Iounlal of Immunological Methods. t55 (19~2)201-206

© 1992 El~vier SciencePublishersB.V. All righls re~rved 0022-1759/92/$0.5.00

JIM 06473

A complement-dependent enzyme immunoassay (C-EIA) with increased sensitivity for IgM-rich rabbit sera Hella-Monika Kuhn, Lore Brade and Helmet Brade Dirisiun of Biochemical Microbiology, Forschut~g~iuslitu!Borstel, Instimt ~r E~rperimentcllelli~ogie end Medizin, D.20~I Borstel. Germany

(Received17 December1991,revisedreceived I January 1992,accepled21 May 1992)

An enzyme immunoassay involving activation of complement (C-EIA) was develoged for rabbit polyclonal IgM antibodies against lipid A and lipiJpolysaeeharide antigens. C-EIA w&s significantly higher in sensitivity for lgM-rich rabbit scra compared to EIA using anti-immunoglobulin seeondaw antibodies. Hence, C-EIAshould be useful for the detection of weak IgM reactivities in rabbit sera, especially after short-time immunizations. Selective inhibition of both complement pathways indicated that C-EIA measures activation of the classical pathway. Key words: En~me immunoassay;IgM antibody,rabbil; Complement;Scnsilivity


Solid-phase enzyme immunoassay (EIA) is a sensitive test system which requires minute amounts of antigen and antibody. However, during our studies with anti-lipid A antibodies, we have observed an underestimation of serum activity with various polyclonal rabbit sera in conventional EIA: high tilers in passive immune hemolysis (PIH) were often accompanied by low reactivity in EIA (Kuhn, 1992). It subsequently became apparent that rabbit lgM antibodies were poorly

detected by anti-immunoglobulin secondary antibodies. Thus, the total activity of IgM-rich rabbit sera was only partially estimated in EIA. Since IgM-antibodies possess high complement-fixing capacity, we have used this property to increase the EIA sensitivity for lgM-rich rabbit sera. The development of a complement-dependent EIA (C-EIA) and the results obtained with rabbit scra arc presented here.

Materials and methods AntigoJs

Correspondence lo: L. Bradc, For~hungsinstitut Borstel. Parkallee 22, D.2061 Borslel, Germany. Tel.: 4537-t0470; Fax: 4537-10209. Al~bre~.iations: BSA, bovine serum albumin; C-EIA, complemenl-depeudeutenzymeimmunoassay;EGTA, ethyleneglycol-17is(~-eminoethytelher)-N,N,N',N'-letraacezicacid;LPS, lipop~Jlysaccharide;mab. monoclonalantibody;NHS, normal human serum;PIH, passiveimmunehemolysis;SRBCs,sheep red bloodcells.

Rough LPS was extracted by the phenol-chloroform-petroleum ether method (Galanos ctal.. 1969) from the following strains: Salmonella minnesota R595 (Re chemotype), R4 (Rdz), R7 (Rd~), R5 (Rc), R345 (Rb2), R60 (Ra), SF1286 (Rz), Escherichia colt F515 (Re), J5 (Re), 1:470 (RI core type), 1:576 (R2 core), F653 (R3 core), 2513 (R4 core) and W2131 (KI2 core), Proteus mirabil[~

202 45R (Re) and Vibrio eholerae 95R. Lipid A was obtained from E. coil 1=515, S. minnesota R595 and Proteus mirabilis 45R by acetate buffer hydrolysis of the respective LPSs. Lipid A of P.

mirabilis 028, M. morganii, 13. lupini, P. aeruginasa, P. rettgeri, R. sphaeroides, ~ capsuiotus, R. gelatinosus, R. palustris, C. vinosum and Chlorobium was kindly provided by H. Mayer, Freiburg. Compound LA-15-PP represents synthetic bisphosphorylated hexaacyl E. coil lipid A (Kusumuto et al., 1985). 16 other synthetic lipid A antigens and partial structures differ from compound LA-15-PP by phosphorylati~n and acylatian pattern (number, type and distribution of fatty acids) and - in the case of monosaccharide partial structures - in the backbone sugar (GleN, 2,3-diamino-2,3-dideoxy-D-glucose).

