Serodiagnosis and Immunotherapy in Infectious Disease (1990) 4, 143-149

Reverse passive haemagglutination with freeze-dried stabilized antibody-coupled red cells for detecting rotavirus in faecal samples

J. J. Gray’, Alicia M. Vasquez2, D. R. Kewley2 and R. R. A. Coombs2

‘Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Hills Road, Cambridge and ZImmunology Division, Department of Pathology, Addenbrooke’s

Hospital, Hills Road, Cambridge

A total of 159 samples of faeces collected from patients with gastroenteritis were tested for rotavirus antigen by reverse passive haemagglutination (RPH). When compared with electron microscopy, for detecting rotavirus antigen, RPH had a sensitivity of 96.0% and a specificity of 985%. Simple methods for removing non- specific red cell agglutinins and anti-globulin factors from the faecal samples are reported.

Keywords: Reverse passive haemagglutination, electron microscopy, rotavirus, non- specific reactions.

Introduction

Human rotavirus is an important cause of infantile gastroenteritis’ and has been associated with outbreaks of gastroenteritis in closed communities’. The diagnosis of rotavirus infection can be performed by electron microscopy3, enzyme immunoassay4 or reverse passive (latex) agglutination5.

Cranage et ~1.~ described a reverse passive haemagglutination (RPH) test with monoclonal antibody-linked red cells for detecting rotavirus antigen in faecal samples. We report here on the pre-treatment of the crude faecal sample to remove sheep red cell agglutinating factors and antiglobulin agglutinating factors and the use of this RPH test with stabilized freeze-dried antibody-coupled red cell reagent for diagnosing rotavirus infection over two rotavirus seasons. We compare the results with those from electron microscopy.

Materials and methods

Faecal samples

A total of 159 samples of faeces were collected from patients with gastroenteritis. The samples were examined for bacterial pathogens by routine culture and the presence of virus particles by electron microscopy. A 10% suspension of faeces in phosphate buffered saline (PBS) containing 0.1% sodium azide was used for both electron

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144 J. J. Gray et al.

microscopy and RPH. The 10% extract was well shaken then clarified by low speed centrifugation. The sample supernatant could be stored in this state at 4°C but not frozen.

Electron microscopy

A 5 ml aliquot of the faecal suspension was clarified by low speed centrifugation (5,000 g). The supematants were collected and centrifuged at 150,000 g in an ultracentri- fuge. The resulting pellets were resuspended in 3% potassium phosphotungstic acid (pH 63), placed on a carbon-formvar coated copper grid and examined at a magnification of 50,000 x in a JOEL JEM-100 CX, electron microscope.

Antibodies used

Monoclonal mouse anti-rotavirus, designated A3M4, directed against a group-specific epitope of the virion inner capsid and a control mouse immunoglobulin prepared from the sera of rotavirus antibody-free mice were used to label the sheep red blood cells.

Preparation of antibody-labeled red cells

Chymotrypsin-treated sheep red blood cells were coupled with antibody, stabilized and freeze-dried as described by Cranage et aL6 Rehydration was performed by adding distilled water to the freeze-dried cells and rotating for 2 h at room temperature before use.

Reverse passive haemagglutination test

Initially 30 l.tl clarified faecal suspension (10%) was titrated from 1:2 to 1: 128 in PBS in each of two rows of a round-bottomed microtitre plate. Stabilized sheep red cells, 30 ~1, enzyme-treated and coupled with normal mouse immunoglobulin (control cells), were added to each well of the first row and 30 l.d reconstituted stabilized sheep red cells, enzyme-treated and coupled with monoclonal mouse anti-rotavirus (A3M4, test cells) were added to each well in the second row. The plates were incubated at room temperature until settling patterns could be read (approximately 60-90 min).

The test was subsequently modified to include at least one adsorption with 10% packed freeze-dried gluteraldehyde-stabilized sheep red cells and the addition of 10% virus antibody-free normal mouse serum to the 1:2 dilution of the faecal extract prior to titration.

