods used are summarised in Table I.
Results and discussion
Preparation of haptoglobin The haemoglobin affinity chromatography method proved laborious and inefficient and did not provide the yields quoted by Javid and Liang (1973). One possible reason for this many have been the physical form of the haemoglobin. The mechanism of haptoglobin.haemoglobin binding has been the subject of much study and the structure of the h~emoglobin appears critical for binding to take place. It is tholaght (Nagel and Gibson, 1971) that dissociation into dimers is necessary for the binding sites to become accessible and that the tetramer is in fact incapable of binding haptoglobin. Increasing the contact time between guanidine and bound haptoglobin failed to improve yields. However, a small quantity of putative haptoglobin was obtained (Table II). SDS-PAGE analysis showed this to be electrophoretically pure, having a single stained band of - 100 kDa. Commercial human haptoglobin, used as a comparator, gave a major band in this position, and also three higher molecular weight components, presumably resulting from polymerisation. The material isolated
TABLE II RELATIVE YIELDS OF HAPTOGLOBIN FROM THREE AFFINITY COLUMNS Ligand
mg haptoglobin eluted per mg ligand immobilised
Haemoglobin Rabbit anti MHp IgG Rabbit anti MHp antibody
0.122 0.059 0.372
from haemoglobin-Sepharose also gave a positive reaction in single radial diffusion against goat anti-mouse haptoglobin, and was accordingly deemed to be haptoglobin of sufficient purity to be used as an immunogen. Ion exchange chromatography was considered as an alternative method to provide more material, but this gives a mixed product, which then requires a further purification step, such as preparative isoelectric focusing (Baseler and Burrell, 1983). Affinity chromatography using immobilised anti-haptoglobin, either as the IgG fraction or specific antibody (Table II), proved the most simple method, bound material being effectively eluted using alkaline conditions. The low capacity of the IgG column was no doubt a reflection of the low antibody content of the serum. Haptoglobin isolated by antibody affinity chromatography gave a single stained band of - 100 kDa on SDS-PAGE, a positive reaction in single radial diffusion against the unrelated goat antimouse Hp, and was positive in two ELISA systems, methods 2 and 3 (Table I). On plates coated with rabbit ant-i-MHp antibody, haptoglobin had a Ds0 (dilution at 50% of maximal response on the titration curve) equivalent to 0.1 /~g/ml. The working range of the curve was 0.03-2 /~g/ml. The assay outlined in method 3 was designed to demonstrate the functional binding of haptoglobin to haemoglobin simultaneously with recognition by antibody conjugated to alkaline phosphatase. As haemoglobin binding is a property unique to haptoglobin, only this species would be expected to react in this assay system. A positive result was obtained, but the sensitivity was decreased (/)so = 1.25 /~g/ml). This result also indicates that the binding of haptoglobin to immobilised haemoglobin does not prevent the antihaptoglobin antibody recognising its antigen.
57
Preparation of rabbit anti-mouse haptoglobin antibodies The availability of quantities of mouse haptoglobin provided an efficient ligand for the purification of anti-haptoglobin antibodies. Rabbit anti-haptoglobin antiserum had a titre of 3.7 × 10 4 when assayed by method 1 (Table I). This did not increase appreciably after further booster injections. The goat antiserum was regularly used in single radial diffusion, but its titre was not determined by ELISA. Protein A chromatography was used to isolate the IgG fraction from rabbit sera. However, as the binding of goat IgG to protein A is variable and not well characterised (Langone, 1982) the initial preparative step for goat antisera was ammonium sulphate precipitation. The capacity of the haptoglobin column was ca. 25 mg antibody and after exhaustive chromatography the serum antibody concentrations were determined as being 0.34 m g / m l for rabbit and 0.78 m g / m l for goat.
Reactivity of anti-haptoglobin antibodies (1) ELISA. Rabbit anti-mouse Hp serum showed marked cross-reactivity with MSA and haemoglobin (Table III) when the purified serum proteins were immobilised on the plate, as in ELISA method 1 (Table I). Goat anti-mouse Hp sera, which gave multiple bands on RID tests, also cross-reacted with MSA in the same assay system (Table III). The cross-reactivity with haemoglobin could be explained by possible carry over of haemoglobin complexed to the apparently pure TABLE III ANALYSIS OF THE REACTIVITIES OF ANTIHAPTOGLOBIN ANTISERA WITH IMMOBILISED PURIFIED SERUM PROTEINS Irnmobilised protein
% Cross-reactivity Goat anti-mouse Hp serum 100 65 0.9
Haptoglobin MSA Haemoglobin Transferrin (human, Sigma) ND a a ND = not detectable.
Rabbitanti-mouse Hp serum 100 46 23 0.4
2"
1.5-
1-
0-~.
, 1
. . . . . . . .
