J. Biosci., Vol. 10, Number 4, December 1986, pp. 475-480. Printed in India. An indirect haemagglutination test to detect serum antibodies to Giardia lamblia K. N. JALAN, TUSHER MAITRA and RITA DAS Kothari Centre of Gastroenterology, The Calcutta Medical Research Institute, 7/2, Diamond Harbour Road. Calcutta 700 027, India Abstract. We used an indirect haemagglutination test with Giardia lamblia trophozoites as the antigen to detect anti-giardia lamblia antibodies in serum, the soluble tritonated Giardia lamblia antigen being used for detecting anti-giardial antibodies in sera of 60 human subjects. Titers in some of these subjects were 1 : 80-1 : 2560. whereas titers in some subjects were negative button to 1 : 20. The results indicated that Giardia lamblia, an intestinal parasite, induced a systemic antibody response and the indirect haemagglu tination test for anti-giardia lamblia antibodies is a simple specific and reproducible system which may be useful in epidemiologic and immunologic studies of giardiasis. The specificity of the anti-bodies was demonstrated by the ability of live Giardia lamblia trophozoites, but not Entamoeba histolytica to absorb the antibody activity. Keywords. Haemagglutination; giardiasis. Introduction Giardia lamblia is now recognised as a frequent gastrointestinal pathogen all over the world (Morbid Mortal Weekly Report Centers for Diseases Control, 1978). The parasitological diagnosis of giardiasis involves repeated examination of faeces and in certain cases examination of duodenal aspirate or jejunal biopsy (Petersen, 1972; Yardley et al., 1972). The application of these methods in large scale epidemiological studies is not practical. Detection of circulating antibody to G. lamblia has recently been demonstrated both by indirect immunofluorescence (IFA) (Ridley and Ridley, 1976) and an enzyme linked immunosorbent assay (ELISA) (Smith et al., 1981). These methods however are not always suitable for epidemiological studies, since IFA technique is subject to observer variation and requires expensive equipment and for the ELISA, live axenic giardia cultures are needed. This study describes a simple test involving indirect haemagglutination (IHA) as the basis for the detection of circulating antibody to G. lamblia both in human subjects as well as in rabbits immunised with whole giardial antigen. Materials and methods Sera Human: Blood was collected from 60 subjects, including both symptomatic and nonsymptomatic human subjects, and sera were prepared. Abbreviations used: IFA, Immunofluorescence; ELISA, enzyme linked immunoabsorbent assay; IHA, indirect haemagglutination; PBS, phosphate buffered saline; SRBC, sheep red blood cells. 475
476 Jalan et al Rabbit: Blood was also obtained from 4 rabbits, each immunised with 3 injections of whole homogenate of giardia antigen at 3 weekly intervals, and from 4 control rabbits which were similarly injected with Freund's adjuvant only without any giardia antigen. Giardia antigen: Giardia antigen, was prepared from axenically maintained portland strain of G. lamblia obtained from Dr. Louis Diamond, National Institute of Health, USA. Organisms were cultured in a modified Diamond's TPS-1 medium supplemented with 3% vitamins and 10% bovine serum (Diamond, 1968; Meyer, 1976). Actively growing, 3 4 days old G. lamblia organisms were dislodged from the walls of the borosilicate (16 125 mm) glass tube by immersing in ice water for 5 min and were then centrifuged (250 g for 10 min). The harvested cells were used as antigen. Antigen for IFA assay: The G. lamblia cells harvested were diluted with 50 mm phosphate buffered saline (PBS) so that 1 drop contained between 1000-2000 organisms per 100 microscopic field. One drop of this suspension was placed in the centre of each glass slide which was then dried at room temperature. The slides, when just moist, were immersed in 90% alcohol for 10 min for fixation. These slides were then usded in IFA assay (Visweswara et al., 1980) using the human serum and fluoreceinisothiocyanate conjugated rabbit anti-human globulin pre pared in this laboratory. Serially diluted human sera samples were used. The same sera dilutions were also evaluated by the IHA test described subsequently. Antigen for IHA test: The harvested G. lamblia cells were suspended in 3 5 ml of 50 mm PBS (ph 7 2) containing 1% Triton X-100. The cells were homogenized using Potter Elvejhem homogenizer. The homogenates were then centrifuged at 9220 g for 30 min (in a Sorval RC 5-SS34 rotor). After centrifugation, the supernatant containing the antigen was dialysed against 50 mm PBS at 4 C overnight. On completion of dialysis, protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. The antigen preparation with a protein concentration adjusted to 2 mg/ml was then used for coating 50% formalised tanned sheep red blood cells (SRBC). Another batch of antigen was prepared in the above way but without the addition of Triton X-100 (non-tritonated). IHA assay: Using the above antigen coated SRBC, haemagglutination assays were performed on microtiter plate.(kessel et al., 1965). Both human and rabbit sera were tested in serial dilutions from 1/20 to 1/10240. With rabbit sera, two sets of haemagglutination tests were done, one using tritonated antigen and the other prepared without triton. Three to four repeat IHA tests were performed on the same sera samples using different batches of antigen, freshly prepared as well as stored in liquid nitrogen. Determination of antibody specificity: Antibody specificity of the indirect haemagglutination assay was evaluated by preabsorbing human and rabbit antibody positive sera alongwith control negative sera with equivalent numbers of trophozoites of G. lamblia. In separate experiments, 1 ml from each of the above sera was incubated for 24 h at 4 C with 1 ml containing 59.2 10 5 washed G.
