A glycophospholipid (GPL) antigen isolated from Plasmodium falciparum culture supernatant hasbeen tested for its antigenicity. Detection of malaria positive known blood samples and unknown fieldsamples from endemic and non-endemic areas were compared. In this study laser light scattering immunoassay(LIA) was used for the detection of P. falciparum malaria. Test results of control (malaria negativesamples from Surat) were compared with known positive samples and unknown malaria positivefield samples. A positive correlation has been observed (97%) in falciparum positive samples from laboratoryand unknown samples from endemic area (Haldwani) by LIA method using GPL antigen. Fromthe results of the study it was found that GPL antigen has a better antigenic property and can detectalmost all the cases of Pf malaria by LIA method

A. Roy, S. Biswas, M.M. Mya, R.K. Saxena & K.B. Roy

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J Vect Borne Dis 40, September & December 2003, pp 105–108Testing of newly developed glycophospholipid antigen for thedetection of P. falciparum malaria by laser light immunoassayin endemic and non-endemic areasA. Roya*, S. Biswasa, M.M. Myab, R.K. Saxenab & K.B. RoycaMalaria Research Centre(ICMR), 22 Sham Nath Marg, Delhi; bCentre for Biomedical Engineering, Indian  Institute  of Technology, New Delhi;  cCentre for Biotechnology, Jawaharlal Nehru University, New Delhi, IndiaA glycophospholipid (GPL) antigen isolated from Plasmodium falciparum culture supernatant hasbeen tested for its antigenicity. Detection of malaria positive known blood samples and unknown fieldsamples from endemic and non-endemic areas were compared. In this study laser light scattering immu-noassay (LIA) was used for the detection of P. falciparum malaria. Test results of control (malaria neg-ative samples from Surat) were compared with known positive samples and unknown malaria positivefield samples. A positive correlation has been observed (97%) in falciparum positive samples from lab-oratory and unknown samples from endemic area (Haldwani) by LIA method using GPL antigen. Fromthe results of the study it was found that GPL antigen has a better antigenic property and can detectalmost all the cases of Pf malaria by LIA method.Key words Laser light immunoassay – microscopic examination – per cent positivity – Pf malariaPlasmodium falciparum (Pf ) malaria is a commoncommunicable disease of third world countries andcauses high morbidity and mortality.  The disease canbe controlled if it is diagnosed and treated at an earlystage of onset. Several methods for the diagnosis ofmalaria are available and the most common method ismicroscopic examination of blood smears. Althoughmicroscopic method provides a reliable diagnosis ofthe disease but some times due to poor quality ofblood smears the results may not be accurate and italso depends on expertise and diligence of technicians.Several other diagnostic techniques for malaria havebeen developed such as fluorescent dye technique,buffy coat technique, Kamamoto fluorochrome tech-nique etc1,2. All these methods have certain limitationsas that of light microscopy.Among immunological methods the enzyme linked im-munoabsorbent assay (ELISA) has been found to bequite sensitive2,3. Several other antigen and monoclonalantibody based methods for the diagnosis of malariahave been developed such as Immunochromatographictest4, ParaSight ‘F’ test5, OptiMAL assay6 and rapiddipstick antigen capture assay7, but all these immuno-logical methods are expensive and beyond the reach ofpoor developing countries where malaria is highly en-demic. Methods based on the identification of parasitespecific nucleic acids by use of the polymerase chainreaction8-11 have been used for parasite detection. Un-fortunately all these methods are costly, needs skilledtechnical manpower and sophisticated laboratory.Therefore, it is felt that there is still a need to develop asimple, rapid and economical method for diagnosis ofmalaria.  Recently, laser light scattering immunoassay *Corresponding  authorJ  VECT  BORNE  DIS  40, SEPTEMBER & DECEMBER  2003 106(LIA) method has been reported to be very sensitivetechnique for the detection of Pf malaria12 and hasbeen found more sensitive than enzyme immunoassay.LIA technique for the detection of Pf malaria is a sim-ple method and can diagnose 95% of all Pf cases.Several antigens had been used for the detection ofmalaria but none had been found to provide very highrate of immunoreactivity with positive blood samples.We have isolated and purified a new kind of Pf anti-gen—glycophospholipid (GPL) (inositol free) from invitro culture supernatant of P. falciparum.  