,
10
,
. . . . . .
~
100
. . . . . . . .
,
. . . . . . . .
,
1000
[XLJTIO~
Fig. 1. Cross-reactivity of affinity purified anti-haptoglobin antibody with serum proteins. Goat or rabbit anti-haptoglobin antibody (10/lg/ml) immobilised on plate. Analytes: II, MHp titrated from 5 ~tg/ml; I~, MSA titrated from 100 /~g/ml; e, MHb titrated from 100 /Lg/ml. Detected by goat or rabbit anti-haptoglobin-alkafinephosphatase conjugatewith pNPP as substrate (see Table 1, method 4). Transferrin and ax-acid-glycoprotein gave similar responses to MHb. haptoglobin used as immunogen. In contrast the cross-reactivity of the affinity purified antibodies was negligible, using ELISA method 4 (Table I and Fig. 1) in which the serum proteins were the analytes. (2) Crossed IEP. Results were similar for both antibodies. Mouse Hp, isolated by antibody affinity chromatography, gave a single peak against specific antibodies and also against antiserum to total mouse serum proteins. Normal mouse serum showed a weak response against both anti-haptoglobin preparations, but multiple peaks against rabbit anti-mouse serum proteins. Acute-phase serum gave a single very large peak against purified antibody, superimposed on the haptoglobin standard, and an intensified pattern of peaks against antiserum proteins.
Development of ELISA for haptoglobin in serum samples ELISA method 2 gave reasonably consistent results for affinity purified haptoglobin (/)so = 0.159 _+ 0.05 /~g/ml, n = 12). The working range of the standard curve was 0.02-0.5 /~g/ml. The same antibody preparation could be used for both
58
plate coating and as enzyme conjugate, indicating that the antibody recognises more than one epitope on the haptoglobin molecule. However, when test serum samples were assayed, very high, unreadable, responses were obtained, even when samples were diluted in excess of 1/10 000. It was not possible to identify the reacting species, although the most obvious serum proteins, albumin and haemoglobin did not cross-react. There was also no apparent cross-reaction with transferrin or al acid-glycoprotein, although these assays were performed using purified human rather than mouse proteins. These very high responses were eliminated when the goat antibody was immobilised on the plate. Using rabbit antibody-enzyme conjugate, the haptoglobin standard response compared well with the original system (/)so = 0.154 _+ 0.022 /,g/ml, n = 4). An alkaline phosphatase conjugate of the goat antibody proved equally satisfactory, and is now used routinely in the assay for test samples (ELISA method 5). The sensitivity of the assay could be increased approximately five-fold by decreasing the plate coating concentration to I # g / m l . It was found to be very important that samples be diluted so that their response fell within the working range of the assay. For mouse sera, this usually involved two serial dilutions of 1/100, to give a final dilution of 1 / 1 0 000. The use of 1% BSA in carbonate buffer as a blocking step after plate coating was also evaluated. This gave a marginal increase is sensitivity (results not shown), but was usually unnecessary as background readings were very low (OD410 < 0.05 AU).
Determination of serum haptoglobin levels The ELISA was validated by analysing normal and acute phase B A L B / c mouse sera in two ways: exhaustive antibody affinity chromatography and ELISA (method 4). A known volume of serum was recycled over the antibody column until no further material could be eluted. The total haptoglobin in the eluate was determined and the concentration in the serum calculated. A good correlation was found between the
TABLE IV COMPARISON OF SERUM HAPTOGLOB1N CONTENT AS DETERMINED BY AFFINITY CHROMATOGRAPHY AND ELISA Serum sample
Normal Acute phase
Serum haptoglobin mg/ml Affinity chromatography
ELISA
0.97 5.30
0.87 5.40
haptoglobin content of serum samples determined by this method and by ELISA (see Table IV). A wide range of haptoglobin levels was found in normal B A L B / c mice (see Table V). As they had not been housed under barrier conditions, the higher haptoglobin levels may be an indication of the animals' health status. Outbred mice tended to have higher levels than B A L B / c (results not shown). Using ELISA method 5 it was possible to detect a ten-fold increase in haptoglobin levels in mouse sera taken 24 h after administration of various doses of concanavalin A (Table VI). Good rank order correlation (r = 0.97) was also achieved between these results and the diameter 2 responses obtained by single radial diffusion for the same samples (see Table VI).
Cross species reactivity It was noted during the early development work that some batches of commercial h u m a n haptoglobin reacted in the ELISA while others did not. As these preparations all showed multiple bands on SDS-PAGE it was assumed that differing degrees of polymerisation prevented epitope recognition by the antibody. In crossed IEP, puri-
TABLE V ANALYSIS BY ELISA OF NORMAL AND ACUTE-PHASE SERA FROM MOUSE AND RAT Serum haptoglobin (mg/ml)
Mouse (BALB/c) Rat (SpragueDawley)
Normal
Acute phase
0.50-1.50 0.99-1.25
3.00-9.25 3.02-7.52
59 TABLE VI CORRELATION BETWEEN RESULTS OBTAINED BY ELISA (METHOD 5) AND SINGLE RADIAL DIFFUSION
assay reported here provides a simple, rapid and highly specific measure for rodent haptoglobin.