Detection of giardiasis 477 lamblia organisms and 23.9 10 5 /ml of washed Entamoeba histolytica organisms. The axenic E. histolytica was cultured in Diamond's TYl-S-33 medium (Diamond, 1968). On completion of incubation, the sera were centrifuged at 660 g for 20 min and used for IHA assay using tritonated giardial antigen coated SRBC. Results Rabbit sera It was noted that non-tritonated giardia antigen had no detectable haemagglutination activity, whereas the tritonated fraction gave a positive haemagglutination reaction with rabbit sera immunised with G. lamblia. Sera from control animal showed no haemagglutination (figure 1). Figure 1. IHA titres of the rabbit antigiardia antiserum. IHA assay of this rabbit anti-giardia antisera was done with two types of giardial antigen, (i) Tritonated antigen (ii) Nontritonated antigen i. e. soluble supernatant of homogenized giardia cell( ). All the sera collected from 60 human subjects were subjected to indirect haemagglutination assay test using tritonated giardial antigen coated SRBC. In 38 samples the indirect haemagglutination titres were found to be 1/80 and above and when the stool of these subjects were tested it was found that in 3 repeated stool examinations G. lamblia could be detected. In 18 cases, the sera did not show any haemagglutination activity (titre was very low, 1 : 20, 1 : 40). The stools of these subjects did not show the presence of G. lamblia. However, 4 of the 60 human subjects gave a titre of 1 : 80 or higher but their stool examination gave a negative
478 Jalan et al. result. However, the presence of G. lamblia infection in these 4 patients can not be excluded since neither their duodenal aspirate nor jejunal biopsies were examined (figure 2). No. of stool positive subject No of stool negative subject Figure 2. IHA titres and the stool reaction of 60 subjects. Specificity of the anti-g. lamblia antibody was evaluated by absorbing the sera (figure 3) in two separate sets with E. histolytica and G. lamblia. The E. histolytica absorption did not reduce the indirect haemagglutination titre of the sera, but absorption with G. lamblia reduced the indirect haemagglutination titre signifi cantly. To evaluate the reproducibility of IHA, the same sera were tested by IHA using 3 different batches of antigens and the resultant IHA titres were similar. Correlation of IHA and IFA The human sera tested for IHA were also titred for antibody to G. lamblia using an IFA technique. There was a good correlation (figure 4) between IHA and IFA titres. The correlation coefficient between IHA amd IFA is 0 5601197. Discussion A simple diagnostic tool for detecting giardiasis is needed for epidemiological studies. Epidemiological study involving parasitological evaluation of stool can be difficult. The existing methods of IFA and ELISA for determining antigiardial antibody are too sophisticated and expensive to be within the reach of ordinary
Detection of giardiasis 479 Figure 3. Efficacy of sera of IHA positive and IHA negative subjects after adsorption with G. lamblia and E. histolytica cells.( ),IHA titre without absorption;, IHA titre after absorption with E. histolytica cells;( ), IHA titre after absorption with G. lamblia cells. Figure 4. Correlation plot of indirect haemagglutination assay and indirect fluorescence assay titres of 60 human subjects. Equal dilutions of these human sera samples were used in both the assays. The IHA and IFA titres are indicated in terms of dilutions of the sera samples of these subjects. ( ), Point of coincidence of the result by IHA and IFA assay methods.
480 Jalan et al. laboratories in developing countries. This study undertaken to devise a simple diagnostic method for detecting giardiasis has achieved the following: (i) An indirect haemagglutination test for the detection of antibodies to G. lamblia has been developed. (ii) Sera from rabbits immunised with giardial antigen have confirmed the validity of this haemagglutination test. (iii) The pattern of antigiardial antibody activity in human subjects both positive or otherwise for G. lamblia in stool has been established. The IHA developed is quantitative and reproducible. It is simple and requires only 1a small amount of serum. The antigen once prepared can be stored in liquid nitrogen and used as and when needed. Employing the IHA test, it has been shown that none of the patients with a positive stool reaction had a negative IHA titre and majority of them had titres above 1/80. However, 4 of the control patients with negative stool reaction had titres above 1/80. However, in these cases the presence of giardia in the duodenum can not be excluded. High titres in these patients could be a reflection of an infection in the near past. The haemagglutinating antibody is probably of the IgG type, since antibodies of other types are not expected to have haemagglutinating activity. References Diamond, L. S. (1968) J. Parasitol., 54, 1047. Kessel, J., Lewis, W. and Pasquel, C. (1965) J. Trop. Med. Hyg., 14, 540. Lowry, O. H., Rosebrough, N. J., Farr. A. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265. Meyer, Ε. Α. (1976) Exp. Parasitol., 39, 101. Morbid Mortal Weekly Rep. Centers for Diseases Control (1978) 27, 167. Petersen, Η. ( 1972) Scand. Gastroent., 7 (Suppl. 14), 1. Ridley, M. J. and Ridley, D. S. (1976) J. Clin. Pathol., 29, 30. Smith, P. D., Gillin, F. D., Brown, W. R. and Nash, T. E. (1981) Gastroenterology, 80, 1476. Visweswara, G. S., Smith, P. D., Healy, G. R. and Brown, W. R. (1980) Ann Internal Med., 93, 802. Yardley, J. H., Takano, Τ. and Hendrix, T. R. (1972) Bull. John's Hopkins Hosp., 115, 389.