Latex agglutination assay for human anti-Brucella IgM antibodies

Journal of Immunological Methods, 122 (1989) 169-175

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Elsevier JIM05276

Latex agglutination assay for human anti-Brucella IgM antibodies C.L. C a m b i a s o a n d J.N. L i m e t Universit~ Catholique de Louvain, UnitOde M~decine Experimentale, 75, avenue Hippocrate, 1200 Brussels, Belgium

(Received16 January 1989, revisedreceived6 March 1989, accepted 24 April 1989)

We report here the development of a homogeneous, easy, precise and rapid anti-Brucella IgM antibody (Ab) latex agglutination assay based on particle counting. The interference of IgG Ab was eliminated by the addition of anti-),Fc and free Bru-LPS. Anti-/~Fc mAb enhanced the agglutinating activity of IgM antibodies and improved the sensitivity of the test. The possible interference of rheumatoid factor was eliminated by adding human aggregated IgG. The assay is complete in 45 min, with an interassay variation of 9%. The assay correlates well (r = 0.94) with the accurate but time consuming capture ELISA. Key words: Brucella; IgM antibody; Latex agglutination

Introduction Brucellosis is often diagnosed clinically and by the demonstration of antibodies (Ab) in serum. The pathogen may be isolated from blood but the bacteraemia is short and intermittent. The diagnosis is therefore often based on serology. The presence of a high level of IgM Ab is recognized as the indication of a recent primary infection in various diseases. For the diagnosis of brucellosis serum agglutination, complement fixation and the indirect anti-human globulin-assisted agglutination tests have been widely used. Immunofluorescence (IF) is often combined with the Correspondence to: C.L. Cambiaso, Universit6 Cathofique de Louvain, Unit6 de M&iecine Experimentale, 75, avenue Hippocrate, 1200 Brussels, Belgium. Abbreviations: Ab, antibody or antibodies; mAb, monoclonal antibody; LPS, lipopolysaccharide; Bru-LPS, Brucella lipopolysaccharide; Bru-LPS-Lx, Brucella lipopolysaccharide coated latex particles; Ig, immunoglobulin; IgG, immunoglobulin G; IgA, immunoglobufin A; IgM, immunoglobulin M; Ig(A, M or (3) Ab = anti-Brucella Ig(A, M or (3) antibodies; GBS, glycine-bufferedsaline; EDTA, ethylene-diamine-tetraacetic acid; BSA,bovine serum albumin.

slow agglutination in tube a n d / o r the Rose Bengal test (for a review see Heizmann et al., 1985). Using the classical tests, the presence of a high IgM Ab titer may not be diagnostic of a recent infection, because both I g G and IgM Ab are concomitantly detected. IF results with specific antisera for IgG, IgA or IgM, are easier to interpret but nevertheless competition between the Ab of the different classes may lead to false negative results for IgM and IgG Ab (S. Poncelet and J.N. Limet, personal communication). More sophisticated methods have been described for anti-Brucella IgM Ab (Bru-IgM Ab) estimation, including radioimmunoassay (RIA) (Hewitt and Payne, 1984) and enzyme-linked immunoadsorbent assay (ELISA) (Marmonier et al., 1979; Sippel et al., 1982; De Klerk and Anderson, 1985; Araj et al., 1986). These techniques require an extraction step and several washing procedures which make the techniques time consuming. Moreover, relatively unstable reagents such as an: tibody-coated tubes or microplates and labelled or enzyme-conjugated antibody are needed. The immunoassays based on latex particle agglutination (Masson et al., 1981) do not require

0022-1759/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)

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complex and unstable reagents in either the separation or washing steps. Moreover, latex particles are easily coated with Bru-LPS (Limet et al., 1988). Such particles are strongly and very rapidly agglutinated in the presence of IgM Ab. Much higher concentrations of IgG Ab and longer incubation are needed to give equivalent agglutination. Nevertheless when both types of Ab are present in a serum the resulting agglutination depends on the concentration of both IgG and IgM Ab. The present paper describes the development of an easy, precise, rapid, homogeneous and reproducible assay for IgM Ab using Brucella lipopolysaccharide-coated latex particle (Bru-LPS-Lx).