Sera taken 24 h after injection of Concanavalin A (n = 5). ConA
ELISA
RID response
(/z g)
Hp (mg/ml)
Diameter 2
0 12.5 25 50 100
0.624 + 0.28 1.886 + 0.904 3.528 + 0.592 8.494 + 1.964 9.331 _ 1.374
38.1 + 1.44 98.1 + 5.7 125.8 + 8.5 192.5 + 4.4 197.4 + 4.6
fied mouse Hp was only weakly recognised by antiserum to human serum proteins. A stronger response was obtained with anti-rat serum but still not so great as mouse Hp - anti-mouse. The ability of the ELISA (method 5) to detect haptoglobin in the sera of different species was evaluated. Samples of rat serum gave strong responses in the assay, with clear differentiation between normal and acute-phase measurements (Table V). However, there was no clear reactivity with normal or rheumatoid human serum, nor did purified human haptoglobin give a titratable response from 10 /xg/ml. In contrast a maximal response was obtained with mouse haptogiobin at 2 # g / m l . These results are interesting in view of the recently reported use of a commercially available anti-human haptoglobin immunodiffusion kit to measure rat haptoglobin (Gilbertsen 1986). It has not been possible to perform the comparable experiment to establish whether purified mouse haptoglobin is recognised by a specific anti-human haptoglobin antiserum.
Conclusion ELISA methods have been reported for measuring many different serum proteins, and of the acute-phase reactants assays have already been published for human rheumatoid factor (Zrein et al., 1986) and serum amyloid A in experimental animals (Zuckerman and Surprenant, 1986). The
Acknowledgements The authors would like to thank Dr. M.E.J. Billingham for helpful discussions, Dr. E.A. Kitchen for performing the single radial diffusion tests and Mrs. E.G. Paculabo and Mrs. P.W. Whiteling for typing the manuscript.
References Baseler, M.W. and Burrell, R. (1981) Acute phase reactants in experimental inhalation lung disease. Proc. Soc. Exp. Biol. Med. 168, 49. Baseler, M.W. and Burrell, R. (1983) Purification of haptoglobin and its effects on lymphocyte and alveolar macrophage responses. Inflammation 7, 387. Engvall, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigencoated tubes. J. Immunol. 109, 129. Gilbertsen, R.B. (1986) Rat haptoglobin: Method of quantitation and response to arthritic therapy in collagen arthritis. Immunopharmacology 11, 69. Javid, J. and Liang, J.C. (1973) The hemoglobin-haptoglobin bond. 1. Dissociation of the complex and recovery of the native haptoglobin in an affinity chromatography system. J. Lab. Clin. Med. 82, 991. Langone, J.J. (1982) In: F.J. Dixon and H.G. Kunkel (Eds.), Advances in Immunology, Vol. 32. Academic Press, New York, p. 157. Mancini, E., Carbonara, A.O. and Heremans, J.F. (1965) Immunological quantitation of antigens by single radial diffusion. Immunochemistry 2, 235. Nagel, R.L. and Gibson, Q.H. (1971) The binding of hemoglobin to haptoglobin and its relation to subunit dissociation of hemoglobin. J. Biol. Chem. 246, 69. Putnam, F.W. (1984) In: F.W. Putnam (Ed.), The Plasma Proteins. Structure, function and genetic control. Academic Press, New York, p. 89. Zrein, M., De Marcillac, G. and Van Regenmortel, M.H.V. (1986) Quantitation of rheumatoid factor by enzyme immunoassay using biotinylated human IgG. J. Immunol. Methods 87, 229. Zuckerman, S.H. and Surprenant, Y.M. (1986) Simplified microELISA for the quantitation of murine serum amyloid A protein. J. Immunol. Methods 92, 37.
53
JIM 04677
Purification of mouse haptoglobin by antibody affinity chromatography and development of an ELISA to measure serum haptoglobin levels C.H. Marsden and R.G. Simmonds Lilly Research Centre Ltd., Eli Lilly & Company, Erl Wood Manor, Windlesham, Surrey GU20 6PH, U.K.
