Open Access Short Research Article

New Monoclonal Antibodies to Human IgA: Obtaining and Study of Biological Properties

Alexander Galkin, Alex Dugan, Valentine Solovjova, Larisa Bondarenko

Biotechnology Journal International, Page 1-9
DOI: 10.9734/BBJ/2015/18955

Aim: Preparation of monoclonal antibodies to human IgA, investigation of their properties and selection of the most appropriate McAbs for highly sensitive and specific immunoassay tests.

Methodology: Balb/c mice were used for monoclonal antibodies (McAbs) production. Animals were immunized subcutaneously with purified preparation of human IgA. Immunization duration - 7 days, B cells source - regional lymph nodes. Hybridization of immunocompetent cells and myeloma cells was performed with polyethylene glycol as a fusogen. Screening and subsequent hybridoma clones selection was performed by ELISA-test using human IgA and IgA Fc-fragments, IgG and human IgM. To determine the isotype of McAbs, titer, affinity constants, and to identify its comparative epitopic specificity appropriate modifications of ELISA-test were used. 

Results: In our experiments new hybrid clones selection scheme to define the most appropriate McAbs for highly sensitive and specific immunoassay tests was developed. It was established that the most prospective were hybridoma clones producing antibodies against Fc-region of IgA molecule. It was proposed to have a comprehensive description of antibodies, which included the establishment of its isotype, titer, and affinity constants. In view of the further use of obtained McAbs for development of highly informative immunoassay methods, only high titer and affinity antibodies were selected. Since IgM-antibodies are characterized by low specificity and affinity, McAbs of such isotype have not been chosen for further study. Focusing on more efficient McAbs purification on protein A based sorbents, antibodies with IgG2a, IgG2b, and IgG3 isotypes were selected. It was established that there is a correlation between the antibody titer in the culture fluid and its affinity constant: antibodies with titer more than 1:500 characterized by an affinity constant not less than 109 М-1.

Conclusion: A set of 14 new monoclonal antibodies to human IgA has been obtained. Following biological properties of obtained McAbs has been studied: Activity in the indirect ELISA, specificity within IgA molecule (Fab- or Fc- fragment), cross-reactivity with other classes of serum immunoglobulins (human IgG, and IgM), titer in the culture fluid, affinity constant, and relative epitope specificity. Criteria of McAbs selection for their further use in highly sensitive and specific immunoassay methods have been developed. McAbs with following characteristics are the most promising for these purposes: they should  be directed to the Fc-fragment of IgA, have a high signal in the indirect ELISA, absence of cross-reactivity with other classes of immunoglobulins, titer in the culture fluid not less then 1:500, and affinity constant not less then 8.0×109 M-1. McAbs of IgM isotype  are not suitable for effective biotechnological approaches.

Open Access Short Research Article

Effect of Cooking Time on Starch and Cyanide Contents of Freshly Harvested Cassava Tubers Used for Tapioca Production

O. R. Ezeigbo, M. U. Ekaiko, Z. O. Ibegbulem

Biotechnology Journal International, Page 1-6
DOI: 10.9734/BBJ/2015/16944

This study investigated the effect of cooking time on starch and cyanide contents of freshly harvested cassava tubers used for tapioca production. Tapioca is a cassava meal commonly consumed in most part of the world and prepared from freshly harvested cassava tubers. The cassava tubers used for tapioca production are first boiled and sliced, before soaking in water overnight. The cassava tubers used for this study were harvested from the eastern farm of National Root Crops Research Institute, Umudike in Abia State, Nigeria. Some freshly harvested cassava tubers were subjected to 90 minutes cooking in clean water and at intervals of 15 minutes, samples were collected for starch and cyanide analyses. The result showed that increase in cooking time decreased the starch and cyanide contents of the cassava samples. Starch was reduced from 13.37% to 4.65% for every 2 g of starch tuber given 65.22% reduction after 90 minutes. The cyanide content was reduced from 36.65±0.16 to 2.49±0.08 mg/kg after 90 minutes of boiling, given a 93.2% reduction. The statistical analysis on the effect of cooking time on starch and cyanide contents showed significant mean difference (P < 0.0001) at both 5% and 1% levels of the various cooking time of the cassava. It is recommended that cassava used for tapioca production should be adequately cooked to reduce the cyanide content which could result in food poisoning.

Open Access Original Research Article

Bio-bleaching of Anthocephalus cadamba Kraft Pulp through Direct Fungal Treatment by FEQP Sequence

Mohan Lal, Dharm Dutt, Archana Gautam

Biotechnology Journal International, Page 1-13
DOI: 10.9734/BBJ/2015/18845

Aims: The study aims at mitigating pulp kappa number before bleaching to minimize pollution load.

Study Design: An experimental study.

Methodology: The various parameters of direct fungal (Coprinellus disseminatus MLK01) treatment (F-stage) of unbleached kraft pulp of Anthocephalus cadamba were optimized and compared with the results of enzymatically pre-bleaching. Finally, the pulp was bleached by EQP three-stage and XECEHH six stages bleaching sequences.