Materials and methods

Brucella abortus biovar 3 (field strain isolated at National Institute for Veterinary Research) was cultivated in Roux flasks according to Alton et al. (1975). Cells were inactivated by heating to 80 °C for 1 h, washed with saline and water and either used as such or lyophylised. Brucella LPS (Bru-LPS) was extracted by the water-phenol procedure of Westphal et al. (1965) and further purified according to Moreno et al. (1979). Finally, LPS was dialysed against water, clarified by centrifugation at 10000 × g for 10 rain and lyophylised. Such extracted material was subsequently quantitatively expressed in terms of its dry weight. Bru-LPS latex particles (Bru-LPS-Lx) were coated as previously described (Limet et al., 1988). Reagents. Glycine-buffered saline (GBS), 0.1 M glycine, 0.17 M NaC1 and 6.15 mM NaN 3, pH 9.2 was used singly or supplemented with 50 mM EDTA (GBS-EDTA) or with 50 mM EDTA and 10 mg/ml bovine serum albumin (GBS-BSAEDTA). BSA was obtained from Calbiochem-Behring Corp., La Jolla, CA. Human IgG was purified by combined etha~ridine lactate (Siegried Zofingen, Switzerland) and ammonium sulfate precipitation, dialysed against isotonic saline and stored frozen at - 20 ° C. Briefly, a pool of blood donors' sera was five-fold diluted in isotonic saline and precipitated using 0.66 vols. of saturated ammonium sulfate. The pellet was resuspended in water and dialysed

against isotonic saline. Remaining contaminating protein was then precipitated by mixing the dialysed IgG solution with 4 vols. of 0.4% ethacridine lactate in water at pH 7.5. After 15 min agitation the mixture was centrifuged at 10 000 × g for 10 rain. Ethacridine lactate was then precipitated by the addition of saturated KBr and removed by centrifugation. The IgG of the dialysed supernatant was concentrated by precipitation with one volume of saturated ammonium sulfate and dialysed against isotonic saline. Aggregated IgG was obtained by heating a 10 mg/ml solution of IgG for 30rain at 63°C. D T T reduction of IgM Ab. To 50/11 serum were added 170/~l of GBS-EDTA, pH 9.2 and 15/xl of dithiothreitol (DTT) (10 mg/rnl). The mixture was incubated at 37°C for 15 min and the excess of Dq-T was destroyed by the addition of 15 /xl of 0.2% hydrogen peroxide. Polyclonal and monoclonal Ab (mAb). Anti-7Fc antisera: the Fc fragment of human IgG was prepared by papain digestion as described by Cerotthai (1968). A goat was repeatedly injected intradermally every 3 weeks. After four injections, booster doses were repeated every month and the goat bled monthly 1 week after each injection. Anti-/~Fc and aFc mAbs were prepared according to classical techniques (Van Snick and Coulie, 1982). Mice were immunized with the Fc fragment of the Ig. Anti-LPS mAb was prepared as described (Limet et al., 1987). Latex as immunosorbent. Human serum albumin (HSA) or anti-aFc mAb were covalently coupled to carboxylated latex as previously described (Limet et al., 1982). Latex agglutination assay. Agglutination reaction was performed in 3 ml polystyrene tubes and reagents were dispensed manually with the help of micropipettes. Reagents were vortex mixed in a specially designed incubator-agitator thermostated at 37 °C by circulating water. The general assay procedure was as follows: 50/~1 of a 1/50 dilution of test sera in GBS-BSA-EDTA were vortex mixed for 10 min with 25/~1 of saline with or without the various anti-Ig. 25 /~1 of heat aggregated IgG (10 mg/ml) were then added to the mixture which was incubated again for 10 min. 50 /tl of this mixture were then taken and vortex mixed for 10 rain with 25 /tl of Bru-LPS-Lx. The reaction was