(Received 15 July 1987, revised received 12 October 1987, accepted 27 October 1987)
Mouse haptoglobin was isolated from acute-phase serum initially by affinity chromatography on haemoglobin-Sepharose. This proved inefficient, but sufficient material was obtained for use as an immunogen. Rabbit anti-haptoglobin antibodies were used as immunoabsorbents to isolate larger quantities of haptoglobin. Subsequently, specific anti-haptoglobin antibodies were prepared by affinity chromatography on haptoglobin-Sepharose. A direct sandwich ELISA for mouse serum haptoglobin was developed, using affinity purified reagents. The working range of the haptoglobin standard curve was 0.02-0.5 ~tg/ml. The reagents did not cross-react with albumin or haemoglobin and the antibody also recognised rat haptoglobin. Key words: Haptoglobin; Anti-haptoglobin antibody; Affinity chromatography; ELISA, sandwich
Introduction
Haptoglobin is a serum a2-glycoprotein of approximately 100 kDa. It exists as a tetramer, comprising two smaller identical a chains and two larger identical fl chains. The a chains are linked to each other by a disulphide bond and each fl chain is similarly linked to an a chain. Human
Correspondence to: C.H. Marsden, Lilly Research Centre Limited, Erl Wood Manor, Windlesham, Surrey GU20 6PH, U.K. Abbreoiations: Hp, haptoglobin; M, mouse, Hu, human; R, rabbit; G, goat; MSA, mouse serum albumin; BSA, bovine serum albumin; IgG, immunoglobulin G; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; FCA, Freund's complete adjuvant; ELISA, enzyme-linked immunosorbent assay; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; IEP, immunoelectrophoresis; -AP, alkaline phosphatase conjugated to protein; RID, radial immunodiffusion; pNPP, p-nitrophenylphosphate.
haptoglobin is usually pol~cmeric b.ut it is thought that animal haptoglobins are rardy so (Putnam, 1984). The most characteristic property of haptoglobin is its ability to form stable complexes with extra-corpuscular haemoglobin and is thus thought to prevent iron loss through urinary excretion. As the haemoglobin binding property is unique, it has become a functional definition for haptoglobin and the basis for isolation and assay procedures (Javid and Liang, 1973; Baseler and Burrell, 1981). Haptoglobin is also one of that group of plasma proteins known as acute phase reactants, the serum levels of which show a marked increase after trauma and during inflammatory disease. Measurement of haptoglobin levels may therefore be used to monitor the progress of inflammatory reactions and assess the efficacy of test drugs. Serum haptoglobin may be measured by single radial diffusion (Mancini et al., 1965) using ex-
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
54
perimentally produced antisera or commercially available kits designed for the evaluation of clinical samples (e.g., M-Partigen, Calbiochem-Behring, La Jolla, CA). The conventional single radial diffusion test is time consuming, requiring several days to obtain a result and is difficult to quantitate. ELISAs are designed to handle large numbers of samples, results are usually available within the working day and, by using purified standardised reagents, will yield reproducibly quantitative results. The aims of this study were to prepare purified mouse haptoglobin for use as immunogen and assay standard, and subsequently to develop a simple ELISA to measure serum haptoglobin levels.
Materials and methods
Immunoabsorbents The following ligands were coupled to CNBractivated Sepharose 4B (Pharmacia, Milton Keynes, U.K.) according to the manufacturer's instructions: mouse haemoglobin (Sigma, Poole, U.K., H-5633), mouse serum albumin (Sigma, A-
3139), rabbit anti-mouse haptoglobin, IgG fraction, rabbit anti-mouse haptoglobin antibody, mouse haptoglobin. Routinely, 3 g of Sepharose were used for each preparation. The imrnunoabsorbents were resuspended in PBS and packed into 1.5 × 10 cm Econocolumns (Bio-Rad, Watford, U.K.). Chromatography was monitored by absorbance at 280 nm using a Uvicord 2 system (LKB, Croydon, U.K.).
Immunogen Haptoglobin was isolated from acute-phase mouse serum (taken 24 h after injection with 1/~g LPS) by affinity chromatography on immobilised mouse haemoglobin, using a modification of the method of Javid and Liang (1973). The clarified serum was absorbed on to the column, washed in with PBS, and bound material eluted using 2.5 M guanidine in 0.2 M sodium acetate pH 5.0. Because of low column capacity, the serum was repeatedly recycled. The pooled neutralised eluates were quantitated by absorbance at 280 nm using E1lcm mg/r,d 1.232, determined for human haptoglobin (Sigma, H-1511). The material was also characterised by SDS-PAGE and reactivity with goat
TABLE I SUMMARY OF ELISA METHODS USED ELISA method
Assay for
Plate coating
Analyte
Detection
Substrate
1
Antibody
Hp (M or Hu)
R or G anti-MHp
G anti-RIgG-AP or R anti-GIgG-AP
pNPP
2
Hp identity
R anti-MHp
MHp
R anti-MHp-AP
pNPP
(IgG) 3 4
5
Hp function
Mouse haemoglobin
MHp
R anti-MHp-AP
pNPP
R anti-MHp-AP
pNPP
G-anti-MHp-AP
pNPP
Recognition
Anti-Hp
MHp
of serum proteins
antibody
HuHp MSA Haemoglobin Transferrin al-Acid glycoprotein
Test samples
G anti-MHp antibody
MHp (standards) Test samples (replicates at 1/10 000)
55 anti-mouse haptoglobin in a single radial diffusion test.
