Evaluation of anti-histidine-rich protein 2 monoclonal antibodies, developed by using poly (N-isopropylacrylamide) as an adjuvant for malarial diagnostic application.

OBJECTIVE: To evaluate the sensitivity and the stability of the monoclonal antibodies (Aa3c10, b10c1), against truncated Histidine-rich protein 2 (PfHRP2), developed using smart polymer, poly N-isopropylacrylamide, as adjuvant for malarial diagnostic applications in comparison with the available commercial antibodies. METHODS: Two hybridoma clones (Aa3c10, b10c1) were used for the production of ascites in BALB/c mice. Purification of monoclonal antibodies from the ascites was carried out using affinity columns. The thermal stability study of monoclonal antibodies was done by storing it at 37°C and 45°C for thirty days. The stored antibodies were analyzed using SDS-PAGE and flow-through device where the antigenantibody interaction was visualized by Protein A colloidal gold solution. Sensitivity was determined by endpoint dilution ELISA and the dissociation constant by competitive ELISA. Sensitive pair optimization was done by sandwich ELISA using biotinylated antibodies. Prototype preparation for lateral flow assay had a colloidal gold-based detection system. RESULTS: Thermal stability experiments showed that both mAbs (Aa3c10; b10c1) are stable up to thirty days at 45°C while the commercially available mAbs were stable up to fifteen days only. Compared to commercial antibodies, the mAb Aa3c10, showed the highest sensitivity in end-point titre. In sensitive pair optimization, it was observed that the mAb, b10c1, as a detector and the mAb, Aa3c10, as a capture antibody showed the highest absorbance to detect 50pg/ml PfHRP2 antigen. The prototype formulation of lateral flow assay using the mAbs (Aa3c10; b10c1) showed good reactivity with WHO panel and no false-positive results were observed with twenty clinically negative samples and five P. vivax positive samples. CONCLUSIONS: The novel monoclonal antibodies (Aa3c10, b10c1) against truncated PfHRP2, could be a strong potential candidates that can be included in making RDTs with better sensitivity and stability.

Evaluation of anti-histidine-rich protein 2 monoclonal antibodies, developed by using poly (N-isopropylacrylamide) as an adjuvant for malarial diagnostic application

@#Objective: To evaluate the sensitivity and the stability of the monoclonal antibodies (Aa3c10, b10c1), against truncated Histidine-rich protein 2 (PfHRP2), developed using smart polymer, poly N-isopropylacrylamide, as adjuvant for malarial diagnostic applications in comparison with the available commercial antibodies. Methods: Two hybridoma clones (Aa3c10, b10c1) were used for the production of ascites in BALB/c mice. Purification of monoclonal antibodies from the ascites was carried out using affinity columns. The thermal stability study of monoclonal antibodies was done by storing it at 37°C and 45°C for thirty days. The stored antibodies were analyzed using SDS-PAGE and flow-through device where the antigenantibody interaction was visualized by Protein A colloidal gold solution. Sensitivity was determined by endpoint dilution ELISA and the dissociation constant by competitive ELISA. Sensitive pair optimization was done by sandwich ELISA using biotinylated antibodies. Prototype preparation for lateral flow assay had a colloidal gold-based detection system. Results: Thermal stability experiments showed that both mAbs (Aa3c10; b10c1) are stable up to thirty days at 45°C while the commercially available mAbs were stable up to fifteen days only. Compared to commercial antibodies, the mAb Aa3c10, showed the highest sensitivity in end-point titre. In sensitive pair optimization, it was observed that the mAb, b10c1, as a detector and the mAb, Aa3c10, as a capture antibody showed the highest absorbance to detect 50pg/ml PfHRP2 antigen. The prototype formulation of lateral flow assay using the mAbs (Aa3c10; b10c1) showed good reactivity with WHO panel and no false-positive results were observed with twenty clinically negative samples and five P. vivax positive samples. Conclusions: The novel monoclonal antibodies (Aa3c10, b10c1) against truncated PfHRP2, could be a strong potential candidates that can be included in making RDTs with better sensitivity and stability.