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stopped by the addition of 2 ml of GBS and unagglutinated particles were counted in an optical counter which discriminated the unagglutinated from the agglutinated latex particles (Masson et al., 1981). The concentration of agglutinant Ab was therefore inversely proportional to the number of unagglutinated particles. Capture ELISA for IgM Ab. This assay will be described in detail elsewhere (Poncelet et al., in preparation). Briefly, microplates were sensitised by incubation with the anti-/~Fc mAb. IgM present in 50-fold dilutions of serum were captured and specific Ab revealed by incubation with a mixture of Bru-LPS and anti-Bru-LPS mAb coupled to horseradish peroxidase. Sera. The sera used to develop the assay were from a patient whose disease was monitored using the ELISA. Two sera were used and were IgM Ab and IgG Ab rich, respectively. The first corresponded to a serum taken when symptoms first appeared (acute brucellosis, only IgM Ab) and the second to a serum having the maximal anti-Bru IgG titer. This serum was reduced with DTT in order to inactivate remaining IgM Ab. The sera used for the correlation study were obtained from patients with acute and chronic brucellosis (n = 31), kindly provided by Prof. Audurier from the Trousseau Hospital from Tours (France) and the sequential sera from the patient in whom the evolution of antibodies was followed (n = 18).

Results

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DTT reduction as described in the materials and methods section. Higher anti-TFc concentrations tended to increase the agglutination mediated by IgG Ab. A good inhibition was already observed after 10 min of incubation.

Interference of IgG Ab-anti-TFc on IgM Ab activity: correction with free LPS When IgG Ab and IgM Ab were mixed together in the presence of anti-TFc Ab, the agglutination of LPS-coated latex particles was weaker than that observed with IgM Ab alone suggesting that IgM Ab are inhibited by the IgG Ab-anti-TFc immune complexes. This inhibition of the IgM Ab increased with the concentration of IgG Ab (Fig. 2) and was clearly attenuated at high Bru-LPS-Lx concentration (Fig. 3). This inhibition could, however, be completely avoided by the addition of free Bru-LPS to the latex suspension. As shown in Fig. 4, in the pres-

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Fig. 1 shows that polyclonal anti-TFc Ab at a concentration of 1.25 mg/ml inhibited completely the agglutination observed with the IgG Ab rich serum in which IgM Ab had been inactivated by

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Fig. 1. Neutralization of IgG Ab by anti-TFc. A 1/50 dilution of the IgG Ab rich serum was tested in the presence of various concentrations of anti-'rFc according to the protocol described in the materials and methods section.

Enhancement of the agglutinating activity of IgM Ab by anti-#Fc mAb A mAb anti-/tFc (up to 60/xg/ml) was found to enhance, by a factor of 3-5, the agglutination initiated by IgM Ab, the effect clearly depending on the anti-#Fc mAb concentration. Incubation times greater than 10 min did not improve the agglutination.

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Fig. 2. Inhibition of IgM Ab by anti-yFc-IgG Ab complexes. A 1/50 dilution of the IgM Ab serum was tested diluted twice either in normal serum 1/50 ( 0 ) or in the IgG Ab rich serum 1/50 (ra) or 1/100 (11) after a 10 rain incubation with anti-TFc (1.25 mg/ml).

112 100 60

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agglutination assay was only weakly influenced by increasing the total IgG or IgM concentration up to 300% of the normal values. The IgM antibody titer fell by only 9% when the serum contained three times the normal IgM concentration. Such an increase in the normal level of IgG gave rise to less than 10% agglutination by the IgG Ab rich serum.

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Rheumatoid factor interference Sera containing RF with titers higher than l/500 in the latex IgG agglutination test (Behring Institute, Marburg, F.R.G.) were found to agglutinate the Bru-LPS-Lx only in the presence of high concentration of IgG Ab. The latter interference could be eliminated by the addition of human heat aggregated IgG at a final concentration of 2.5 mg/ml but it was essential that this aggregated IgG be added after the first incubation of the sera with anti-yFc and before the addition of the Bru-LPS-Lx. A 10 min incubation was sufficient to neutralize the strongest rheumatoid factor found.

