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| Year : 1985 | Volume
: 31
| Issue : 3 | Page : 146-9 |
Detection of amoebic antigen by enzyme linked immuno-sorbent assay (ELISA).
Bhave GG, Wagle NM, Joshi UM
How to cite this article:
Bhave G G, Wagle N M, Joshi U M. Detection of amoebic antigen by enzyme linked immuno-sorbent assay (ELISA). J Postgrad Med 1985;31:146 |
The diagnosis of amoebiasis is usually based either on direct demonstration of vegetative forms of Entamoeba histolytica in the stool or pus samples or indirectly by demonstrating high antibody levels. But, vegetative forms are difficult to demonstrate and antibodies to E.H. persist for a longer time even after recovery from the infection. Antigen detection is a recently developed specific, diagnostic method and gives a rapid and accurate diagnosis of the active stage of the disease. It has been used for a number of bacterial, viral and fungal infections but not much tried for parasitic infections.[1],[3],[4],[10]
Different types of techniques liks counterimmunoelectrophoresis or ELISA have been used for the detection of antigen.[1],[3],[10] In the present study we have used ELISA technique for the detection of amoebic antigen in the supernatent of the pus and serum samples from clinically suspected cases of amoebiasis.
A total of 100 samples were tested, of which 56 were pus samples and 50 were serum samples. Out of 50 pus samples, 25 were from amoebic liver abscesses and 25 were control samples from cases of purulent peritonitis, empyema etc. Out of 50 serum samples, 25 were from healthy blood donors and 25 were from cases of clinically suspected hepatic amoebiasis with clinical signs and symptoms of fever, anorexia, tenderness in the liver region etc. The liver function tests in these cases were not grossly abnormal. Pus samples were centrifuged at 4°C for 1 hour at 4000 r.p.m. and the supernatent was used for the test. Serum samples were used without further treatment.
Soluble amoeba antigen (ICN Laboratory, USA) was conjugated with enzyme penicillinase EC 3-5-2-6. (HA Limited, India) using glutaraldehyde as a coupling agent.[2] The conjugate was purified by passing it on the Sephadex G-200 (1.5 cm x 30 cm column). Specific antibody was raised in rabbit by injecting soluble amoebic antigen mixed with Freund's adjuvant (1:1) at multiple sites every week for four weeks. Antibody was further purified by saturated ammonium sulphate precipitation. 'U' shaped polyvinyl microtitre ELISA plates were used for adsorption of antibody. The optimum dilutions of coating antibody (diluted in 0.075 M Barbitone buffer, pH 9.6) and conjugate (diluted in 0.1 M phosphate buffer saline. PBS, pH 7.2) were found out by checkerboard titration using known positive and negative controls.
The test was performed as follows: Specific antibody (diluted appropriately with PBS) was adsorbed onto the plate by overnight incubation at 4°C. After rinsing with 0.05 M NaCl, 200 µl of test samples were added in each well on the plate and were incubated at 37°C for half an hour. Plates were rinsed again, and 200 µl of appropriately PBS diluted conjugate were added, followed by incubation as earlier. Plates were rinsed once again and starch-iodine penicillin V reagent* was added. When the known negative sample decolourised, the reaction was stopped by addition of 50 µl of 5 N HCl.
Colour change of the indicator from blue to colourless indicated negative result. No change in colour indicated positive reaction for the presence of amoebic antigen. Known positive and negative controls were accompanied with each batch of the test.
Out of 25 pus samples aspirated from amoebic liver abscess cases, 23 were positive for amoebic antigen and 2 were negative. Out of these 23 positive cases, 22 patients received antiamoebic treatment; they responded well and were subsequently discharged. One patient died and at post mortem a huge liver abscess occupying almost ¾th of the liver was seen. The scrapping from the abscess wall and histopathology sections showed vegetative forms of E. histoloytica. Amongst two negative cases of pus samples, one case was false negative. This was a 36 year old female, a known case of amoebic liver abscess. She was tapped many times, and vegetative forms of E. histolytica were demonstrated in the pus. The pus tested by ELISA was greenish in colour and showed growth of Pseudomonas. The second negative case was a true negative. This was a post mortem pus sample from a liver abscess of a 12 year old child who died of septicaemia and showed pyemic liver abscess at autopsy. The culture from liver abscess showed Staphylococcus aureus. Unfortunately, serum samples of all these patients could not be tested by ELISA.
Out of 25 serum samples from the test group, 6 samples were positive for amoebic antigen. In these 6 cases, 3 were pediatric cases and two were adult cases which were treated with antiamoebic drugs and discharged. The remaining case was of a 50 year old female admitted for vaginal ulcers. She was operated in the recent past for prolapse of the uterus and gave history of colitis. Biopsy from the ulcer showed large number of amoebae. The final diagnosis in all the remaining 19 negative cases was other than amoebiasis [Table 1].
Incidence of amoebiasis reported from selected population of India and many other countries is more than 20% and in food handler families it is upto 50%.[5] Liver is the commonest site for metastatic amoebiasis. Amoebic liver abscess carries high morbidity and mortality. Mahajan et al[9] have reported the utility and reliability of amoebic antigen detection in pus and serum samples but they have used counterimmunoelectrophoresis test.
Joshi et al[6] were the first to establish the use of enzyme penicillinase in ELISA but they used this technique for hormone assay. The enzyme penicillinase is quite suitable for use as it is locally available, cheap and conjugate is stable at 4[0]C for a year. The final colour differentiation is distinct as it is very easy to differentiate between dark blue and colourless appearance. There was no false positive test in the control or the study group. The one false negative in the test group was due to secondary Pseudomonas infection. The other negative case was a true negative as it turned out to be a case of pyemic liver abscess. The chances of finding. E. histolytica in the pus from a case of liver abscess varies from 20 to 40%.[8] Hence antigen detection appears to be a more reliable and specific diagnostic method for amoebic liver abscess. In the serum samples the positivity was much less as clinical presentation of hepatic amoebiasis is often vague. Antigen detection in serum may prove to be of great help in differential diagnosis of hepatic amoebiasis.
Further evaluation of the technique for the detection of amoebic antigen in stool, homogenate from rectal biopsies and material from other foci of amoebiasis might prove to be of extreme importance for the diagnosis and prognosis of amoebic infection.
Authors are thankful to Dean, Seth G.S. Medical College and K.E.M. Hospital for permission to publish the paper.
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