An anti-genic property of GPL has been evaluated by LIAmethod using different known positive blood samples.Material & MethodsPreparation of glycophospholipid (GPL) antigenfrom Pf culture supernatant : This antigen was iso-lated from P. falciparum in vitro culture supernatantwhich was made free from erythrocyte and parasitedebris by differential centrifugation, concentrated, dial-ysed, lyophilised and extracted with chloroform-meth-anol-water mixture according to our published proto-col13. Chloroform extract was found out to be glyco-phospholipid. This antigen contains galactose, glucose,xylose and mannose. It is completely soluble in etherand chloroform. It is a sticky material, difficult to han-dle and its TLC mobility is slow indicating presence oflipid.Study groupMalaria positive blood sample collection :  Activefever case blood samples (50) were collected in Sep-tember to October 1999 during transmission seasonfrom endemic area of Haldwani. Blood sampleswere collected with the informed free consent of thepatients and stored on filter paper.Negative control blood collection : Humans subjectsaged between 20 and 40 years of both sexes were se-lected after careful medical examinations and thosehaving no history of malaria were used as negativecontrol.  Blood samples negative for malaria (negativecontrol) were collected from Surat. Finger prick bloodwas collected on filter paper. Requisite approval wastaken for collection of blood from the donors. Bloodsamples on filter paper were stored at 20oC in deepfreezer.Testing of blood samples by LIAPreparation of antigen coated latex beads for LIAstudy : 25 µl of 1 mg/ml of the antigen solution and 1ml of 1% latex beads in carbonate-bicarbonate buffer(pH 9.6) were mixed and kept over night at 4oC. Lat-er, latex bead suspension was incubated at 37oC forone hour in an incubator. Then the bead suspensionwas washed three times with distilled water and col-lected by centrifugation at 20,000 rpm at 25oC for 30min. Washed beads were resuspended in 100 µl of1% BSA (bovine serum albumin) solution . Suspendedbeads were reincubated for one hour at 37oC. Afterincubation the beads were washed again three timeswith distilled water and centrifuged at 20,000 rpm at25oC for 30 min. After final washing, the beads weresuspended in 1 ml carbonate-bicarbonate buffer (pH9.6) and kept at 4oC. 18 µl of antigen coated latexbeads and 2 µl of 1:100 diluted blood samples [2punched discs (5 µl blood in each) from filter paperwas eluted in 0.5 ml 0.1M PBS (pH 7.2)] was thor-oughly mixed in a 1.5 ml eppendorf tube. The mixturewas then immediately drawn into a 6 cm long and 3mm diameter glass capillary tube so that the mixturestays in the lower one-third part of the capillary tube.Both ends of the tube were closed with paraffin waxand kept for one hour at room temperature. After one-hour, sample capillary tube was placed in the sampleholder of LIA. LIA measurements of blood sampleswere done as per published procedure14.Study of blood samples from endemic and non-en-demic field areas by LIA methodStudy subjectsIn laboratory : About 50 finger prick blood sampleswere collected during transmission season from villag-ROY et al :  GPL ANTIGEN FOR PF  MALARIA BY LIA 107es Shaulana and Lalpur, District Ghaziabad in theyears 2000 and 2001 on filter papers from micro-scopically identified Pf patients and 50  samples fromhealthy individuals as negative control. Sample bloodwas eluted from filter paper with PBS buffer just be-fore laboratory testing.  P. falciparum specific antigenmolecules like GPL113 were used and studied by LIAtechniques and their results were compared with thatof  standard microscopic method.Control group : Blood samples were collected fromhealthy donors (Surat, Gujarat) and served as negativecontrols. About 50 samples of finger prick blood werecollected on 3 mm thick Whatman filter paper. Thickand thin blood smears on glass slides were also pre-pared for microscopic examination to confirm the ab-sence of malarial parasites.Endemic area : About 50 active fever cases fingerprick blood samples from Haldwani, District Nainital,Uttaranchal (identified endemic area) were collectedon 3 mm thick Whatman filter paper for LIA study.Also thick and thin blood smears on slides were madefor microscopic confirmation of the disease.Results & DiscussionAntigenic sensitivity of isolated P. falciparum glyco-phospholipid, devoid of inositol has been measured bylaser light immunoassay in blood samples of patientsand healthy individuals to test its efficiency in detectionof malaria parasite. Percentage positivity values of LIAwere compared with the results of microscopy. Thesensitivity of LIA on latex agglutination in terms of se-ropositivity has been calculated in terms of a cut-offvalue decided as mean value of control (healthy) sera+2 SD  and found to be 96% with GPL when com-pared with microscopy in laboratory  (Fig.1 & Table 1).Immunopositivity was calculated. Significance of val-ues was calculated by student ‘t’ test and comparedwith control for the determination of  ‘p’ value. Allcomparisons of values showed p < 0.0001 in all thesets of measurements. Filter paper blood samplesfrom field studied by LIA method showed 97.36%detection rate with GPL antigen, whereas samples ofthe control group were found negative.  