Results

Incidence of sheep red cell agglutinating factors and agglutinating activity against immunoglobulin-linked red cells

Initially 48 faecal samples were titrated against gluteraldehyde-treated, but uncoupled sheep red cells to determine the proportion of samples with sheep red cell agglutinating factors, which would need removing or neutralizing before a diagnostic test could be performed. Out of 48 samples, 37 (77.08%) showed no haemagglutinating activity, while 11 (22.9%) had this activity. (Figure 1).

Reverse passive haemagglatination for detecting rotavirus 145

0 2 4 8 16 32

Titre of sheep cell agglutinating factor

64

Figure I. Levels of sheep red cell agglutinating factors seen in 48 faecal samples tested with glutaraldehyde- treated sheep red blood cells.

As an adsorption would thus be necessary prior to any test, the 48 samples were adsorbed en bloc with 10% packed gluteraldehyde-stabilized sheep red cells to remove this red cell agglutinating factor. A second adsorption was necessary to remove this factor completely from 8% of the samples.

These 48 samples (adsorbed with sheep red cells) were then titrated against stabilized sheep red cells coupled with normal mouse immunoglobulin (control cells). A total of 29/48 (60.4%) agglutinated these cells to the titres shown in Figure 2. The haemaggluti- nating factor or factors in these samples was presumed to be directed against the coupled mouse immunoglobulin. This was found to be the case as the reaction could be inhibited with a solution of mouse immunoglobulin added to the test system in all but five of the samples. This was done by adding 10% virus antibody-free normal mouse serum to the faecal extract prior to titration. Reactive samples were titrated in PBS containing 0.1 mg ml-‘, O-01 mg ml-’ and 0.001 mg ml-’ mouse immunoglobulin in order to determine the optimum concentration of mouse immunoglobulin required to inhibit this haemagglutinating factor. The mouse immunoglobulin had no effect at a concentration of O-001 mgmll’ whereas 0.01 mg ml-’ reduced the titre of haemagglutinin and 0.1 mg ml-’ eliminated it.

Thus in 43/48 (89.5%) of faecal samples “non-specific haemagglutinating factors” could be eliminated or neutralised by adsorption with fresh or glutaraldehyde-stabilized red cells and also adding normal mouse immunoglobulin to the test system to neutralize antiglobulin activity. (Levels of this activity are shown in Figure 2). The remaining five (10.5%) samples still haemagglutinated to some degree the reconstituted freeze-dried cells coupled with normal mouse immunoglobulin. However these samples also hae- magglutinated uncoupled reconstituted freeze-dried red cells but not glutaraldehyde- stabilized red cells. This suggested a selective agglutinating activity to reconstituted freeze-dried red cells. It was found that this activity could be adsorbed with 10% packed freeze-dried red cells.

146 J. J. Gray et al.

0 <2 2 4 8 18 32 64

Tlfre of sheep cell agglutinating factor

I Glutaraldehyde - fixed Eil Freeze-dried

Figure 2. Sheep red cell agglutinating factors in samples adsorbed with glutaraldehyde-treated red cells and then tested with glutaraldehyde-treated sheep red blood cells coupled with virus antibody free mouse immunoglobulin.

J

There were thus three “non-specific” agglutinating factors which had to be removed or neutralized before crude faecal samples could be screened with freeze-dried antibody- coupled red cells. These were (i) sheep red cell agglutinating factors, (ii) agglutinating factors selective for freeze-dried red cells and (iii) antiglobulin factors reacting with the coupled mouse monoclonal antibody. The sheep red cell agglutinating factors and the agglutinating factors selective for freeze-dried red cells could both be adsorbed with freeze-dried sheep red cells. The antiglobulin factors could be neutralized by adding normal mouse immunoglobulin, free of rotavirus antibody to the test system.

Bacteriology results

Bacteria including Escherichia coli, Clostridium d@cile, Salmonella spp., Campylobacter spp., Shigelia sonnei, and Streptococcus viridans were grown from several of the samples. Giardia intestinalis cysts and bacterial enterotoxins were also found. None of these organisms or toxins interfered with the RPH test.

