Journal of Immunological Methods, 135 (1990) 257-261

257

Elsevier JIM05794

A one-step two-particle latex immunoassay for the detection of Salmonella typhi endotoxin Pak-Leong Lim Department of Microbiology, Unioersityof Hong Kon~ Pokfulam Road, Hong Kong

(Received7 June 1990, revisedreceived20 August 1990, accepted 14 September1990)

A simple and rapid test (LIMM, short for latex immunoassay) is described for detecting Salmonella typhi endotoxin. It involves the simultaneous binding of the antigen by two types of reagent particles contained in a micro-tube: an indicator latex particle coated with a monoclonal antibody specific for the 0 - 9 determinant on the endotoxin, and a magnetic bead coated with another monoclonal antibody specific for a different O-determinant. At the end of-the test, the magnetic beads are sedimented by use of a magnet, and the result is read based on the turbidity of the indicator latex suspension. Compared to a similar assay developed previously which uses only a single particle reagent (i.e., a tube agglutination system), L I M M was found to be slightly more sensitive especially when using short ( < 30 rnin) incubation times, and was at all times easier to read. The sensitivity of LIMM, in fact, increased with increasing time of incubation. When compared to the sensitivity (25 n g / m l ) of a conventional slide latex agglutination test performed using the same indicator latex reagent, this parameter was 0-, 4.9-, 12.5- and 28.7-fold better after 5, 15, 30 and 60 min of incubation in the LIMM. Key words: Latex immunoassay; Salmonella typhi endotoxin; Magnetic bead

Introduction

We are interested in developing simple immunoassays for the detection of Salmonella typhi endotoxin, to assist in the diagnosis of typhoid fever. We have previously described a tube latex agglutination method (TA(3G) which was found to be more sensitive than the conventional slide method (SAGG) (Lim and Fok, 1987). The latex particles used in these tests were coated with a monoclonal antibody (Abl) specific for the 0 - 9 determinant on the endotoxin. We showed subse-

Correspondenceto: P.-L. Lira, Department of Microbiology, University of Hong Kong, Pokfulam Road, Hong Kong.

quently that the results of T A G G could be objectively read using a spectrophotometer (Lim and Choy, 1988). This study describes an improvement of T A G G (LIMM, abbreviated for latex immunoassay), in which a second reagent particle is used simultaneously to capture the antigen. This second particle (Dynabead) is coated with a monoclonal antibody (Ab2) directed against another determinant (N-acetyglucosamine-glucose) on the endotoxin (Tsang et al., 1987). The Dynabead has magnetic properties which allow it, as well as any indicator particle linked with it by endotoxin, to be sedimented at the end of the experiment (Fig. 1). Thus, when endotoxin is present in sufficient quantities, all the particles are removed to the bottom of the tube leaving a clear supernatant. In the absence of antigen, the latex

0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

258 POSITIVE (clear supernatant)

Abt-latex--~ ~

NEGATIVE (turbid supernatant)

~

LPS---4=-~

Fig. 1. Principleof LIMM. suspension remains turbid while the Dynabeads are sedimented.

Materials

Abl was an IgM mouse monoclonal antibody produced in our laboratory (Lim and Ho, 1983). Ab2, also an IgM mouse monoclonal antibody, was a gift from Dr. R.S.W. Tsang (Department of Microbiology, University of Hong Kong, Hong Kong). Both antibodies were purified from mouse ascites fluid by cryoprecipitation following fractionation of the crude material with 50% cold saturated ammonium sulphate. A b l was coupled to latex particles (Rhone-Poulenc, Paris) (Lim and Fok, 1987) and Ab2 to Dynabeads (Dynal, Oslo) (Lira et al., 1988). S. typhi endotoxin was purchased from Difco Laboratories, Detroit. Pooled normal urine or serum was prepared from five healthy laboratory workers.