Highest sensi-0100200300400500600700800900Ghaziabad Surat HaldwaniAREAMEANPARTICLESIZEINNANNOMETRESFig. 1: P. falciparum positivity detected by laser lightimmunoassayTable 1. Comparison of malaria positivity by microscopy and laser light immunoassayMicroscopic LIA of laboratory    LIA of field  samplesexamination samplesSurat (control, n= 50)   Haldwani (Pf 38/50)Pf +ve (50) Pf + ve 96% (48) 0 (0) 37 (97.36)Mean + SD 804.96 + 182.23 98.7143 + 3.8527 699.047 (237.309)p-value < 0.0001                                                  < 0.0001Figures in parentheses indicate samples detected positive; Cut-off value —106.J  VECT  BORNE  DIS  40, SEPTEMBER & DECEMBER  2003 108tivity for detection of malaria was seen with LIA andresults are compared with earlier results describedelsewhere12,14. Blood samples showed that LIA withGPL antigen is a sensitive and reliable method for thedetection of falciparum malaria. Present study pro-vides information on the antigenicity of GPL and its usein detection of Pf infected blood by LIA method.AcknowledgementAuthors are thankful to the laboratory staff for theirexcellent technical/field support.References1. Ward Law SC, Levine RA.  Quantitative buffy coat analy-sis : a new laboratory tool function as a screening com-plete blood cell count. J Amer Med Assoc  1983; 249 :617–20.2. Piper RC, Vander Jagt DL, Holbrook JJ, Makler M.  Malar-ia lactate dehydrogenase target for diagnosis and drugdevelopment. Ann Trop Med Parasitol 1996; 90 : 433.3. Palmer CJ, Lindo JF, Klakala W, Quesada J, Kaminsky R,Ager AL.  Evaluation of the Optimal test for rapid diag-nosis of Plasmodium vivax and Plasmodium falciparummalaria. J  Clin Microbiol 1998; 36 : 203–6.4. Taylor D, Voller A. Development and validation of a sim-ple antigen detection ELISA for Plasmodium falciparummalaria. Trans R  Soc  Trop Med Hyg 1993; 87 : 29–31.5. Verle P, Bink L, Lieu TT, Yen PT, Coosemans M. Para-Sight F-test to diagnose malaria in hypo-endemic areasand epidemic prone regions of Vietnam. Trop Med IntnatlHlth 1996; 1 : 794–6.6. Peyron FG, Martet JP, Vigler A.  Dipstick antigen-captureassay for malaria detection. Lancet 1994; 1 : 1502–3.7. A rapid dipstick antigen capture assay for the diagnosisof falciparum malaria. Bull WHO 1996; 74 : 47–54.8. Snounou G , Viriyakosol S, Jarra W, Thaithong S, BrownKN. Identification of the four human malarial species infield samples by the polymerase chain reaction and de-tection of a high prevalence of mixed infections. Mol Bio-chem Parasitol 1993; 58 : 283–92.9. Humer A, Ohrt C, Harrington MA, Pillai D, Kain KC.ParaSight F test compared with their polymerase chainreaction and microscopy for the diagnosis of Plasmodi-um falciparum malaria in travellers. American J Trop MedHyg 1997; 56 : 44–8.10. Kawamoto F, Miyake H, Kaneko O, Kimura M, NguyenQ, Liu TD, Zhou M, Le DD, Kawai S, Isomura S, WatayaY.  Sequence variation in the 18S rRNA gene, a target forPCR-based malaria diagnosis in Plasmodium ovale fromsouthern Vietnam. J Clin Microbiol 1996; 34 : 2287–9.11. Oliveira DA, Shi YP, Oloo AJ, Boriga DA, Nahlen BL,Hawley WA, Holloway BP, Lal AA. Field evaluation of apolymerase chain reaction-based non-isotonic liquid hy-bridization assay for malaria diagnosis. J Infect Dis 1996;173 : 1284–7.12. Bhakat P, Roy Arati, Roy Kunal B. Laser light scatteringimmunoassay for malaria. J Immunoassay 1999; 20 : 103–44.13. Mya MM, Roy Arati, Saxena RK, Roy KB. Isolation, partCharacterization, Immunogenicity, and specificity studyof Plasmodium falciparum culture supernatant. Japa-nese J Infect Dis 2002; 55 : 150–6.14. Mya MM, Saxena RK, Roy A. Sensitivity and specificityof isolated antigen from Plasmodium falciparum culturesupernatant. Int J Clin Biochem 2002; 17 : 75–82.

Journal of Vector Borne Diseases

Testing of newly developed glycophospholipid antigen for the detection of P. falciparum malaria by laser light immunoassay in endemic and non-endemic areas

Testing of newly developed glycophospholipid antigen for the detection of P. falciparum malaria by laser light immunoassay in endemic and non-endemic areas

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