RPH test for detecting rotavirus in faecal samples

With the above information 159 clarified faecal extracts (10% in PBS/Azide) were adsorbed at least once with freeze-dried sheep red blood cells. The samples were then centrifuged and 30 ~1 titrated from 1:2 to 1:256 in the presence of normal mouse immunoglobulin (5% in PBS). A test was regarded as positive when haemagglutination was observed with the test cells in the absence of agglutination with the control cells or there was a four-fold or greater difference in titre between test and control cells.

A total of 146/159 (91.8%) samples did not react with the control cells. Of these 146 samples, 23 agglutinated the test cells at a dilution of 1:4 or greater (Figure 3) indicating the presence of rotavirus antigen in those samples. Of the remaining 13 samples, seven failed to react with the control cells after a second adsorption with freeze-dried sheep red blood cells. Two were rotavirus positive and five were negative when tested with test cells. The remaining six (3.77%) samples agglutinated both test and control cells at a dilution of 1:4.

Reverse passive haemagglutination for detecting rotavirus 147

2 4 0 16 32 64 128 256

Rotavirus antigen tiire

Figure 3. Rotavirus antigen titres in 25 faecal samples positive in the RPH test.

Comparison of the RPH and EM results

There was good correlation between RPH and electron microscopy. Of the 25 RPH rotavirus positive samples, 23 were positive by electron microscopy. Two samples were RPH positive and electron microscopy negative and one sample was RPH negative and electron microscopy positive (Table 1).

Confirmation of the RPH test results

A specific RPH reaction can be inhibited by adding free specific antibody to the test systems, normal immunoglobulin having no effect. Of the 25 samples positive by RPH only 23 were positive by electron microscopy. To determine if the results seen with the remaining two samples were specific, haemagglutination inhibition was attempted with a 1: 10 dilution of the anti-rotavirus monoclonal antibody A3M4. An irrelevant monoclo- nal antibody was included as a control. Haemagglutination in four presumed RPH positive samples, confirmed by electron microscopy was inhibited when A3M4 anti- rotavirus antibody was added to the test system whereas the irrelevant monoclonal antibody had no inhibitory effect. The haemagglutination seen with the two electron microscopy negative samples was not inhibited in the RPH test with specific rotavirus antibody (Table 2). Thus the RPH test had given a false-positive result with these two samples.

Table 1. Comparison of results from RPH and electron microscopy (EM)

EM Positive

EM Positive

RPH EM RPH Positive Negative Positive

23 2* ~____-._---____

RPH EM RPH Negative Negative Negative

1 133

* RPH reaction not inhibited by A3M4 anti-rotavirus monoclonal antibody indicat- ing non-specificity.

148 J. J. Gray et al.

Table 2. Confirmation of RPH specificity by antibody-inhibition

RPH titre

Specimen _

Inhibition with normal mouse

immunoglobulin

Inhibition with - anti-rotavirus

antibody (A3M4)

Electron microscopy

I 40 < 10 2 640 < 10 3 640 < 10 4 320 < IO 5 40 40 6 40 40

Rotavirus seen Rotavirus seen Rotavirus seen Rotavirus seen Virus not seen Virus not seen

Comparison of freshly prepared antibody-coupled sheep red blood cell reagent and stabilized freeze-dried reagent

Chymotrypsin-treated sheep red blood cells were coupled, stabilized and resuspended to 1% in PBS/Azide for use or in PBS containing 1.5% sucrose (w/v) and 1% bovine plasma albumin for freeze-drying. Freeze-dried cells were reconstituted in distilled water with continuous agitation for up to 2 h.