Methods

In the new latex immunoassay (LIMM), 50 ~1 buffer (0.1 M glycine - 0.9% sodium chloride, p H 8.2) or diluted normal serum or urine, contained in small (40 mm X 6 ram) polystyrene tubes (Sterilin, Feltham, U.K.) were spiked with a known amount of S. typhi endotoxin. A mixture (50 /~1) of Abl-conjugated latex particles (0.01%, final concentration) and Ab2-sensitized Dynabeads (2 X 10 6 particles, final) was added and the suspension was incubated on a roller mixer (Coulter

Electronic; Luton, U.K.) at room temperature for a defined period. The Dynabeads were sedimented by use of a magnet and the results were read visually according to the turbidity of the mixture, and by determining the absorbance (ODal 0 nm) of the supernatant (80 /xl) contained in a microELISA plate (Immunon-2, Dynatech Labs., Alexandria, VA) in an ELISA reader (Dynatech Model M R 710). The end-point of the assay was determined at the 80% absorbance level of the control reaction mixture which contained indicator latex particles alone incubated for the same time. This was also the visual cut-off between positive and negative reactions. The tube latex agglutination test ( T A G G ) was performed in a similar manner with the omission of the Dynabeads (Lim and Choy, 1988). The slide latex agglutination test (SAGG) was performed on a reaction card using 0.5% (final) latex suspension (Lim and Fok, 1987) and incubated for 5 rain on a flatbed (clinical) rotator (Arthur H. Thomas Co., Philadelphia, PA).

Results

Fig. 2 shows the results obtained with LIMM, T A G G and SAGG, when these were performed at the same time using the same preparations of reagents. The results are representative of repeat experiments and show the relative sensitivities of the assays for detecting S. typhi endotoxin in buffer. Thus, the limit of detection for S A G G was found to be 25 n g / m l (final) of antigen. Improved sensitivities were obtained with L I M M and T A G G depending on the period of incubation, which increased with increasing time in both cases. Thus, the limits of detection for L I M M at 5, 15, 30 and 60 min of incubation were 25, 5.2, 2.0 and 0.9 n g / m l respectively. Compared to the sensitivity of SAGG, these were 0-, 4.9-, 12.5- and 28.7-fold better at the respective times of incubation. L I M M was 2.2-fold more sensitive than T A G G at 15 rain incubation, but this decreased to 1.5-fold at 30 min, and at 60 min, there was little difference between them. However, at all times (particularly at 15 rain), the reactions in L I M M were more pronounced and easier to read than the corresponding reactions seen in T A G G .

259

(a)

(b)

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I •

0.4

~

•

•

0.

o O°

~

~

0.1 -

~

~

m

I 25.0

I

I

I

6.25

I

I

I. 56

I

I

0

25.0

Antigen (ng/ml)

I

I

I

6.25

I

I

I. 56

I 0

Antigen (ng/ml)

Fig. 2. Comparison of L1MM (b) with TACd3 (a). Times of incubation: 5 rain (11), 15 rain (e), 30 mill (A) and 60 rain (O). The vertical arrow indicates the limit of detection of SAGG, the horizontal arrow, the 80% cut-off of the mean absorbance of the control latex suspensions (no antigen) obtained at different times. Mean absorbance (+ SD) for (a) = 0.515 + 0.005; (b) = 0.524±0.010.

We have previously demonstrated that TAGG r e s e m b l e s a classical p r e c i p i t a t i o n r e a c t i o n in t h a t t h e r e is a n e q u i v a l e n c e p o i n t b e y o n d w h i c h , at a n t i g e n excess, t h e r e a c t i o n b e c o m e s i n c r e a s i n g l y i n h i b i t e d ( L i m a n d C h o y , 1988). I n t h e p r e s e n t s t u d y , w e i n v e s t i g a t e d w h e t h e r t h e use o f D y nabeads would change the pattern of reaction. The r e s u l t s s h o w n in Fig. 3 i n d i c a t e t h a t L I M M is still l a r g e l y a p r e c i p i t a t i o n a s s a y u n d e r the c o n d i t i o n s u s e d , a l t h o u g h it is less (50%) s e n s i t i v e to i n h i b i t i o n b y excess a n t i g e n t h a n T A G G .