Results: Direct fungal treatment (F-stage) delignified the Anthocephalus cadamba kraft pulp more selectively with Coprinellus disseminatus MLK01 compared to xylanase prebleached pulp from the same fungus and oxygen delignification. F-stage mitigated the unbleached pulp kappa number by 55.0% and improved brightness and viscosity by 17.3 and 7.63% respectively. Kappa number reduction and brightness improvement were 22.1 and 6.3% more in F-stage compared to XE-stage. The kappa number and pulp brightness of oxygen delignified were 0.9 and 5.2% less compared to F-stage. The viscosity of oxygen delignified pulp reduced drastically due to alkaline peeling reactions compared to XE-stage (‒26.86%) and F-stage (‒27.09%). The brightness and viscosity of XECEHH bleached pulp were 80.1% and 7.4 cps at a chlorine demand of 4.3% while FEQP bleached pulp produced brightness of 79.7% and viscosity 8.2 cps. COD and colour values in effluent generated during FEQP bleaching were 53.29% and 54.36% less compared to CEHH bleaching.

Open Access Original Research Article

Production of Amylase by Arthrobacter kerguelensis VL-RK_09 Isolated from Mango Orchards

Rajesh Kumar Munaganti, Kumar Munaganti, Krishna Naragani

Biotechnology Journal International, Page 1-10
DOI: 10.9734/BBJ/2015/19383

Objectives: To optimize the cultural parameters for improved production of amylase by Arthrobacter kerguelensis VL-RK_09 isolated from Mango orchards of Vissannapet, Krishna District, A.P., India.

Methods: The strain A. kerguelensis was screened initially for amylase production on Inorganic salts starch agar medium (ISP-4). The enzyme assay was performed as per the procedure described by Bernfield (1955). One amylase unit equals to that amount of enzyme needed to release 1 mg of reducing sugar (maltose as standard) for 15 min at 37°C. Attempts were also made to optimize cultural parameters such as pH, temperature, carbon and nitrogen sources affecting the production of amylase by the strain.

Results: Maximal yields of amylase were recorded after 4 days of incubation in Inorganic salts starch medium with initial pH 7.0 and temperature 35°C. ISP-4 broth amended with sorghum flour (2%) and yeast extract (0.5%) with initial pH 7.0 inoculated with Arthrobacter kerguelensis VL-RK_09 and incubated at 30°C for 96 h resulted in improved production of amylase from initial 4.0 U to 10.4 U/mL.

Conclusion: This is the first report on the production and optimization of amylase by A. kerguelensis and further studies on purification and characterization of the enzyme are in progress.

Open Access Original Research Article

Assessment of Genetic Diversity in Ethiopian Sesame (Sesamum indicum L.) Germplasm Using ISSR Markers

Mohammed Abate, Firew Mekbib, Amsalu Ayana, Mandefro Nigussie

Biotechnology Journal International, Page 1-13
DOI: 10.9734/BBJ/2015/18481

Aim: This study aimed to uncover the diversity and population structure of 128 sesame genotypes using ISSR markers and identify highly diverse genotypes for the purposes of broadening the genetic base of sesame landraces grown in Ethiopia.

Place and Duration of Study: The study was conducted in Botany research laboratory of Kasetsart University, Thailand, from April to July, 2013.

Methodology: Genomic DNA of 128 sesame genotypes were subjected to PCR amplification and electrophoresis using seven ISSR markers and a binary data matrix prepared for each primer by scoring clear bands. The data generated were used to calculate the number of total bands (TB), polymorphic bands (PB), polymorphism percentage (P %) and polymorphic information content (PIC) for each locus. The number of different (Na) and effective (Ne) alleles, polymorphic loci (%), Shannon’s information index (I) and Nei’s gene diversity (He) for each population were calculated using GenAlEx 6.5 software. The data were also subjected to analysis of molecular variance (AMOVA) and principal coordinate analysis (PCoA) via distance matrix. Fixation index (Fst) was computed to measure genetic differentiation among populations. Genetic associations among individual genotypes were determined based on dissimilarity matrix using Darwin version 5.0 and a Neighbour-Joining hierarchal tree was constructed based on UPGMA.

Results: The 7 ISSR primers in 128 sesame genotypes yielded 96 reproducible amplified bands. The number of amplified bands varied from 7 to 19. Out of 96 bands, 89 (92.2%) were polymorphic. Average number of bands and polymorphic bands per primer were 14 and 12.6 respectively. The polymorphic information content (PIC) value ranged between 0.26 and 0.76, showing the high informativeness of the selected primers. The overall gene diversity and Shannon’s information index were 0.37 and 0.54 respectively. Average dissimilarity value among the genotypes was 0.39. Maximum dissimilarity (0.88) was observed between genotypes Amr-NW6 and Amr-NG9 and less dissimilarity (0.014) was recorded between Amr-NW1 and Amr-NG1. SNNP-7 was the most diverse of all genotypes with highest average dissimilarity value of 0.77. AMOVA showed lower genetic divergence between populations (6%) than within population (94%) with average Fst of 0.061 across populations. The high intra-population variation could be because of large number of genotypes included and due to high out-crossing nature of sesame. Clustering and PCoA analyses clustered the genotypes into individual groups where most of the landraces were grouped in separate clusters irrespective of their geographic origins, while the cultivars were grouped in one cluster, suggesting less variability within the released varieties than the landraces. Accessions no. 56, 73, and 105 were out grouped from the rest.

Conclusion: There exist considerable variations among sesame genotypes collected from different geographical regions of Ethiopia. Genotypes Amr-NSh-6, Benishangul-6 and SNNP-7 exhibited a good amount of genetic divergence and hence can be used in crossing program for genetic improvement of sesame in Ethiopia.