Letter to the editor. Unilateral thalamic infarction in vein of galen and straight sinus thrombosis.

Deep cerebral venous thrombosis is an uncommon event. It usually involves the halami and adjacent areas on both sides. Unilateral thalamic infarction is extremely rare subsequent to deep cerebral venous thrombosis. Imaging findings of a female in the postpartum period with unilateral thalamic infarction and basal ganglia edema are presented.

Production, purification and diagnostic application of filarial recombinant protein WbSXP-1 expressed in salt inducible Escherichia coli.

Wuchereria bancrofti protein WbSXP-1 was identified and established as a potential candidate for the diagnosis of lymphatic filariasis. For the economic production of rWbSXP-1, osmotically (salt) inducible Escherichia coli GJ1158 was preferred. Cultivation and expression was optimized in 3 L airlift bioreactor (ALB) and was successfully extended to 30 L ALB. Purification of rWbSXP-1 his-tag protein was optimized in technical scale using FPLC and the maximal recovery of rWbSXP-1 with significant level of purity was achieved using the combination of IMAC and gel filtration. Quality criteria for immuno-reactivity of purified rWbSXP-1 were established for diagnostic applications. Enhancement of sensitivity in rapid diagnostic format was optimized to effectively detect weak to strong antibody reactivity in individuals exposed to lymphatic filariasis. Performance of the rapid format during field evaluation was successful. The accelerated stability assessment of the rapid format satisfied the requirements of WHO-cGMP norms. This investigation presents a successful technical scale production and purification of rWbSXP-1 considering the future industrial application and an enhanced rapid flow through antibody assay for the diagnosis of human lymphatic filariasis.

Quality control of culture media in a microbiology laboratory.

The nature of reporting of a microbiology laboratory depends upon the quality of the culture media used. Quality of media directly affects the observations and inferences drawn from the cultural characteristics of microorganisms. Checking of different parameters of media such as growth supporting characteristics, physical characteristics, gel strength and batch contamination can help to assess their quality. There are different methods to check all these parameters systematically. The meticulous performance of quality control of culture media can assure precision in reporting.

Periventricular hemorrhage in a developing world. Is drug intervention appropriate?

This study examined the factors associated with the development of periventricular hemorrhage (PVH) in black low-birth weight infants from a disadvantaged community. In addition, the use of drugs as a preventive strategy for the development of PVH was addressed. A total of 177 babies weighing less than 1.5 kg were studied. Eighty (45.2%) were found to have PVH. Grade I PVH was diagnosed in 33 (41.3%), grade II in 37 (46.3%) and grade III in 10 (12.4%) of the babies studied. The overall mortality was 32% (57/177). Mortality of babies with grade I was 33% (11/33), grade II 63% (22/35) and 70% (7/10) for grade III. In those with normal scans the mortality was 18% (17/96). Babies with a lower weight had the severer grade of PVH (P < 0.05). Univariate analyses suggested that lower weight and gestational age, mode of delivery, asphyxia, RDS, shock, hypothermia and the need to administer plasma were the risk factors for both mortality and PVH. However, multiple logistic regression analysis revealed that lower weight (P < 0.001, RR 6.135), asphyxia (P < 0.0004, RR 6.849) and the administration of plasma (P < 0.0026, RR 6.024) were the major factors contributing to death. For the development of PVH weight (P < 0.0001, RR3.719) and the need for plasma (P < 0.03,RR2.079) were crucial. Obstetric complications, place of delivery and booking status did not play a significant role in PVH. The findings in this study suggest that drug intervention in the future may not be appropriate and should be considered when care of the small baby has improved.

Sickle cell syndromes in and around Bardoli.