Antisera aM monoclonal antibodies Murine monoclonal antibodies A6 (IgG) and S11-7 (lgM) are directed against lipid A (Kuhn et al., 1992), IgA mab Rc878 against J5-LPS. Kdospecific mab A20 has been described previously (Brade et at., 1987a), Polyclonal rabbit sera nos. 64, 81, 46, 66, 43, 47, 65, 82, 56, 57, 59, 61, F12, FT, I=3 (Brade et al., 1985, 1986, and 1987b) and sera has. 123, 124, 115, R1, R2, 155, 156, 154, 122 were obtained by immunization with various lipid A immunogens including fully synthetic examples. Sera against S. minnesota R5 bacteria (serum no. 6) were taken from the same rabbit after a short and long immunization period. All rabbit scra were complement-inactivated (56°C, 30 rain).

Isolation of lgG and lgM fractions lgG and IgM of rabbit sera were separated with ready-made columns according to the manufacturer's instructions (Viramed, Martinstied, Germany).

Buffers Buffer A, 0.01 M sodium phosphate buffer, pH 7.3, with 0.9% saline; milk buffer, 10% skim milk in buffer A; gelatine buffer, 0.2% gelatine in buffer A; citrate buffer, 0.1 M sodium citrate, pH 4.5; veronaI-BSA, 145 mM NaCI, 1.8 mM sodium diethylbarbiturate, 3.1 mM diethylbarbiturie acid, 0.5 mM MgCIz, 0.15 mM CaCI 2, supplemented with 3% bovine serum albumin.

Human C3 and secondary antibodies Purified human C3 was purchased from Calbioehem, San Diego (USA). Horseradish peroxidase-conjugated goat antibodies directed against human C3, human IgG(H + L) and IgM(~), n~ufine IgG(H + L) and IgM(p.), and rabbit IgC~H + L) wcre ob~alned from Dianova, Hamt'urg (Germany).

Enzyme substrate ABTS [2,2 '- azino-bis(3-ethyl-benzthiazoline6-sulfonic acid)] was obtained from Sigma, Munich (Germany).

Serological assays Passive immune hemolysis (PIH) was performed as previously described (Brade et al., t987b), A conventional enzyme iinmunoassay (EIA) for amphipathie molecules.using anti-lg secondary antibodies was developed in our lab (Kuhn et al., 1992).

Standardization of complement-dependent EIA (CEtA) All incubations were performed at 37°C with gentle agitation, all washing steps with an automatic washer (Dynateeh, Germany). Coating, Polyvinyl pla;es (Falcon 3911, Beeton Dickinson, USA) were coated overnight at 4°C with 100 ng antigen per well (511 p.1) diluted in buffer A. Excess antigen was removed by two washes with buffer A. Blocking. Unreaetive sites were blocked with milk buffer for 1 h. Antibodies. Plates were incubated for 1 h with serial two-fold dilutions of antibodies in milk buffer (25 pA/well) and then washed four times with gelatine buffer. Complement. Normal human serum (NHS, aliquots stored at -70°C) as complement source, was diluted 1/250 with ice cold veronaI-BSA. 25 ~l of the dilution were applied per well and incubated for I h. Plates were washed three times with gelatine buffer, soaked for 10 rain and washed for another three times. Cot~iugare. Anti-human C3-secondary antibody was diluted 1/'750 in milk buffer; 25 /zl were added per well Incubation for 1 h was

203 followed by four washes with gelatine buffer and two additional washes with citrate buffer. Enzyme substrate. 25 ~t substrate (2 mg ABTS and 25 #1 0.1% H 2 0 z per ml citrate buffer) was added. After 30 rain at 37°C, the enzymatic reaction was stopped with 25 ~1 2% oxalic acid. Colour development was measured at 405 n m ( < 0.2 was regarded as negative). Controls without antigen and without antibody were included on each plate.