Antibodies Male New Zealand White rabbits were injected twice, with a 4 week interval, with 100/~g putative haptoglobin in 0.5 ml PBS, emulsified with an equal volume of FCA. After a further 2 weeks, 100 ~tg haptoglobin in saline (0.5 ml) were given intravenously. Blood samples of up to 40 ml were taken from the central ear artery at intervals starting 7 days after the third injection. The presence of anti-haptoglobin antibodies was determined by ELISA (method 1, see Table I). The IgG fraction was obtained by chromatography on Protein A-Sepharose (Pharmacia). The serum was washed in using PBS and bound material eluted using 0.1 M citrate-phosphate pH 3.5. After dialysis against PBS, the eluate was absorbed against immobilised MSA. Specific antibodies were then isolated on mouse haptoglobinSepharose. Bound material was eluted using 1 M potassium thiocyanate/0.5 M ammonia (KCNS/ NHaOH ), neutralised, dialysed against PBS and quantitated by absorbance at 280 nm, using Elmg/ml_ 1.43. lcrn Goat anti-mouse haptoglobin serum was kindly donated by Mr. M. Smith (I.C.I. Ltd., Alderley Edge, U.K.). The IgG fraction was obtained by precipitation with 50% ammonium sulphate. The precipitate was washed with 50% ammonium sulphate solution, dissolved in and then dialysed against PBS. After absorption against MSA, the antibody was isolated on haptoglobin-Sepharose as above. Both antibody preparations were tested for reactivity with mouse haptoglobin and other serum proteins using ELISA method 4 (see Table I).
Preparation of haptoglobin Rabbit anti-mouse haptoglobin IgG fraction was absorbed against mouse serum albumin and then immobilised on CNBr-Sepharose. This immunoabsorbent was used to isolate mouse haptoglobin directly from acute-phase serum. The column was run in PBS and bound material eluted using KCNS/NH4OH. The neutralised, pooled eluates were dialysed against PBS and characterised by SDS-PAGE, single radial diffu-
sion and ELISAs for identity and functional binding (Table I, methods 2 and 3). A portion of the haptoglobin so isolated was in turn immobilised on CNBr-Sepharose for routine isolation of anti-haptoglobin specific antibodies. Finally, when sufficient antibody had been prepared, a rabbit anti-mouse haptoglobin-CNBrSepharose immunoabsorbent was made, to provide a more efficient, direct isolation procedure for haptoglobin, using the method described above.
Electrophoretic techniques All electrophoresis experiments were performed using an LKB Multiphor and associated equipment. Procedures were run according to the manufacturer's relevant application notes. SDS-PAGE was carded out under non-reducing conditions using 5% acrylarnide in 0.1 M phosphate buffer pH 7.0. Crossed IEP. Samples of mouse haptoglobin (4 /~g), normal and acute-phase mouse serum were first run in 1% agarose in Tris-barbiturate buffer pH 8.6 and then perpendicularly into 1% agarose containing rabbit or goat anti-haptoglobin antibody (10 /~g/ml), or rabbit antiserum to mouse, rat or human serum proteins (Nordic, Maidenhead, Berkshire, U.K.) at 1/200. Electrophoresis was carried out for 1.5 h in each direction at 10 V/cm. The gels were processed as described in the application notes and stained using Coomassie blue.
General ELISA protocol Flat bottom rnicrotitre plates (Nunc Immunoplate II, Gibco, U.K.) were coated with target protein, usually at 10 # g / m l in 0.1 M sodium carbonate pH 9.3, 200 #l/well, and incubated for 3 h at 37 °. Plates were stored sealed at 4°C, still containing the coating solution. Between each stage of an assay the plates were washed six times by immersion in wash buffer (0.15 M sodium chloride, 0.05% Tween 20, 0.025% sodium azide) and then emptying. Samples and reagents were diluted in 1% BSA in 0.15 M sodium chloride, 0.05% Tween 20, 0.05% azide. Sample dilutions were prepared in Eppendorf tubes, then 100/zl aliquots pipetted per well, and incubated for 2 h at room temperature.