The study comprised of 2 groups. In group I sickling test was done in students studying in a school which mainly caters to the educational needs of the backward community. Out of 130 students examined 24 were found to be sicklers. The distribution of this cases among various castes/tribes were as follows--Choudharys (Cd)-13, Gamits (Gt)-4, Dhodhia Patels (DP)-4, Koknis (K)-2 and Koli Patel (KP)-1. In group II, patients admitted in the hospital between Jan '81 to June '82 were studied. The prevalence of sickle cell syndrome was 1.74%. The most common mode of presentation were limb pains and weakness. Hemoglobin values ranged from 3.0 gram% to 12 gms%. 35 cases of HbSS, 149 cases of HbAS and 1 case of Sickle Beta thalassemia were seen. The distribution of the cases amongst the various tribes and castes were as follows-Cd-93, Gt-56, DP-23, KP-7, K-4 and Rathods (R)-2. No cases were found in Anavil Brahmins or Patidar Patels. Clinical and pathological observations included palpable splenomegaly in 54 cases, splenic abscess in 1 case, isothenuria in large number of patients, microscopic hematuria in 6 cases and frank hematuria in 1 case. Osteomyelitis and cholecystitis were seen in one case each.

Detection of HIV-1, HBV and HCV antibodies in blood donors from Surat, western India.

PIP: HIV continues to spread in India mainly through heterosexual intercourse, but also among homosexual men and through blood transfusion. The government of India has mandated since 1992 that donor blood from the larger cities be screened for hepatitis B (HBV) and HIV infections. It is expected that this policy will be extended to other cities. Surat is a town 250 km north of Bombay. Approximately 50% of blood is obtained from professional donors to meet the requirement of blood for transfusion. The authors investigated the extent to which blood from professional and voluntary blood donors was infected with HIV-1, HIV-2, HBV, and hepatitis C virus (HCV). They tested 85 blood samples from professional blood donors and 94 samples from voluntary donors from Surat and Pune, respectively, using ELISA and immunoassay. 56 of the professional blood donors were HIV-1-seropositive, 2 of whom were also seropositive for HIV-2. The voluntary donors were seronegative for both HIV-1 and HIV-2. With regard to infection with HBV, 9 samples from professional donors were reactive for HBsAg, 62 of the total 85 samples were reactive for anti-HBs, and 1 sample was borderline reactive. Among the voluntary donors, 6 were reactive for HBsAg, 3 were borderline reactive for HBsAg, and 5 were reactive for anti-HBs. 47 samples from professional donors were reactive for antibodies to HCV, while 13 samples were indeterminate. 4 voluntary donors were reactive for anti-HCV and 5 were indeterminate. The professional blood donors typically were of lower socioeconomic level, among whom awareness about HIV infection and AIDS is incomplete. The voluntary donors, however, were of relatively higher socioeconomic status, and donated blood to help society or as replacement donors. This latter group was more aware of HIV transmission and AIDS. Many blood banks in India still procure blood from professional donors, most likely the reason why parenterally-transmitted HIV infection still takes place in India.^ieng

Cranial ultrasonography, CK-BB and neurological assessment as predictors of outcome in hypoxic ischaemic encephalopathy.

A comparison of cranial ultrasonography, CK-BB and neurological assessment was undertaken in 30 (16 severely, 14 moderately) asphyxiated newborns. In the 16 severely asphyxiated newborns, cranial ultrasonography proved to be of value when changes of 'cerebral oedema' persisted for longer than 48 hrs. Although the mean creatine-kinase (CK-BB) activity at 12-33 hrs after birth was higher compared with that in controls of the same age, the numbers were too small in the different age categories for statistical analysis. The 81.25% specificity of ultrasound as compared with 76.47% of CK-BB suggests that the former investigation is a better indicator of cerebral injury. Phenobarbitone administered as an anticonvulsant in eight patients did not influence the CK-BB levels and the outcome in these babies. The persistence of neurological signs for more than 2 weeks predicted a poor outcome. Sixty-two per cent of the severely asphyxiated babies were light-for-dates, indicating that a chronic condition may have contributed to the poor overall prognosis in these neonates. From a combination of persisting abnormal appearances on cranial ultrasonography, elevated CK-BB activity and neurological impairment, death or significant neurological damage in nearly 90% of the severely asphyxiated babies could be predicted.