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Fig. 3. Influence of the Bru-LPS-Lx concentration on the inhibition of IgM Ab by anti-y Fc-IgG Ab complexes. As in Fig. 2, a l/50 dilution of the IgM serum was tested diluted twice either in normal serum l/50 (+) or in the IgG Ab rich serum l/50 (o), in the presence of anti-yFc, with Bru-LPS-Lx at various concentrations: 0.1% (top panel), 0.02% (middle panel) and 0.01% (w/v) (bottom panel).

ence of Bru-LPS at a concentration of 5 pg/ml the agglutination due to IgM Ab was the same in presence or absence of IgG Ab. Effect of total IgG and IgM serum concentration on the IgM Ab titer An increase in the total IgM concentration of test sera will change the relative proportion of anti-PFc to IgM Ab. The effect of the anti+Fc may therefore be decreased. When the total IgG concentration of the serum increases, more antiyFc is consumed and part of the IgG Ab may not be inactivated. When this was tested the latex

Role of IgA Ab The influence of IgA Ab on the agglutination process was studied using a serum containing the three classes of Ab. This serum was depleted of its IgA by immunoadsorption with anti-aFc latex. This immunoadsorption was controlled with an HSA-coated latex immunosorbent and the in-

TABLE I REPRODUCIBILITY OF LATEX AGGLUTINATION ASSAY FOR HUMAN ANTI-BRUCELLA IgM ANTIBODIES Serum no. Within day 1 2 3 4 Between days 5 6 I 8

Mean a (arbitrary units)

SD

CV (W)

321 159 112 31

21 1.9 5.53 2.56

8.5 4.9 4.9 6.8

1184 1442 697 128

64.46 114.56 55.11 8.61

5.4 1.9 1.9 6.7

’ Values were calculated from the mean of three dilutions.

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Fig. 4. Inhibition of the activity of anti-yFc-IgG Ab complexes by free LPS. IgM Ab sera diluted 1/200 mixed with normal serum 1/50 ( , ) or with IgG Ab rich serum 1/50 (D) was tested in the presence of anti-#Fc (60/~g/ml) and anti-yFc (1.25 m g / m l ) and various concentrations of free LPS in the latex suspension: A (0/xg/ml), B (0.5/~g/ml), C (1.25 # g / m l ) and D (5 # g / m l ) .

fluence of anti-aFc mAb on the agglutination of Bru-LPS-Lx studied. As shown in Fig. 5, with the serum adsorbed on HSA-coated latex, agglutina100 80

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tion was decreased by both anti-yFc and anti-aFc and the agglutination was still weaker when both anti-Ig were added together. With the IgA-depleted serum, anti-aFc did not influence the agglutination, either in the presence or the absence of anti-yFc. The agglutination observed in the presence of anti-yFc approximately corresponded to that observed with the sera adsorbed on HSAcoated latex in the presence of anti-yFc alone. Therefore the reduction of the agglutination due to anti-aFc mAb was not due to the inactivation of the IgA Ab but rather to an inhibition of IgM Ab by the immune complexes formed between the IgA Ab and the anti-aFc mAb. It was concluded that in the absence of anti-aFc, IgA Ab does not modify significantly the agglutination mediated by IgM Ab. The agglutination observed in the presence of the anti-yFc was therefore directly proportional to the activity of IgM Ab.

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Fig. 5. Effect of IgA A b on the agglutination of Bru-LPS latex in the presence of IgG and IgM Ab. A serum containing high AB titers of the three Ig isotypes was adsorbed either on anti-aFc (bottom panel) or HSA (top panel) latex immunosorbents and tested in the absence of anti Ig (D), in the presence of anti-aFc (60 # g / m l , II) or anti-yFc (1.25 m g / m l , 0 ) and of both anti-Fc (O). Free LPS (5 ?tg/ml) was added t o the latex suspension.