T h e s u i t a b i l i t y o f L I M M f o r use w i t h b i o l o g i c a l fluids was also e x a m i n e d . P o o l e d n o r m a l u r i n e o r s e r u m s u i t a b l y d i l u t e d in b u f f e r a n d s p i k e d w i t h e n d o t o x i n was u s e d f o r t h e s e studies. A s s h o w n in T a b l e I, u r i n e u s e d at a c o n c e n t r a t i o n as h i g h as 1 / 4 b e h a v e d like b u f f e r w h i l e s e r u m , o n t h e o t h e r h a n d , i n t e r f e r e d w i t h b o t h L I M M a n d T A G G at a concentration of 1/40. Thus, the sensitivity of L I M M in this d i l u t i o n o f s e r u m w a s r e d u c e d b y a b o u t t w o - f o l d at 30 m i n i n c u b a t i o n ( d a t a n o t s h o w n ) . N o i n h i b i t o r y effect, h o w e v e r , w a s o b -

TABLE I EFFECT OF URINE AND SERUM ON THE ASSAYS a Assay Abl-latex + Ab2-Dynabeads Abl-latex + Ag b Abl-latex + C-Dynabeads c + Ag Abl-latex + Ab2-Dynabeads + Ag

Urine dilution

Serum dilution

1/4

1/8

1/16

Buffer

1/40

1/160

buffer

(93.4) 3 + (40.5) 2 + (44.4) 3+(16.0)

- (96.4) 3 + (50.0) 3 + (46.6) 3+(15.2)

- (92.2) 3 + (40.8) 3 + (46.9) 3+(15.4)

- (93.2) 3 + (48.0) 3 + (48.0) 3+(15.1)

- (91.3) + (69.8) + (85.1) 2+(37.4)

- (83.6) 3 + (45.4) 3 + (49.5) 3+(19.3)

- (94.3) 3 + (51.0) 3 + (38.0) 3+(18.0)

-

a Reaction mixtures were incubated for 30 min. Results were read visually ( - , negative; + to 3 +, increasing reactivity) or using a reader (in parentheses). The latter are expressed as % turbidity (O1)410 rim) of that of control mixture which contained Abl-latex only. Experiments on urine and serum were performed on separate days using different preparations of Ag. b S. typhi endotoxin (25 ng/ml, final). c Untreated Dynabeads.

260

0.5

g

0 15000

I 3000

I 600

I 120 Antigen

I 24

I 4.8

I 0.96

I 0

(rig/ml)

Fig. 3. Inhibition of LIMM (O) and TAGG (11) by excess antigen. Both assays were performed for 30 min at the same time using the same preparations of reagents (e.g., antigen and latex particles). served when serum was used at 1/160. Stronger solutions of either serum (1/5) or urine (1/2) could not be used in LIMM when the results were electronically read because of their high natural absorbance at 410 nm. The difference in reactivity between LIMM and T A G G was similar whether the test was performed in buffer or in biological fluid.

Discussion

In the development of LIMM, we have retained the simplicity (namely, the one-step procedure) of S A G G while improving its sensitivity and specificity. The latter was achieved using a second reagent particle (Dynabead) which is magnetic, in order to enhance the separation of reacted from unreacted primary (indicator) particles. The idea of using Dynabeads is derived from our previous study (Lim et al, 1988) in which these particles were used in a modified T A G G assay to detect specific IgM antibodies. In this system, however, the reagent (antigen-coated) Dynabeads were first incubated with the test sample prior to incubation with the indicator (anti-g coated) particles in a two-step procedure. Particle agglutination as employed in SAGG and T A G G depends on the formation of