Left main coronary artery aneurysm following percutaneous transluminal angioplasty: a report of a case and review of the literature.

Since its introduction in 1977, the number of PTCAs and its indications have grown. Along with more frequent usage, newer complications have been reported. Aneurysm of left main coronary artery is rare. This report describes the formation of a new non-obstructing aneurysm in the left main coronary artery after PTCA of left circumflex artery. The patient has had 7 yr of follow-up with a benign clinical course.

Purification and characterization of E. histolytica antigen for diagnosis of amoebiasis.

Entamoeba histolytica soluble crude antigen was fractionated by gel filtration on Sephacryl S-300 into four fractions, viz. F1(669 kDa); F2(51.2 kDa); F3(25.1 kDa) and F4(10.5 kDa). F1 fraction was observed to be more sensitive and specific for the detection of antibody in amoebiasis than the crude and other fractions of purified antigens employing IHAT and ELISA. ELISA was found to be better than IHAT since it could detect antibody in the sera (3/6) of asymptomatic cyst passers. The cross reaction of crude antigen with toxocariasis (1/4) and toxoplasmosis (2/5) sera were associated with F4 fraction. F3 and F4 were having low molecular weight and were not sensitive in detection of antibody in amoebiasis. Biochemical characterization revealed glycoprotein nature of the specific (F1) antigen fraction.

Development of simple and stable ELISA for detection of copro antigen in intestinal amoebiasis.

A simple, sensitive and stable ELISA (enzyme linked immunosorbent assay) was developed using rabbit antibody to fractionated Entamoeba histolytica antigen for the detection of copro antigen in the faeces of individuals with intestinal amoebiasis. In this test none of the healthy individuals, all trophozoite positive, 40% cyst passers and 6% individuals living in endemic area showed the presence of copro antigen. ELISA using polyclonal rabbit antibody could detect 1-5 trophozoites/well and 20-50 ng protein per well of NIH-200 strain of E. histolytica and the sensitivity of the test was comparable with that using monoclonal antibody. Cross reaction was observed only with E. invadens when faeces having other parasites were screened. The reagents of ELISA were stabilized and found to be stable for more than 6 months when stored at 4 degrees C.

Mannitol in neonatal cerebral oedema.

Twenty-five full term severely asphyxiated newborn babies with cerebral oedema were divided into 2 groups and treated on a randomised basis with either Mannitol or no Mannitol. We report our findings and conclude that Mannitol is of no benefit in the management of cerebral oedema in severely asphyxiated term infants.

Cholesteryl ester storage disease: pathologic changes in an affected fetus.

The prenatal diagnosis of cholesteryl ester storage disease, a rare autosomal recessive disorder, was made by demonstration of deficient lysosomal acid lipase activity in cultured amniocytes from an at-risk fetus. The histochemical and ultrastructural changes in the affected fetus (at 17 gestational weeks) are described and compared to findings in liver and duodenal biopsy specimens from a 9-year-old homozygous female. Massive lysosomal cholesterol and lipid accumulation was demonstrated in fetal hepatocytes, adrenal cells and syncytiotrophoblasts. Of particular note was the observation of extensive necrosis in the fetal adrenal glands. Necrosis of the adrenals may precede the calcification observed in some patients with cholesteryl ester storage disease and in most patients with Wolman disease, an allelic variant due to lysosomal acid lipase deficiency. Fibrosis of the liver and lipid accumulation in macrophages in liver and duodenum, which were present in the 9-year-old homozygote, were not observed in the affected fetus, and therefore, may represent later manifestations of the disease.

Detection and differentiation of metabolic acidosis in parturients.

Base deficit and blood glucose were measured in 36 laboring women in whom metabolic acidosis was clinically suspected. Of 15 parturients, 11 showed presumptive evidence of lactacidemia, two ketoacidemia, and two a combination of both. Differential diagnosis is important since therapy differs: ketoacidosis is treated with intravenous glucose, lactacidosis is treated with continuous segmental extradural block.