56
Enzyme conjugates were prepared by coupling anti-haptoglobin antibodies to alkaline phosphatase (Sigma, P-5521) by glutaraldehyde (Engvail and Perlmann, 1972). 100/zl of an appropriate dilution were added per well and the plates incubated for 1 h at room temperature. p-Nitrophenylphosphate (Sigma 104 phosphatase substrate) was used at 1 mg/ml in 0.1 M sodium carbonate, 0.002 M magnesium chloride, pH 9.3. 100/~1 were added per well and incubated at 37 °C for about 1 h, until the development of maximum readable coloration. The reaction was stopped by the addition of 50 /~1 5 N sodium hydroxide per well and the absorbances read at 410 nm as sample wavelength and 630 nm as reference, using an MR600 microplate reader (Dynatech, Billingshurst, U.K.). A reagent blank value was automatically subtracted from all sample readings. The five ELISA methods used are summarised in Table I.
Results and discussion
Preparation of haptoglobin The haemoglobin affinity chromatography method proved laborious and inefficient and did not provide the yields quoted by Javid and Liang (1973). One possible reason for this many have been the physical form of the haemoglobin. The mechanism of haptoglobin.haemoglobin binding has been the subject of much study and the structure of the h~emoglobin appears critical for binding to take place. It is tholaght (Nagel and Gibson, 1971) that dissociation into dimers is necessary for the binding sites to become accessible and that the tetramer is in fact incapable of binding haptoglobin. Increasing the contact time between guanidine and bound haptoglobin failed to improve yields. However, a small quantity of putative haptoglobin was obtained (Table II). SDS-PAGE analysis showed this to be electrophoretically pure, having a single stained band of - 100 kDa. Commercial human haptoglobin, used as a comparator, gave a major band in this position, and also three higher molecular weight components, presumably resulting from polymerisation. The material isolated
TABLE II RELATIVE YIELDS OF HAPTOGLOBIN FROM THREE AFFINITY COLUMNS Ligand
mg haptoglobin eluted per mg ligand immobilised
Haemoglobin Rabbit anti MHp IgG Rabbit anti MHp antibody
0.122 0.059 0.372
from haemoglobin-Sepharose also gave a positive reaction in single radial diffusion against goat anti-mouse haptoglobin, and was accordingly deemed to be haptoglobin of sufficient purity to be used as an immunogen. Ion exchange chromatography was considered as an alternative method to provide more material, but this gives a mixed product, which then requires a further purification step, such as preparative isoelectric focusing (Baseler and Burrell, 1983). Affinity chromatography using immobilised anti-haptoglobin, either as the IgG fraction or specific antibody (Table II), proved the most simple method, bound material being effectively eluted using alkaline conditions. The low capacity of the IgG column was no doubt a reflection of the low antibody content of the serum. Haptoglobin isolated by antibody affinity chromatography gave a single stained band of - 100 kDa on SDS-PAGE, a positive reaction in single radial diffusion against the unrelated goat antimouse Hp, and was positive in two ELISA systems, methods 2 and 3 (Table I). On plates coated with rabbit ant-i-MHp antibody, haptoglobin had a Ds0 (dilution at 50% of maximal response on the titration curve) equivalent to 0.1 /~g/ml. The working range of the curve was 0.03-2 /~g/ml. The assay outlined in method 3 was designed to demonstrate the functional binding of haptoglobin to haemoglobin simultaneously with recognition by antibody conjugated to alkaline phosphatase. As haemoglobin binding is a property unique to haptoglobin, only this species would be expected to react in this assay system. A positive result was obtained, but the sensitivity was decreased (/)so = 1.25 /~g/ml). This result also indicates that the binding of haptoglobin to immobilised haemoglobin does not prevent the antihaptoglobin antibody recognising its antigen.
57
Preparation of rabbit anti-mouse haptoglobin antibodies The availability of quantities of mouse haptoglobin provided an efficient ligand for the purification of anti-haptoglobin antibodies. Rabbit anti-haptoglobin antiserum had a titre of 3.7 × 10 4 when assayed by method 1 (Table I). This did not increase appreciably after further booster injections. The goat antiserum was regularly used in single radial diffusion, but its titre was not determined by ELISA. Protein A chromatography was used to isolate the IgG fraction from rabbit sera. However, as the binding of goat IgG to protein A is variable and not well characterised (Langone, 1982) the initial preparative step for goat antisera was ammonium sulphate precipitation. The capacity of the haptoglobin column was ca. 25 mg antibody and after exhaustive chromatography the serum antibody concentrations were determined as being 0.34 m g / m l for rabbit and 0.78 m g / m l for goat.