suitably-sized aggregates of the indicator particles. In the conventional tube method (see Derman et al, 1981), this formation requires a long incubation period (90 rain) and the reaction mixture must be undisturbed so that large aggregates can form and sediment under gravity at a rate proportional to their size. We demonstrated previously in T A G G that the reaction can be accelerated using continuous agitation in the incubation,but aggregate settlement was still dependent on gravitational force (Lim and Fok, 1987; Lim and Choy, 1989). Here, we have shown that this dependency can be obviated, in part at least, by the use of magnetic particles, to enhance the separation of reacted from unreacted indicator particles (Fig. 2). Aggregate formation, in fact, is theoretically not required in LIMM, since any individual indicator particle associated with the Dynabeads can be sedimented by magnetic force. However, in practice under the conditions used, L I M M is not completely independent of this requirement. Thus, the assay was found to be irthibitable by excess antigen, although this effect was less pronounced than the effect seen in T A G G (Fig. 3; Lim and Choy, 1988). As expected the inclusion of the Dynabead reagent in the system improves the speed, resolution and sensitivity of the test (Fig. 2). More importantly, it also enhances its specificity. In theory, this would be expected if the Dynabeads and the indicator particles bind to different determinants on the target antigen. Moreover, as apparent from our study (Figs. 2 and 3, Table 1), when LIMM is used in conjunction with T A G G , a specificity check is provided which is not possible when LIMM, T A G G or S A G G are used alone. That is, under suitable test conditions the reaction seen in LIMM should be significantly more pronounced than the corresponding reaction of T A G G if the reaction is real, whereas, if the effect is non-specific, e.g., due to the sheer acidity of the test material, the two reactions should be of similar intensity. Thus, L I M M may be used as a simple and rapid (e.g., 15 min) test not only for detecting S. typhi endotoxin, but potentially, for a wide range of applications including the detection of bacteria and antibodies. Results may be read objectively or, if preferred, by the naked eye. A limitation of

261 the system, however, is that s e r u m samples, unlike urine, have to be used at high dilutions ( > 1/50). A similar interference b y serum has b e e n observed previously i n T A G G a n d S A G G (Lim a n d Fok, 1987).

Acknowledgement I t h a n k Mr. Otis K.H. K o for technical assistance, Dr. R.S.W. T s a n g for gift of the Ab2, a n d Miss Sally Liu for typing the manuscript.

References Derman, R., Corson, S.L. and Horwitz, C.A. (1981) Early diagnosis of pregnancy. A symposium. J. Reprod. Med. 26, 149.

Lim, P.L. and Choy, W.F. (1988) A spectrophotometric method for evaluating a latex agglutination assay of Salmonella typhi lipopolysaccharide. J. Immunol. Methods 115, 269. Lim, P.L. and Choy, W.F. (1989) An improved latex agglutination-inhibition test for the detection of human chorionic gonadotropin in urine. J. Immunol. Methods 117, 137. Lim, P.L. and Fok, Y.P. (1987) Detection of group D salmonellae in blood culture broth and of soluble antigen by tube agglutination using an 0-9 monoclonal antibody latex conjugate. J. Clin. Microbiol. 25, 1165. Lim, P.L. and Ho, Y.M. (1983) Diagnosis of enteric fever by inhibition assay using peroxidase-labeiled monoclonal antibody and Salmonella typhi lipopolysaccharide. Aust. J. Exp. Biol. Med. Sci. 61, 687. Lim, P.L., Ko, K.H. and Choy, W.F. (1989) A two-particle turbidometric latex immunoassay for the detection of specific IgM antibodies. J. Immunol. Methods 117, 267. Tsang, R.S.W., Chan, K.H., Chau, P.Y., Wan, K,C., Ng, M.H. and Schlecht, S. (1987) A murine monoclonal antibody specific for the outer core oligosaccharide of Salmonella lipopolysaccharide. Infect. Immun. 55, 211,