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.

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.

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.

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.