Final procedure for the determination of IgM Ab To 50 #1 of human serum diluted 1/50 in GBS-BSA-EDTA, was added 25 #1 of isotonic saline containing 1.25 m g / m l goat anti-human yFc IgG and 60 # g / m l of anti-#Fc monoclonal IgG Ab. After 10 rain vortexing at 37°C, 25 #1 of aggregated human IgG at a concentration of 10 mg/ml were then added and the mixture further

174

vortexed for 10 min at 37 o C. Finally, to 50/xl of this mixture were added 25 gl of Bru-LPS latex (0.05% w/v) containing 5 g g / m l of free Bru-LPS. After a further 10 min incubation, the reaction was stopped by the addition of 2 ml of GBS. The resulting mixture was then passed through the cell of the optical counter to measure the concentration of unagglutinated particles. The concentration of IgM Ab was inversely proportional to the concentration of the free latex particles.

Reproducibility Four sera with different titers of IgM Ab were tested 20 times each on the same day (within a day) and 20 times on 20 different days in a month (between days). Titers in arbitrary units were estimated by comparison with an IgM Ab rich serum to which a value of 1000 arbitrary units was ascribed. These results are summarized in Table I. Correlation with ELISA A close correlation (r = 0.94) was observed, on 49 sera, between the finalised latex agglutination assay and a capture ELISA for IgM Ab developed in the laboratory. The results were related by the regression equation y = 0.998x + 35.87.

Discussion Various techniques for the estimation of IgM Ab have been reported. Isokinetic-sucrose gradient fractionation of the test serum is probably the most reliable technique but is expensive, time consuming and requires sophisticated equipment (Desmyter et al., 1971). Alternative procedures for removing IgG by the addition of anti-human IgG have been reported (Gispen et al., 1975) but liquid phase absorption is not recommended because of the likelihood of specific IgG:anti-IgG-soluble complex formation which could interfere in the subsequent immunoassay. Solid-phase anti-IgG absorbents are preferable but in practice they often lack the capacity to effectively remove all the IgG (Chantler and Diment, 1981). Anti-3,Fc-IgG complexes effectively inhibited the agglutination of Bru-LPS-Lx by IgM Ab. This inhibition could be neutralized by the addition of free LPS. The affinity of IgG Ab being in general

higher than that of IgM Ab, complexed IgG Ab probably interacts more easily with free LPS than IgM Ab which prefer the polymerized LPS on the latex particles. The concentration of LPS needed to completely inhibit the residual activity of IgG Ab produces an inhibition of IgM Ab which results in a loss of about 10% of agglutinating activity. Nevertheless this loss of sensitivity is largely compensated by the enhancement of the agglutinating activity of IgM Ab by anti-gFc mAb. The adsorption experiment with anti-aFccoated latex particles indicates clearly that IgA Ab do not modify significantly the agglutination of the Bru-LPS-Lx when they are in competition with IgM. This is probably due to the fact that IgA are generally poor agglutinators and that the incubation time for the assay is very short (10 min). Moreover we have never found IgA Ab alone in the sera of brucellosis patients which we have analysed by capture ELISA for IgM and IgA Ab (Poncelet et al., in preparation). IgA Ab could be assayed by their inhibitory activity (in the presence of anti-aFc) toward the agglutination mediated by the IgG a n d / o r IgM Ab. Coefficients of correlation greater than 0.9 have been obtained with a capture ELISA for IgA Ab (data not shown). However the value of titrating IgA Ab is unclear and the latex agglutination assay procedure rather complicated. The good correlation observed between the latex agglutination assay and the capture ELISA for IgM Ab was also a strong indication that IgM Ab are correctly titrated when anti-TFc and free LPS are added to the serum before the addition of the Bru-LPS-Lx. The capture ELISA was chosen as the reference test because of its accuracy and sensitivity as well as the absence of competition between antibodies of the various classes. In conclusion, anti-Brucella IgM Ab are easily, accurately titrated by the latex agglutination assay described. The assay is faster than ELISA and uses very stable reagents. If necessary, it could also be fully automated since it does not involve extraction and washing steps.

Acknowledgements This work was supported by Grants nos. BR 1/4 15009/014 from the Institut pour la Re-

175 c h e r c h e S c i e n t i f i q u e d a n s l ' I n d u s t r i e et l ' A g r i c u l ture and by the Commission of the European Communities, contrast BAP-0123-B, 1986-1989. The excellent technical assistance of Claire Vander M a e l e n a n d J o h a n n V a n B r o e c k was g r e a t l y a p preciated.

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