Treatment of complement Aliqunts of NHS were treated as follows: (i) incubation at 56°C for 30 rain to inactivate con~ponents of the classical and alternative pathwa:,s (Miiiler-Eberhard, 1961; Harrison and Lachman, 1986), and prevent the formation of C3 and C5 convertases; (it) incubation at 500C for 20 rain and immediate cooling to 4°C (inactivation of factor B) allowing only classical pathway activation (Whaley, 1985; Harrison and Lachman, 1986); (iii) dilution in veronal-BSA containing 10 mM E G T A and 7 mM MgCI 2, which inactivates the classical pathway by selective chelation of Ca 2. but leaves the alternative pathway intact (Whaley, 1985).


Assay specif+cily Sin(~ normal human sera may contain specific antibodies to the antigens to be tested in C-EIA, the NHS used as a complement source was first checked in conventional EIA with anti-lg secondary antibodies. At best only marginal titers (maximum titer of 40) were found and false positive results in CoEIA by antibodies of the complement source itself were excluded. We also excluded the possibility that antibodies against rabbit lg present in NHS influence the specificity. In the NHS dilution used (1/250) no reactMty was observed when the assay was developed with anti-human IgG and lgM. Standardization of CEIA was performed with constant dilutions of antibodies. Controls without complement established the absence of cross-reactivity between the anti-C3 secondary antibody and the murine monoclonal IgG, IgM or rabbit serum. Moreover, no


o B T A I N E D WITi-t{A) CON-

TROLS, (B) AFTER HOMOLOGOUS AND (C) HETEROLOGOUS REACTIONOF MLIRINEMARS.AND (D) BY DIRECT BINDINGTO ANTIGENS Constantdilutionof antilx'glics:mabsA6 and A20, I / Ifi01gl0; mab RcS78. I/I(R~ rabbit serum (RS) 81, 1/300 Antigen

Serum or mab A LipidA A6 RSqS-LPS A20 LipidA 81 A6 A2fl 81 B

Iso- Complement NHSdilution

type (NHS) IgM RS IgG + IgM + RS + IgG

A6 lgG + RSq5-LPS A20 lgM + J5-LPS Rc878 lea + Lipid A

C R595-LPS Ab D LipidA RSq,5-LPS J5-LPS Lipid A R595+LPS J5-LPS

tgG +

yielding A.avj> 0.2 < 20 0.2 EIA

IgM-rich IgM-rich igM-rieh

46 64 sl 46 155 156 123

lgM-rich tgM-rich



s2 122 47


It5 56

IgG-rieh IgG-rich

,,3 RI 59

IgG-rich IgG-rieh IgG-rieh


57 4+

lgM-rieh IgM-rieh



lgM-rieh lgM-rieh

IgG-rich IgG.rich


IgG-rieh lgG-rieh

c~l F7 F3 F6 R2

IgG-rfeh IgG-rieh IgG-rich IgG-rieh IgG-rieh



3200 3 200 32011 3 200 3200 3200 6400 f~4110 64110 6 4110 6 400 128fl0 32000

12800 12800 256011 2fi600 25600 51200 2560O 25 600 25600 512110 51200 51 2110 1280110

3 200 6400 6 400 6400 .6400 6400 128011 64000 641YJ0 800OO 320OO0 320(F,)0 1280000 25600il0

1600 6400 3 2(Ill 3200 6400 6400 6400 641JOe 16000 :~{Im)(I flflt~XI 1600L)0 fi4fl()On 6411000

TABLE V TITERS IN PIH, EIA AND C-EIA OF SERUM b (AS) ~.FTER SHORT- AND LONG-TERM IMMUNIZATION AND OF THE RESPECTIVE IgM AND IgG FRACTIONS Repre~nlalive tilers with RS-LPSof at least three tilrations (maximalvariationof I dilution slep). Highestdilutionslysing 50% of antigen-sensitizedSRBCs tn PIH (100 p,g antigen/4 ml 5% SRBCs)or yielding A4qt~> 11.2in EIA and C.EIA ( 10(I ng antigen/well)are shown

column-separated lgG and lgM fractions of both sera revealed the contribution of immunoglobulin classes in the assays ITable Vk whereas reactivity in conventional EIA was almost exclusively derived from the lgG-fraction, the C-EIA liter increase in a serum following short-term immunization was dearly due to lgM.

