February 1, 2023

Production of pseudorabies virus recombinant glycoprotein B and its use in an agar gel immunodiffusion (AGID) test for detection of antibodies with sensitivity and specificity equal to the virus neutralization assay.

  • Pseudorabies virus (PrV) causes Aujeszky’s disease (AD), which affects mainly swine, but also cattle, sheep, and wild animals, resulting in substantial economic losses due to animal mortality and lost productivity worldwide. To combat PrV, eradication programs using PrV strains lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable, easy-to-use, and sensitive tests that can detect PrV infection in pigs infected with either wild-type virus or vaccine strain (gE-deleted) virus.
  • To meet this demand, we used the baculovirus-insect cell system to produce recombinant glycoprotein B (gB) as antigen for an immune assay. The high GC-content (70% average) of the gB gene from the Argentinian PrV CL15 strain necessitated the use of betaine as a PCR enhancer to amplify the extracellular domain. Recombinant gB was expressed at high levels and reacted strongly with sera from PrV infected pigs.
  • We used the recombinant gB to develop an agar gel immunodiffusion (AGID) test for detection of PrV antibodies. Compared to the gold standard virus neutralization (VN) assay, the AGID sensitivity and specificity were 95% and 96.6% respectively. Thus, recombinant gB produced in the baculovirus-insect cell system is a viable source of antigen for the detection of PrV antibodies in AGID tests. Considering its relatively lower cost, simplicity of use and result interpretation, our AGID is a valuable alternative tool to the VN assay.

Agar gel immunodiffusion assay to detect antibodies to Type A influenza virus.

The agar gel immunodiffusion (AGID) test is used to detect antibodies to Type A influenza group-specific antigens, i.e., the ribonucleoprotein (RNP) and matrix (M) proteins. Therefore, this test will detect antibodies to all influenza A virus subtypes. AGID is commonly used to screen poultry flocks for avian influenza virus infection. The AGID is a simple and economical serological test.
All serological testing has its advantages and disadvantages which should be considered before choosing the optimal test for the laboratory needs. Each laboratory must evaluate the laboratory’s resources, the volume of testing, the goal of testing, how the test results are used and what types of samples are being tested in order to select the optimal test.

Use of the single cell gel electrophoresis (comet assay) for comparing apoptotic effect of conventional antibodies versus nanobodies.

  • The large molecular size of antibodies is considered one major factor preventing them from becoming more efficient therapeutically. It is well established that all camelids have unique antibodies circulating in their blood called heavy-chain antibodies (HcAbs). Unlike antibodies from other species, these HcAbs contain a single variable domain and two constant domains (CH2 and CH3). HcAbs are a novel type of immunoglobulin-like, antigen binding protein with beneficial pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. Since the antigen-binding site of dromedary HcAb is comprised in one single domain, it was referred to as nanobody.
  • In the present work, the different IgG subclasses from immunized camel (Camelus dromedairus) were purified employing their different affinity for protein A column (PA) and protein G column (PG). Characterization of IgG subclasses was done by using 12% SDS-PAGE under reducing conditions. Protein bands were visualized after staining with Coomassie Brilliant Blue, showing two bands at 50 kDa and 30 kDa in case of IgG1 while IgG2 and IgG3 produce only one band at 46 kDa and 43 kDa respectively.
  • The induction of apoptosis by either conventional or nanobodies was evaluated on two different cell lines, Colon and Hepatic cancer cell (HCT116 and HepG2), using the comet assay. Induced apoptosis were confirmed by visualizing DNA fragmentation bands on 2% agarose gel, and the gel was photographed under UV light. This study demonstrates the successful targeting of human cancer colon cell lines by nanobodies in vitro. It may open perspectives for their future use as tumor target vehicle, due to their small size, soluble behavior and they interact with epitopes that are less antigenic for conventional antibodies.

Performance of an automated solid-phase red cell adherence system compared with that of a manual gel microcolumn assay for the identification of antibodies eluted from red blood cells.

