Year : 1989 | Volume
: 37 | Issue : 1 | Page : 8--9
ELISA (enzyme linked immuno sorbent assay) as a diagnostic tool in retinoblastoma
PK Kar, R Singh, OPS Maurya
C - 7 New Medical Enclave, Banaras Hindu University, Varanasi - 221005, India
C - 7 New Medical Enclave, Banaras Hindu University, Varanasi - 221005
Estimation of retinoblastoma antigen by double antibody sandwich ELISA technique was carried out in 20 histopathologically confirmed cases of retinoblastoma, 20 cases of pseudoglioma and 20 normal cases. The method of detection was done on the lines of Voller et al. (1976) with slight modification. The number of seropositive cases was more in cases with retinoblastoma.
|How to cite this article:|
Kar P K, Singh R, Maurya O. ELISA (enzyme linked immuno sorbent assay) as a diagnostic tool in retinoblastoma.Indian J Ophthalmol 1989;37:8-9
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Kar P K, Singh R, Maurya O. ELISA (enzyme linked immuno sorbent assay) as a diagnostic tool in retinoblastoma. Indian J Ophthalmol [serial online] 1989 [cited 2020 May 27 ];37:8-9
Available from: http://www.ijo.in/text.asp?1989/37/1/8/26097
Early diagnosis of retinoblastoma may change the outlook of the disease and reduce the mortality. Although thorough history and clinical examination by direct and indirect ophthalmoscopy with scleral indentation, ultrasonography, CT scanning are all helpful in the diagnosis of retinoblastoma, certain other serological and biochemical tests like serum LDH activity, estimation of carcinoembryonic antigen (CEA) and Alphafetoprotein etc. have shown encouraging results.
ELISA is being utilized for the diagnosis of various viral, bacterial and parasitic diseases either for the detection of antibodies or antigens. The present study was carried out to detect the soluble antigen of retinoblastoma in cases with proved retinoblastoma at various stages, in pseudoglioma cases and in the apparently normal cases.
MATERIAL AND METHODS
In the present study circulating retinoblastoma antigen was detected by using double antibody sandwich ELISA technique. At first our antibody to retinoblastoma tissue extract was prepared by injecting it into healthy rabbits. When the antibody titre was sufficiently raised as detected by gel diffusion method, the rabbits were bled and antibodies were separated from the sera of the rabbits and were made to react with the test sera of patients by double antibody sandwich ELISA method.
A total number of 60 cases of both sexes in the age group from 1 year to 60 years were-included in the study. The cases were divided into three groups.
Group A: Control cases (Normal Cases)
Group B: Diagnosed retinoblastoma cases at different stages of the disease.
Group C: Pseudoglioma cases (with white pupillary reflex).
Group A (Control cases):
There were 20 patients who attended the Bhuwalka Eye Hospital for the treatment of refractive error, glaucoma, strabismus or other disorders. They were grouped as test controls.
Group B (Retinoblastoma Cases):
This group included 20 patients with retinoblastoma in its different stages which included early stage (Group B 1) late stage (Group B2) and late stage with metastases (Group B3).
Group C (Pseudoglioma cases):
This group included 20 patients comprising of cases simulating retinoblastoma causing a white reflex such as congenital cataract, persistent hyperplastic primary vitreous, iridocyclitic, cyclitic membrane, endophthalmitis etc.
In all these groups only those patients who did not suffer from any chronic disease like tuberculosis, systemic malignancy, rheumatism etc. were included. All the patients were subjected to the following examinations before proceeding to the estimation of circulating retinoblastoma antigen by ELISA.
(i) Routine medical checkup
(ii) Visual acuity
(iii) Oblique illumination
(iv) Intraocular tension
(v) Direct and indirect ophthalmoscopic examination (where ever possible)
(vi) Biomicroscopic examination(where ever possible) and special investigations such as x-ray of skull, orbit, optic foramen chest and biopsy of tumor were made.
Estimation of circulating retinoblastoma antigen by double antibody ELISA was done in the line of Voller et al. (1976) with slight modification in the Department of Microbiology Institute of Medical Sciences, Banaras Hindu University.
Group A Control Cases
Total number of ELISA seropositive cases was 10, out of these 1.0 positive cases, 3 had traumatic cataract, 4 had ametropia, 2 had glaucoma and 1 had strabismus. Only one case of traumatic cataract was seropositive both at 1:100 and 1:200 dilution.
Group B (Retinoblastoma cases)
Total number of seropositive cases was 16. Four were in the retinoblastoma B 1 stage, 10 were in B2 stage and 2 were in retinoblastoma B3 stage. Out of the 16 seropositive cases, 9 were positive at 1:100 dilution, 4 were positive at 1:200 dilution and only 1 was positive both at 1:400 and 1:800 dilution of serum.
Group C (Pseudoglioma Cases)
Total number of seropositive cases was 15. These 15 seropositive cases comprised of 6 cases of iridocyclitis 5 cases of congenital cataract, 3 cases of endophthalmitis and only 1 case of persistent hyperplastic primary vitreous. Out of these 15 seropositive cases 3 were positive at 1:100 dilution and only 1 was positive at 1:200 dilution.
In the present study our observation shows that 50% of cases in control (Group A) were seropositive though at lower dilution. In case of retinoblastoma (Group B) and pseudoglioma (Group C) seropositivity was 80% and 75% respectively which is comparable with each other statistically, However, the number of seropositive cases at higher dilution was more in case of retinoblastoma (Group B) that Pseudoglioma (Group C) [Table 1][Table 2]. Since no literature on specific retinoblastoma antigen detection by utilising ELISA has been done on comparison of our results with those of other authors can be made.
The reason for many false positive (sero-negative) cases as in control (Group A) - 50% and in pseudogliomas (group C) - 75% may be attributed to the fact that the antigen used in the present study was a crude one [Table 3]. With the facilities available locally it was not possible to use the purified retinoblastoma antigens. Only the soluble part of the retinoblastoma 'tissue which may be containing some substance found in normal eyes also was used in raising the antibody in the rabbit's blood.
In case of retinoblastoma (Group C) 4 patients were seronegative (20%) of which 3 were in subgroup 131 and 1 in subgroup B2 [Table 3]. The reason for this seronegativity is not understood, however, it is felt that since in the earlier stage of retinoblastoma there is less necrosis of the tumor, there may not be any circulating retinoblastoma antigens in the blood of the patient which can be detected by the ELISA.
Thus, we conclude from this study that ELISA may be an adjunct to other methods of diagnosis of retinoblastoma specially in differentiating it from pseudoglioma.
|1||Voller,A.;Bidwell,D.anmd Bartlett,A.: Microplates Enzyme immunoassays for the Immunodiagnosis of virus infections. Chapter 69, Manual of Clinical Immunology 1976.|