A total of 36 samples (10 positive by electron microscopy and 26 negative) was tested with both freeze-dried and fresh antibody-coupled enzyme-treated red cells in the RPHA test. The freeze-dried A3MCcoupled red cells failed to detect rotavirus antigen in two electron microscopy positive samples. Both had a low titre of rotavirus antigen when tested with the fresh antibody-coupled cells. Two rotavirus electron microscopy negative samples reacted with freeze-dried control cells even after three adsorptions with 10% freeze-dried glutaraldehyde-fixed sheep red cells. This reaction was not inhibited by the addition of normal mouse serum. The non-specific factor was removed only after adsorption with freeze-dried chymotrypsin-treated sheep red cells indicating the pres- ence of a haemagglutinating factor or factors in these two samples reacting with a receptor on the red cell which had been unmasked by the chymotrypsin treatment.

Discussion

Reverse passive haemagglutination is potentially a very potent diagnostic reaction because of its technical simplicity. The assay is both homogeneous, requiring no washing procedures, and relatively rapid. End-points can easily be determined in the absence of specialised plate-reading equipment. RPH has a sensitivity of 96.0% and a specificity of 98.5% when compared with electron microscopy for detecting rotavirus antigen in clinical samples.

The present investigation with stabilized freeze-dried reagents shows a satisfactory correlation with the much more laborious electron microscopy techniques. The inability of the RPH test to detect rotavirus antigen in one electron microscopy positive sample may indicate a difference in sensitivity between the two tests or an inability to distinguish between two morphologically similar viruses by electron microscopy. The potential for RPH to determine the sub-group to which any particular rotavirus belongs again offers advantages over electron microscopy6.

This study has shown the feasibility of RPH to test crude faecal extracts in the

Reverse passive haemagglutination for detecting rotavirus 149

routine diagnosis of human rotavirus infection. An even more important contribution has been in determining simple procedures for eliminating non-specific reactants to the carrier sheep red cells and the coupled mouse immunoglobulin in faecal extracts. Antiglobulin factors were effectively neutralised with normal mouse immunoglobulin. Sheep red cell agglutinating factors, including those selected for by freeze-drying or enzyme treatment, were adsorbed with uncoupled sheep red cells which had undergone the same treatments. Once these “non-specific” reactants were removed or inhibited, the RPH assay for detecting rotavirus antigen was performed and its specificity confirmed by inhibition with free anti-rotavirus antibody.

Since this investigation was completed the sensitivity of the RPH for detecting rotavirus in faecal extracts has been greatly increased by means of large volume RPH7.

Acknowledgement

We wish to thank Dr T. H. Flewett of the East Birmingham Hospital for supplying us with the A3M4 monoclonal antibody to the virus.

References

1. Blacklow NR, Cukor G. Viral gastroenteritis. N Eng J Med 1981; 304: 397406. 2. Kapikian AZ, Lam SK, Madeley CR, Mathan M, Middleton PK, Woode GN. Rotavirus and

other viral diarrhoeas. Bulletin of the World Health Organisation 1980; 58(2): 183-198. 3. Flewett TH, Bryden AS, Davies H. Diagnostic electron microscopy of faeces. 1. The viral flora

of the faeces as seen by electron microscopy. J Clin Path 1974b; 27: 603-8. 4. Yolken RH, Miotti P, Viscidi R. Immunoassays for the diagnosis and study of viral

gastroenteritis. Paediatr Infect Dis 1986; 5: 4652. 5. Haikala OJ, Kokkonen JO, Leinonen MK, Nurmi T, Mantyjarvi R, Sarkkinen HK. Rapid

detection ofrotavirus in stool by latex agglutination: comparison with radioimmunoassay and electron microscopy and clinical evaluation of the test. J Med Virol 1983; 11: 91-7.

6. Cranage MP, Campbell AD, Venters JL, Mawson S, Coombs RRA, Flewett TH. Detection and quantitation of rotavirus using monoclonal antibody coupled red cells: comparison with ELISA. J Virol Methods 1985; 11: 273-87.

7. Coombs RRA, Kewley DR, Moon DK. Large volume reverse passive haemaglutination greatly increases the sensitivity of the usual RPH assay. Serodiag Immunother Infec Dis 1988; 2: 201-9.

(Manuscript accepted 5 March 1990)