Reactivity of anti-haptoglobin antibodies (1) ELISA. Rabbit anti-mouse Hp serum showed marked cross-reactivity with MSA and haemoglobin (Table III) when the purified serum proteins were immobilised on the plate, as in ELISA method 1 (Table I). Goat anti-mouse Hp sera, which gave multiple bands on RID tests, also cross-reacted with MSA in the same assay system (Table III). The cross-reactivity with haemoglobin could be explained by possible carry over of haemoglobin complexed to the apparently pure TABLE III ANALYSIS OF THE REACTIVITIES OF ANTIHAPTOGLOBIN ANTISERA WITH IMMOBILISED PURIFIED SERUM PROTEINS Irnmobilised protein
% Cross-reactivity Goat anti-mouse Hp serum 100 65 0.9
Haptoglobin MSA Haemoglobin Transferrin (human, Sigma) ND a a ND = not detectable.
Rabbitanti-mouse Hp serum 100 46 23 0.4
2"
1.5-
1-
0-~.
, 1
. . . . . . . .
,
10
,
. . . . . .
~
100
. . . . . . . .
,
. . . . . . . .
,
1000
[XLJTIO~
Fig. 1. Cross-reactivity of affinity purified anti-haptoglobin antibody with serum proteins. Goat or rabbit anti-haptoglobin antibody (10/lg/ml) immobilised on plate. Analytes: II, MHp titrated from 5 ~tg/ml; I~, MSA titrated from 100 /~g/ml; e, MHb titrated from 100 /Lg/ml. Detected by goat or rabbit anti-haptoglobin-alkafinephosphatase conjugatewith pNPP as substrate (see Table 1, method 4). Transferrin and ax-acid-glycoprotein gave similar responses to MHb. haptoglobin used as immunogen. In contrast the cross-reactivity of the affinity purified antibodies was negligible, using ELISA method 4 (Table I and Fig. 1) in which the serum proteins were the analytes. (2) Crossed IEP. Results were similar for both antibodies. Mouse Hp, isolated by antibody affinity chromatography, gave a single peak against specific antibodies and also against antiserum to total mouse serum proteins. Normal mouse serum showed a weak response against both anti-haptoglobin preparations, but multiple peaks against rabbit anti-mouse serum proteins. Acute-phase serum gave a single very large peak against purified antibody, superimposed on the haptoglobin standard, and an intensified pattern of peaks against antiserum proteins.
Development of ELISA for haptoglobin in serum samples ELISA method 2 gave reasonably consistent results for affinity purified haptoglobin (/)so = 0.159 _+ 0.05 /~g/ml, n = 12). The working range of the standard curve was 0.02-0.5 /~g/ml. The same antibody preparation could be used for both
58
plate coating and as enzyme conjugate, indicating that the antibody recognises more than one epitope on the haptoglobin molecule. However, when test serum samples were assayed, very high, unreadable, responses were obtained, even when samples were diluted in excess of 1/10 000. It was not possible to identify the reacting species, although the most obvious serum proteins, albumin and haemoglobin did not cross-react. There was also no apparent cross-reaction with transferrin or al acid-glycoprotein, although these assays were performed using purified human rather than mouse proteins. These very high responses were eliminated when the goat antibody was immobilised on the plate. Using rabbit antibody-enzyme conjugate, the haptoglobin standard response compared well with the original system (/)so = 0.154 _+ 0.022 /,g/ml, n = 4). An alkaline phosphatase conjugate of the goat antibody proved equally satisfactory, and is now used routinely in the assay for test samples (ELISA method 5). The sensitivity of the assay could be increased approximately five-fold by decreasing the plate coating concentration to I # g / m l . It was found to be very important that samples be diluted so that their response fell within the working range of the assay. For mouse sera, this usually involved two serial dilutions of 1/100, to give a final dilution of 1 / 1 0 000. The use of 1% BSA in carbonate buffer as a blocking step after plate coating was also evaluated. This gave a marginal increase is sensitivity (results not shown), but was usually unnecessary as background readings were very low (OD410 < 0.05 AU).
Determination of serum haptoglobin levels The ELISA was validated by analysing normal and acute phase B A L B / c mouse sera in two ways: exhaustive antibody affinity chromatography and ELISA (method 4). A known volume of serum was recycled over the antibody column until no further material could be eluted. The total haptoglobin in the eluate was determined and the concentration in the serum calculated. A good correlation was found between the
TABLE IV COMPARISON OF SERUM HAPTOGLOB1N CONTENT AS DETERMINED BY AFFINITY CHROMATOGRAPHY AND ELISA Serum sample
Normal Acute phase
Serum haptoglobin mg/ml Affinity chromatography
ELISA
0.97 5.30
0.87 5.40
haptoglobin content of serum samples determined by this method and by ELISA (see Table IV). A wide range of haptoglobin levels was found in normal B A L B / c mice (see Table V). As they had not been housed under barrier conditions, the higher haptoglobin levels may be an indication of the animals' health status. Outbred mice tended to have higher levels than B A L B / c (results not shown). Using ELISA method 5 it was possible to detect a ten-fold increase in haptoglobin levels in mouse sera taken 24 h after administration of various doses of concanavalin A (Table VI). Good rank order correlation (r = 0.97) was also achieved between these results and the diameter 2 responses obtained by single radial diffusion for the same samples (see Table VI).