  • IgG antibodies coating red blood cells (RBCs) can be removed by elution procedures and their specificity determined by antibody identification studies. Although such testing is traditionally performed using the tube agglutination assay, prior studies have shown that the gel microcolumn (GMC) assay may also be used with comparable results. The purpose of this study was to compare an automated solid-phase red cell adherence (SPRCA) system with a GMC assay for the detection of antibodies eluted from RBCs. Acid eluates from 51 peripheral blood (PB) and 7 cord blood (CB) samples were evaluated by both an automated SPRCA instrument and a manual GMC assay.
  • The concordance rate between the two systems for peripheral RBC samples was 88.2 percent (45 of 51), including cases with alloantibodies (n = 8), warm autoantibodies (n = 12), antibodies with no identifiable specificity (n = 2), and negative results (n = 23). There were six discordant cases, of which four had alloantibodies (including anti-Jka, -E, and -e) demonstrable by the SPRCA system only.
  • In the remaining 2 cases, anti-Fya and antibodies with no identifiable specificity were demonstrable by the GMC assay only. All seven CB specimens produced concordant results, showing anti-A (n = 3), -B (n = 1), maternal anti-Jka (n = 2), or a negative result (n = 1). Automated SPRCA technology has a performance that is comparable with that of a manual GMC assay for identifying antibodies eluted from PB and CB RBCs.

The gel microdrop secretion assay: Identification of a low productivity subpopulation arising during the production of human antibody in CHO cells.

The long-term stability of high-level expression is the mostimportant factor to consider when choosing cell lines for the expression of recombinant proteins. Declining volumetricyields in large-scale fermentation can be caused by changes affecting the cell population as a whole such as loss in viability, depletion of nutrients or accumulation of metabolites affecting cell growth. Alternatively, geneticinstability may lead to the outgrowth of a less productive,metabolically favored sub-population. Currently a variety ofparameters are measured to monitor the condition of cells infermenters including glucose uptake, lactate accumulation andoxygen consumption; in addition, periodic viable cell countsallow the determination of the growth rate and viability of the population.
All of these methods measure the condition ofthe cell population as a whole and changes must involve a significantly large proportion of the total culture in orderto be detectable. Here we report on a method that allows theevaluation of the productivity of individual cells. Using the gel microdrop secretion assay, we detected the appearance ofa sub-population of cells with lower productivity. Subsequentanalysis of the culture confirmed the existence of lower productivity cells with a lower vector copy number. Therefore,the single cell secretion assay proved to be a rapid method todetect and isolate a low productivity variant of the producer cell line.

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TC Gel

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Comparison of the enzyme-linked immunosorbent assay (ELISA), haemagglutination inhibition test and agar gel precipitation test for detection of antibodies to avian infectious bronchitis virus.

The immune response after vaccination with infectious bronchitis virus (IBV) under field conditions was measured by the ELISA, haemagglutination-inhibition (HI) and agar-gel precipitin (AGP), tests. Vaccinations were performed in three flocks and one experimental group via the drinking water with the vaccine strains H 120 and H 52. In each flock 40 random serum samples were taken every 2 weeks and tested individually. In the experimental group blood samples were collected every week from each of the 10 chickens. The primary vaccination with H 120 resulted in a rapid increase of antibody titre as detected by ELISA followed by a slow decrease over the next few weeks. By the HI and AGP tests no antibody responses could be seen after this primary vaccination. Revaccination with the H 52 strain provoked a further increase in ELISA titres. In the experimental group, and in flock W, a similar increase occurred by the HI test and precipitating antibodies appeared.
The formation of HI antibodies in flock T (nipple waterers) was somewhat retarded and precipitating antibodies were just detectable. In flock F revaccination did not result in the immediate production of HI and AGP antibodies. However, 6 weeks after revaccination a significant rise in ELISA, HI and AGP antibodies was observed, probably as the result of a field infection. It was demonstrated that, based on the higher sensitivity, the ELISA test is more suitable than HI and AGP to monitor antibody responses to vaccination against infectious bronchitis. Strain specificity in the HI test is discussed as a reason for its failure to detect antibodies after primary vaccination with the highly attenuated vaccine strain H 120.

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