Serum or fraction

]mmunization time

Titcr oblained in assay PIH EIA C-EtA



Short tong Shorl Long Short I.,~ng

5 120 256fl 2011 640 2560 1280


160t~0 1280C~ 16000 128O11O 2000 20110

1280110 32~) 401"10 32C()0 fi40(H) 8C~1

Assay variation Interassay variation was measured by assaying two high titre and low titre IgM-rich and IgG-rich sera each on four different days. The maximum variation by repeated titrations was one dilution step (Table liD.

Comparison o[ rabbit sera in EIA and C-EIA With the standardized C-EIA, a series of either IgG- or IgM-rich rabbit anti-lipid A sera was tested parallel to conventional E1A. With C-EIA, titers of IgG-rich sera were unchanged or slightly reduced (not more than four-fold; Table IV). All liters of lgM-rieh sera~ however, increased 4-16 times in C.EIA in comparison to conventional EIA (Table IV). The increase in reactivity was actually due to the predominance of IgM antibodies as was ascertained with sera taken from the same rabbit after short- and long-term immunization. Although both sera yielded comparable reactivity in PIH (Table V), the liter of .serum obtained after prolonged immunization was much higher in conventional EIA with anti-rabbit IgG secondary antibody (ratio of EIA titer: PIH liter only 3 following short-term immunization but 50 following long-term immunization). With C-EIA, an eight-fold increase in tiler of the IgM-rich serum following short-term immunization was observed compared to conventional E1A. Testing

During our studies with poiyelonat rabbit antilipid A sera, with various sera we noted an underestimation of serum activity in conventional EIA (Kuhn, 1992a). It transpired that IgM antibodies were only poorly detected ~ the anti-rabbit lgG (H + L) secondary antibody used. Unlike highly reactive secondary antibodies e.gainst routine IgM, various commercially available anti-rabbit Ig or lgM antibodies did not yield satisfactory results. None of these secondary antibodies increased the sensitivity compared to the anti-lgG (H + L) antibody used here (data not shown). Thus, we developed an alternative EIA-system for IgM-rieh rabbit sera. The assay is based on the ability of antigenbound IgM antibodies to induce complement activation. It is well known that both lipid A and LPS can also activate complement by mechanisms which do not require the participation of antibodies (e.g., Morrison and Kline, 1977). Such antibody-independent reactivity of lipid A and LPS was also ob~rved in our assay. However, the assay conditions were designed to eliminate false-positive reactions due to direct activation of complement by the antigens. The standardized C-EIA measured activation via the classical pathway by A g / A b complexes and not merely the binding of C3 as shown by inactivation experiments and lack of reactivity with purified C3. Unlike P1H, C-EIA determines only activation of components CI-C3 (anti-C3 secondary antibody) but not the terminal components of the complement cascade forming the membrane attack complex. This difference in detection of complement activation probably accounts for a discrepancy found with mah S11-7 in both assays. Although binding to SRBC-assnciated antigen was clearly proven, this lgM mab did

206 not lyse antigen-sensitized SRBCs and, therefore, mat) S!]-7 was thought to lack complement activating capacity (Kuhn et al., 1992b). In C-EIA, however, complement activation by mab S11-7 was observed. The difference between detection of complement activation in E I A or PIH parallels observations by other authors. Thus polyspeeiflc natural autoantibodics have been shown to activate complement with solid-phase bound antigen, but not lyse antigen-sensitized SRBCs (Poncet et al., 1988). These antibodies w e r e also unable to fix complemenl in the presence of soluble antigen (no inhibition of anti-SRBC derived hemolysis of SRBCs). It i~ feasible thai the complement cascade is interrupted at a stage later than C3 in cases (e.g., mob S] 1-7) where complement activation is detected by the anti-C3 antibody in C-EIA but not in PIH. It is not known, whether the complement activation determined in C-E]A corresponds to any biological activity in vice. However, as shown with a variety of sera, it did significantly increase the sensitivity of E I A with IgM-rich rabbit sera compared to conventional EIA. We feel that our assay will be useful for the detection of weak IgM reactivities in rabbit sera especially after shortterm immunization. It may aiso be used to test lgM-rich sera with antigens which cannot be tested in PIH since they either do not sensitize SRBCs or are water-insoluble. Finally, comparing titers obtained in parallel in conventional EIA and C-EIA, permits a gross, but rapid decision on whether IgG- or IgM-antibodies predominate in a given serum.

