Human Antibodies

Dissociation constant (K_d) is of major significance in immunoassay. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the condition of the assay used.

To employ a rapid and simple ELISA- based method for measuring dissociation constants of two Alkaline Phosphatase (ALP) specific MAbs (A₁G₈F₇ and A₁G₉G₃) established in our laboratory.

A simple and rapid method on enzyme- linked immunosorbent assay (ELISA) was developed for measuring the dissociation constant of antibody – antigen reactions. In this method the ability of increasing amounts of antigen to bind to antibody measured in the fluid phase.

Based on the data obtained from this study, the dissociation constants of A₁G₈F_7 and A₁G₉G₃ MAbs were 3.8 × 10⁻⁹ and 4.3 × 10⁻⁹ M, respectively.

A₁G₈F₇ and A₁G₉G₃ MAbs with reasonably high affinity are suggested for used in very immunoassay such as ELISA, immunocytochemistry and immunihistochemistry (APAAP method).

Immunoglobulin A (IgA) is the second immunoglobulin in human serum. Monoclonal antibodies have many potential uses in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgA are utilized in diagnostic kits.

For production of monoclonal antibodies against human Immunoglobulin A, spleen cells of the immune mouse were fused with SP2/0 as myeloma cells and Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened to determine the antibody by ELISA. The appropriate clones were selected for limiting dilution (L.D). Ultimately, appropriate monoclone was injected intraperitoneally into the mouse that has been primed with Pristane.

In current study, 175 clones were obtained of which 5 clones had absorbance values of about 3 were selected for limiting dilution. Between these clones, 3-D5monoclone with IgG1 subclass was selected as appropriate one and it was reproduced in FCS free RPMI 1640. For large scale production in invivo, the appropriate clone was implanted in the peritoneum of the Balb/c mouse and its titer was determined, which showed 1/100,000 dilution for ascitic fluid, having no cross reactivity with IgM & IgG.

Monoclonal antibody was purified by chromatography, confirmed by SDS-PAGE and then conjugated with enzyme and used for diagnostic kits.

Over the last three decades, monoclonal antibodies have become powerful therapeutics and diagnostics tools. Progress in the antibody engineering, and the appearance of great selection technology such as phage display has made human antibodies production possible against antigens with high affinities.

The purpose of this study was to construct an immune antibody library from a vaccinated donor against tetanus toxin.

A blood sample was drawn from the donor who was vaccinated with tetanus toxoid. PBMC were isolated by using ficoll. After RNA extraction and cDNA synthesis. In order to amplify VH and VL regions, two uniplex PCRs were performed and put considering NcoI & HindIII, MluI & NotI restriction sites respectively for their cloning. These amplicons were cloned to pSEX81 vector and transformed to E. coli XL1blue strain. Eventually, recombinant plasmids were extracted and sequenced.

The result showed acceptable similarity between antibody gene library nucleotide sequences and the antibody genes were deposited in this database.

In this study, the VH and VL genes human antibody library was constructed and confirmed by using DNA sequencing and alignment of sequences with blast database.

Pemphigus and bullous pemphigoid are two autoimmune diseases that have similar pathogenesis. Both have a genetic predisposition, which promotes the production of auto antibodies targeted against different components of the epidermal desmosome and hemidesmosome. Antiphospholipid antibodies (aPL) are heterogeneous group of antibodies found in patients with autoimmune diseases and inflammatory conditions and are associated with thrombotic events.

We sought to determine the expression profile of eight non classical aPLs in ABD patients.

A cohort of 266 serum samples of patients with pemphigus, bullous pemphigoid and controls was screened for the presence of eight aPL antibodies, using the Bio-Rad BioPlex™ 2200 system.

Phosphatidylserine-beta-2-glycoprotein-I (aPS-β2GPI) and anti prothrombin complex (aPT-PT) serum profiles were significantly more prevalent among ABD patients; 20.7% patients with ABD compared to 5.9% of control patients were positive for aPS-β2GPI IgM. In addition, aPT-PT IgM was more prevalent among ABD patients (31% vs. 14.8%).

aPL auto antibodies are more prevalent in ABD. Any clinical association should be further determined.

Multiple sclerosis is a demyeliting autoimmune inflammatory disorder of central nervous system (CNS) with unknown etiology, which afflicts more than 2.5 millions of the world’s population. The aim of this study was to investigate the association between some immunological-related risk factors (tonsillectomy, appendectomy and hepatitis B vaccination) and multiple sclerosis in 2014 in Hamadan city, west of Iran.

As a case-control study, data of filled questionnaire with written consents from 250 patients and 250 age and sex matched controls, were collected and analyzed using SPSS version 16 software and Logistic regression test.

In both groups, the case and the control, with average age of 33 years, 70% of the participants were women. Statistical analysis showed that there was no significant association between tonsillectomy and appendectomy with the multiple sclerosis ( p > 0.05 ). Also hepatitis B vaccination did not increase the risk of MS significantly ( p > 0.05 ), although there was a trend toward more vaccination rate in the patients.

Considering previous global investigations on this topic with the result of our study, it seems more studies are needed to determine the definitive association between tonsillectomy, appendectomy and hepatitis B vaccination with multiple sclerosis.