Cross species reactivity It was noted during the early development work that some batches of commercial h u m a n haptoglobin reacted in the ELISA while others did not. As these preparations all showed multiple bands on SDS-PAGE it was assumed that differing degrees of polymerisation prevented epitope recognition by the antibody. In crossed IEP, puri-
TABLE V ANALYSIS BY ELISA OF NORMAL AND ACUTE-PHASE SERA FROM MOUSE AND RAT Serum haptoglobin (mg/ml)
Mouse (BALB/c) Rat (SpragueDawley)
Normal
Acute phase
0.50-1.50 0.99-1.25
3.00-9.25 3.02-7.52
59 TABLE VI CORRELATION BETWEEN RESULTS OBTAINED BY ELISA (METHOD 5) AND SINGLE RADIAL DIFFUSION
assay reported here provides a simple, rapid and highly specific measure for rodent haptoglobin.
Sera taken 24 h after injection of Concanavalin A (n = 5). ConA
ELISA
RID response
(/z g)
Hp (mg/ml)
Diameter 2
0 12.5 25 50 100
0.624 + 0.28 1.886 + 0.904 3.528 + 0.592 8.494 + 1.964 9.331 _ 1.374
38.1 + 1.44 98.1 + 5.7 125.8 + 8.5 192.5 + 4.4 197.4 + 4.6
fied mouse Hp was only weakly recognised by antiserum to human serum proteins. A stronger response was obtained with anti-rat serum but still not so great as mouse Hp - anti-mouse. The ability of the ELISA (method 5) to detect haptoglobin in the sera of different species was evaluated. Samples of rat serum gave strong responses in the assay, with clear differentiation between normal and acute-phase measurements (Table V). However, there was no clear reactivity with normal or rheumatoid human serum, nor did purified human haptoglobin give a titratable response from 10 /xg/ml. In contrast a maximal response was obtained with mouse haptogiobin at 2 # g / m l . These results are interesting in view of the recently reported use of a commercially available anti-human haptoglobin immunodiffusion kit to measure rat haptoglobin (Gilbertsen 1986). It has not been possible to perform the comparable experiment to establish whether purified mouse haptoglobin is recognised by a specific anti-human haptoglobin antiserum.
Conclusion ELISA methods have been reported for measuring many different serum proteins, and of the acute-phase reactants assays have already been published for human rheumatoid factor (Zrein et al., 1986) and serum amyloid A in experimental animals (Zuckerman and Surprenant, 1986). The
Acknowledgements The authors would like to thank Dr. M.E.J. Billingham for helpful discussions, Dr. E.A. Kitchen for performing the single radial diffusion tests and Mrs. E.G. Paculabo and Mrs. P.W. Whiteling for typing the manuscript.
References Baseler, M.W. and Burrell, R. (1981) Acute phase reactants in experimental inhalation lung disease. Proc. Soc. Exp. Biol. Med. 168, 49. Baseler, M.W. and Burrell, R. (1983) Purification of haptoglobin and its effects on lymphocyte and alveolar macrophage responses. Inflammation 7, 387. Engvall, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigencoated tubes. J. Immunol. 109, 129. Gilbertsen, R.B. (1986) Rat haptoglobin: Method of quantitation and response to arthritic therapy in collagen arthritis. Immunopharmacology 11, 69. Javid, J. and Liang, J.C. (1973) The hemoglobin-haptoglobin bond. 1. Dissociation of the complex and recovery of the native haptoglobin in an affinity chromatography system. J. Lab. Clin. Med. 82, 991. Langone, J.J. (1982) In: F.J. Dixon and H.G. Kunkel (Eds.), Advances in Immunology, Vol. 32. Academic Press, New York, p. 157. Mancini, E., Carbonara, A.O. and Heremans, J.F. (1965) Immunological quantitation of antigens by single radial diffusion. Immunochemistry 2, 235. Nagel, R.L. and Gibson, Q.H. (1971) The binding of hemoglobin to haptoglobin and its relation to subunit dissociation of hemoglobin. J. Biol. Chem. 246, 69. Putnam, F.W. (1984) In: F.W. Putnam (Ed.), The Plasma Proteins. Structure, function and genetic control. Academic Press, New York, p. 89. Zrein, M., De Marcillac, G. and Van Regenmortel, M.H.V. (1986) Quantitation of rheumatoid factor by enzyme immunoassay using biotinylated human IgG. J. Immunol. Methods 87, 229. Zuckerman, S.H. and Surprenant, Y.M. (1986) Simplified microELISA for the quantitation of murine serum amyloid A protein. J. Immunol. Methods 92, 37.