Acknowledgements We thank D. BrOtzmann for expert technical assistance, B.J. Appelmelk (mabs A6 and A20) and F, di Padova (mab Rc878-2/51-9) for providing mouoclonal antibodies, and S. Kusomoto for compound LA-15-PP. This work was supported by the Deutsche Forschungsgemeinschaft (grant Br 731/7 to H.B.).

References Bradc, L. and Brade, H. (1%51 Characterization of two different antibody specificities recognizingdistinct antigenic determinants in frec lipid A oF Escherichia cog Infect. Immun, 48, 776-781. Bradc. L, RiclscheL E.T., gusumoto, S., Shiba,T. and Brade, H. 0986) Immunngenieity and antigenicity of synthetic E.s'cheriehia c'nli lipid A. Infect. Immun. 51, II0-114. Brade. L, Kosma,P., Appclmelk,B,J., Paulsen, H, and Brade, H. (1987a) Use of synthetic antigens to determine Ihe cpitope s~cificities of monodonal antibodies against the 3-deoxy-l)-manno-2-octulosonat¢ region of bacterial lipopolysaccharide, Inject. lmmun. 55, 462-466. Brade, L,, Brandenburg, K. Kuhn, H.-M., Kusumolo, S., Mother, I., Rietschel. E.T, and Brade, H. (1987b) The immunogen[cltyand antigenicily of lipid A are influenced hy its physicochemica[slate and environment. Infect. Iramun. 55~ 2636-2644. Ga]unos, C., Liideritz, O. and Westphal, O. (19fi9) A new method lor the extraction of R lipopolysaccharides.Ear. J. Biochern. 9, 245-249. Haffison, R.A. and Lachman~P.J. (1986) Complement technology. In: D.M. Weir (Ed.), Handbook of Experimental Immunology,4th edn., Vol. L Immunochemistry.Blackwell, Oxford, pp. 39.21-39.23. Kuhn, H.-M (1992) Immune response of rabbits to lipid A: influence of immunogen preparation and distribution of various specificities. Infect. tmmun,, submitted. Kuhn. H.-M., Brade, L.. Appelraelk, B.J.. Kusumoto, S., Rietschel. E.Th. and Brade, H. (1992) Characterization of the cpitop specificity of murinc monnclonal antibodies directed against lipid A. Infect. Iramun., 611,2201-2210. Kusumoto, S., Yoshimura, H., lmoto. M. Shimamoto, T. and Shiba, T. (1985) Chemical synthesis of t-dephospho derivatives of Esc'hc,richia colt lipid A. Tclrahedron Len. 26, 9119-912. Morri~n, D.C. and Klinu, L,F, (1977) Aolivationof Ihe classical and ptoperdin pathways of complement by bacterial lipopolysaccharides(LPS). J. Immunol. 118, 362. Miiller-Eberhard, H.J. and Kunkel, H.G. (1961) Isolation of a thermolabile serum protein which precipitates gammaglobulin aggregates and participat=s in immunhemolysis. Prec. Soc. Biol. Exp, Med. lf16,291. Poncet, P,, Manhes, T., Billecocq, A. and Dighiero. G. (1988) ]mmunochcmiculstudios of polyspccil'icnatural autoanlibodies: charge, lipid reactivity, Fob'2 Iragments activity and complemenl fixation. Mol. Immunol. 25, 981-989. What,, K, (1985) In: K. Wbaley (Ed+), Methods in Complement for Clinical lmmunolnglsls. Churchill Livingstone, Edinburgh, Ch. 3, pp. 84-89.

A complement-dependent enzyme immunoassay (C-EIA) with increased sensitivity for IgM-rich rabbit sera.

An enzyme immunoassay involving activation of complement (C-EIA) was developed for rabbit polyclonal IgM antibodies against lipid A and lipopolysaccha...
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